AU1789400A

AU1789400A - Screening methods

Info

Publication number: AU1789400A
Application number: AU17894/00A
Authority: AU
Inventors: Dario Renato Alessi; Antonio Casamayor; Jeremy William Thorner; Pamela Diane Torrance
Original assignee: Medical Research Council; University of California; University of California Berkeley
Current assignee: Medical Research Council; University of California
Priority date: 1998-12-14
Filing date: 1999-12-14
Publication date: 2000-07-03
Also published as: WO2000036135A2; JP2002532100A; WO2000036135A3; EP1141001A2; CA2355362A1

Description

WO 00/36135 PCT/GB99/04228 1 SCREENING METHODS The present invention relates to screening methods for drugs or lead compounds, enzymes, to polynucleotides encoding enzymes and to uses of enzymes and 5 polynucleotides. Protein kinase B (PKB) [1], also called RAC kinase [2] is the mammalian homologue of the viral oncogene product v-Akt [3] and has therefore also been termed c-Akt. The current interest in this enzyme stems from several 10 observations. Firstly, it is activated within minutes in response to insulin or growth factors and activation is prevented by inhibitors of phosphatidylinositol (Ptdlns) 3-kinase [4-6]. There is increasing evidence that PKB may mediate a number of the actions of insulin, including stimulation of glucose and amino acid uptake, glycogen and protein synthesis and cardiac muscle glycolysis (reviewed 15 in [7, 8]) as well as regulation of the transcription of specific genes [9, 10]. Secondly, the PKBp isoform is overexpressed in a significant percentage of ovarian and pancreatic cancers [11, 12] and the PKBa isoform in some breast cancers [2]. It appears that PKB provides a survival signal that protects cells from apoptosis induced in a variety of ways (reviewed in [8, 13]). The activation 20 of PKB by gene amplification and other mechanisms may therefore contribute to the generation of malignancies that are able to flourish in the absence of extracellular survival signals. PKB phosphorylates proteins and peptides at seine and threonine residues that lie 25 in Arg-Xaa-Arg,-Xaa-Xaa-Ser/Thr- sequences [14]. In insulin signal transduction two physiological substrates of PKB appear to be the protein kinase glycogen synthase kinase-3 (GSK3) [15, 16] and the cardiac isoform of phosphofructokinase-2 (PFK2) [8, 17]. Phosphorylation by PKB inhibits GSK3 activity leading to dephosphorylation and activation of glycogen synthase and WO 00/36135 PCT/GB99/04228 2 protein synthesis initiation factor eIF2B [18]. These events appear to contribute to the insulin-induced stimulation of glycogen synthesis and protein synthesis, respectively. PKB activates cardiac PFK2, which seems to underlie the insulin induced stimulation of glycolysis in the heart. In the protection of cells against 5 apoptosis, BAD appears to be one of the physiological substrates of PKB. This protein, in its dephosphorylated form, interacts with the Bcl family member BclxL, thereby inducing apoptosis in some cells. However, when PKB phosphorylates BAD at Ser136, it dissociates from BclXL, interacts with 14-3-3 proteins instead, and apoptosis is prevented [19]. 10 The activation of PKB by insulin or growth factors requires its phosphorylation at two sites [20]. These are Thr308, which is located in the "activation loop" of the catalytic domain between subdomains VII and VIII, and Ser473 which is very close to the C-terminus in a hydrophobic Phe-Xaa-Xaa-Phe-Ser-Phe motif that is 15 present in a number of protein kinases that play important roles in signal transduction [21]. The phosphorylation of both sites is prevented by inhibitors of PtdIns 3-kinase [20]. Thr308 is phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) [22, 23] and Ser473 by a protein kinase(s) having PDK2 activity. PDK1 may have PDK2 activity, for example when the kinase domain of 20 PDK1 interacts with a region of Protein kinase C-Related Kinase-2 (PRK2) termed the PDK1 Intracting Fragment (PIF) or other polypeptide having a Phe Xaa-Xaa-Phe motif. This converts PDK1 from a form that phosphorylates PKB at Thr308 to a form that phosphorylates PKB at both Thr308 and Ser473 (Balendran et al (1999) Current Biology 9, 393-404). 25 PtdIns 3-kinase converts phosphatidylinositide 4,5 bisphosphate (PtdIns[4,5]P2) to PtdIns[3,4,5]P3 which is then converted to PtdIns[3,4]P2 by one or more 5' phosphatases. PKB can only be activated by PDK1 in vitro in the presence of lipid vesicles containing PtdIns[3,4,5]P3 or PtdIns[3,4]P2 [22]. These 3- WO 00/36135 PCT/GB99/0 4 2 2 8 3 phosphoinositides bind to the pleckstrin homology (PH) domain at the N-terminus of PKB [24, 25], which is thought alters its conformation in such a way that Thr308 becomes accessible to PDK1. PDK1 also possesses a PH domain C terminal to the catalytic domain [23], that interacts with PtdIns[3,4,5]P3 and 5 PtdIns[3,4]P2 even more strongly than does the PH domain of PKB [26, 83]. The interaction of PDK1 with 3-phosphoinositides enhances the activation of PKB in vitro, probably by facilitating the interaction of PDK1 and PKB in lipid vesicles [27]. 10 There is increasing evidence that PDK1 activates a number of protein kinases in vivo, such as p70 S6 kinase [28] and isoforms of PKC [29], by phosphorylating threonine residues that lie in positions equivalent to Thr308 of PKB. These protein kinases also contain the hydrophobic PDK2 consensus sequence that lies 160-165 residues C-terminal to the PDK1 phosphorylation site [8]. Thus other 15 substrates of PDK1 are also likely to be phosphorylated by a protein kinase with PDK2 activity. We have characterised yeast protein kinases that appear to be required for cell growth and that may have mammalian homologues that are able to rescue yeast 20 cells (ie restore their ability to grow) that are not able to express the endogenous yeast protein kinase or kinases. Characterisation of these yeast protein kinases that may be functional homologues of mammalian protein kinases may allow improved methods of identifying compounds that may modulate the activity of signalling pathways in which the mammalian homologues are involved, or 25 compounds that may be useful as antifungal agents. We have characterised non-mammalian proteins that may be functional homologues of PDK1 and human serum and glucocorticoid induced protein kinase (SGK), an enzyme whose catalytic domain is 70 % similar to PKB.

WO 00/36135 PCT/GB99/0422 8 4 Screening methods using yeast cells A first aspect of the invention is a method of identifying a compound which 5 modulates the activity (preferably inhibits) to different extents of (a) a host yeast cell protein kinase or kinases and (b) a protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases, wherein a compound is exposed to 1) a first host yeast cell wherein the yeast cell is capable of expressing the said 10 host yeast cell protein kinase or kinases and is not capable of expressing the said equivalent protein kinase and 2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said host yeast cell protein kinase or kinases and (b) is capable of expressing 15 the said equivalent protein kinase derivable from a source other than the host yeast cell and the effect of the compound on the phenotype of the said yeast cells is measured, wherein either (1) the host yeast cell is a pathogenic yeast (preferably a yeast pathogenic to a 20 mammal, preferably a human) and the source other than the host yeast cell is any source other than the host yeast cell or (2) the host yeast cell is any yeast and the source other than the host yeast cell is not a mammal. 25 It will be appreciated that the said first and second host yeast cells differ substantially only in the features indicated such that apart from these features the cells are essentially the same. This ensures that there is a reasonable expectation that the effect of the compound on the phenotype of the said yeast cells can be WO 00/36135 PCT/GB99/0422 8 5 attributed to the compound's effect on the modulation of the activity to different extents of the protein kinases as said. Thus, the first and second host yeast cells are from the same species and have substantially the same genetic content with the exception of the features indicated ie the capability to express the said host 5 yeast cell protein kinase or kinases and the ability to express the said equivalent protein kinase derivable from a source other than the host yeast cell. It will be appreciated that the said first and second host yeast cells may differ in genetic content relating to the generation or selection of the said first or second host yeast cells (for example, in selectable marker genes used in recombinant techniques, as 10 well known to those skilled in the art). Typically, a compound that affects the viability of the first said yeast cell and the said second yeast cell differently, is identified. More particularly, the compound so identified is selected for further study. 15 It will be appreciated that the phrase "modulates the activity (preferably inhibits) to different extents" includes the meanings that (1) the host yeast cell protein kinase or kinases is/are modulated (inhibited or activated) and the protein kinase derivable from a source other than the said host yeast cell that is equivalent to the 20 said host yeast cell protein kinase or kinases is not modulated (inhibited or activated), (2) the host yeast cell protein kinase or kinases is/are not modulated (inhibited or activated) and the protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases is modulated (inhibited or activated), and (3) that both types of protein 25 kinase are modulated (inhibited or activated), but that one type is modulated (inhibited or activated) more than or differently to the other. It will be appreciated that the modulation is judged by the effect on the phenotype of the said first and second yeast cells. It will be appreciated that the effect of the WO 00/36135 PCT/GB99/0 42 2 8 6 compound on the protein kinase must be sufficient to cause a change in the phenotype being observed in order for any modulation to be detected. Preferably, the effect of a compound that modulates the said protein kinases on the phenotype of a cell is an alteration in the viability or ability of the cell to 5 grow. Thus, it is preferred that the said host yeast cell protein kinase or kinases is desirable or essential for the viability or ability of the cell to grow. By "protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases" (the "equivalent 10 protein kinase") is included a protein kinase derivable from a source other than the said host yeast cell that is able to function in place of the said host yeast cell protein kinase or kinases in at least some aspect (ie in relation to at least one phenotypic characteristic), for example as judged by the viability or ability of a cell in which the host yeast cell protein kinase or kinase is replaced by the said 15 equivalent protein kinase to grow. Thus by "equivalent protein kinase" is included a protein kinase that is able to function in place of the host yeast cell protein kinase or kinase in conferring on a host yeast cell a particular wild-type phenotype, for example the ability to grow under certain conditions. 20 For example, it is preferred that a yeast cell that is not able to express the said host yeast cell protein kinase or kinases is substantially not able to grow under given conditions, and that the said host yeast cell that is capable of expressing the said equivalent protein kinase is capable of growing under the same conditions. In the presence of a compound that inhibits the host yeast cell protein kinase or 25 kinases but does not inhibit the equivalent protein kinase, the said first host yeast cell may not be substantially capable of growing but the said second host yeast cell may be capable of growing. Similarly, in the presence of a compound that inhibits the equivalent protein kinase but does not inhibit the host yeast cell WO 00/36135 PCT/GB99/0 4 2 2 8 7 protein kinase or kinases, the said second host cell may be substantially incapable of growing. Alternatively, it is preferred that a yeast cell that is able to express the said host 5 yeast cell protein kinase or kinases expresses the said host yeast cell protein kinase at a level too low to permit growth of the cell at wild-type rates, preferably at a level at which the said cell is substantially not able to grow under given conditions, and that the said host yeast cell that is capable of expressing the said equivalent protein kinase expresses the said equivalent protein kinase at a 10 level too low to permit growth of the cell at wild-type rates, preferably at a level at which the said cell is substantially unable to grow under the same conditions. In the presence of a compound that activates the host yeast cell protein kinase or kinases but does not activate the equivalent protein kinase, the said first host yeast cell may be more capable of growing but the said second host yeast cell 15 may remain substantially incapable of growing. Similarly, in the presence of a compound that activates the equivalent protein kinase but does not activate the host yeast cell protein kinase or kinases, the said second host cell may be more capable of growing and the said first host yeast cell may remain substantially incapable of growing. Thus, for example, the host yeast cell protein kinase or 20 kinases and the equivalent protein kinase may be expressed from a regulatable promoter and the cells may be grown under conditions in which the activity of the promoter is reduced. For example, the GAL1 promoter may be used, in a manner analogous to that described in Example 1. The GAL1 promoter is repressed in the presence of glucose, as known to those skilled in the art; thus, 25 when cells are grown in the presence of glucose, expression from the GAL1 promoter will be reduced, in a manner analogous to that described in Example 1. Thus, a yeast host cell which is incapable of expressing the said host yeast cell protein kinase or kinases may be substantially incapable of growing unless the WO 00/36135 PCT/GB99/0 4 2 2 8 8 said yeast host cell is capable of expressing the said protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases at an sufficient level. 5 It will be appreciated that a compound that affects the phenotype, for example the viability or ability of the cell to grow, by a mechanism that does not directly involve the said protein kinase or kinases or equivalent protein kinase is unlikely to have a different effect on the first and second said host yeast cells. Thus, a compound that has a similar effect on both the first and second said host yeast 10 cells (for example, no effect on the viability of either type of cell, or, alternatively, toxic to both types of cell) may not be selected for further study, as the compound may not be affecting the activity of the said protein kinase or kinases or equivalent protein kinase. For example, the compound may be a non specific cytotoxic agent affecting, for example, the integrity of the cellular 15 membrane. It will be appreciated that a compound that is capable of modulating the activity of the said host yeast cell protein kinase or kinases and the said equivalent kinase may have a similar effect on both said first and second host yeast cells; however, 20 it will be appreciated that it may not be possible or convenient to attempt to distinguish such a compound from a compound that is affecting the phenotype by a mechanism that does not directly involve the said protein kinase or kinases or equivalent protein kinase. 25 It will be appreciated that the possibility of distinguishing compounds that may be acting directly on the said protein kinase or kinases or equivalent protein kinase from those that may be acting on other cellular components may be beneficial, for example as set out above. Thus, the method of the first aspect of the invention, which may allow such distinguishing, may be beneficial even if, for WO 00/36135 PCT/GB99/042 2 8 9 example in the context of the intended use of any compound identified, it is not considered of great importance that the compound is not able to inhibit the said host yeast cell protein kinase or kinases, so long as it is able to inhibit the equivalent protein kinase (or vice versa). 5 Thus, for example, if it is intended to identify a compound that is capable of inhibiting a particular human protein kinase and that therefore may be useful in treating a human with a disease or condition that may be caused by elevated activity of the said protein kinase, it may not be essential for the medical 10 usefulness of the compound that it does not inhibit an equivalent protein kinase in yeast. Similarly, if it is intended to identify a compound that is capable of inhibiting a particular yeast (for example a pathogenic yeast) protein kinase and that therefore 15 may be useful in treating a human with a disease or condition that may be caused by that pathogenic yeast (which compound may be identified by a screen according to the first aspect of the invention in which the host yeast cell is a non pathogenic yeast cell, for example S. cerevisae and the source from which the equivalent protein kinase is derivable is a yeast (for example Candida) which is 20 capable of being a pathogen), it may not be essential for the compound to be medically useful that the compound does not inhibit an equivalent protein kinase in non pathogenic yeast, such as S. cerevisiae. However, it will be appreciated that in other circumstances it may be desirable or 25 essential for the medical or horticultural usefulness of the compound that the compound is (1) able to inhibit the said host yeast cell protein kinase or kinases, and (2) is not able to inhibit the equivalent protein kinase (or vice versa). For example, when the host yeast cell is a pathogenic yeast cell, a compound that WO 00/36135 PCT/GB99/0422 8 10 inhibits the host yeast cell protein kinase but not an equivalent protein kinase, such as a human protein kinase or a plant protein kinase, may be useful, for example as an anti-yeast (or anti-fungal) drug or plant protection product or in the design of an anti-yeast drug (or anti-fungal) drug or plant protection product. 5 A further aspect of the invention provides a method of identifying a compound which modulates the activity to different extents of (a) a protein kinase derivable from a first source and (b) a protein kinase derivable from a second source, both said protein kinases being equivalent to the same host yeast cell protein kinase or 10 kinases, wherein a compound is exposed to 1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from the first source and 15 2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from the second source and the effect of the compound on the viability of the said yeast cells is measured, and a compound that affects the viability of the first said yeast cell and 20 the said second yeast cell differently, is identified. More particularly, the compound so identified is selected for further study. The method of this aspect of the invention may allow a compound to be identified that may have an effect on cells of the said first source but that may not have an 25 effect on cells of the said second source, or vice versa. Thus, it may be useful if the said first source is a mammal, for example a human, and the said second source is a parasite or pathogen (including an opportunistic pathogen), for example a yeast pathogen such as Candida spp. The terms parasite, pathogen and opportunistic pathogen are well known to those skilled in the art, and include WO 00/36135 PCT/GB99/04228 11 any source that may cause or contribute to a disease or condition of an organism, preferably a mammal, still more preferably a human. It will be appreciated that the parasite or pathogen may be a parasite or pathogen 5 of a plant; thus it may be useful if the said first source is a plant and the second source is a parasite or pathogen that may affect the said plant, for example is capable of causing rust. A compound that does not have an effect on cells of the said first source but that has an inhibitory effect on cells of the said second source may be useful in treating a plant that is affected by the said parasite or pathogen 10 or in preventing the plant being affected by the said parasite or pathogen. Thus, it may be preferred that the first or second source are both not mammalian, or that either the first or second source is a pathogenic yeast cell. It will be appreciated that the first and second source may both be plants; a 15 compound that is capable of affecting cells of the first source and second source differently may be useful as a selective herbicide or a selective growth promoter. Thus, for example, the first source may be a monocotyledenous plant and the second source may be a dicotyledenous plant. 20 By "protein kinase derivable from" is included the meaning that the protein kinase is encoded by a nucleic acid, for example genomic DNA or mRNA, of the source. Thus, for example, a protein kinase derivable from a human may be a protein kinase encoded by a portion of a human genome or by a cDNA copied from human mRNA. It will be appreciated that the first and second source may 25 be first and second tissue from an organism. In such a case, the nucleic acid may be the mRNA of a cell of the tissue. It is preferred that the protein kinase derivable from the first source is not identical to the protein kinase derivable from a second source. The said protein kinases may be tissue-specific or tissue restricted isoforms, as well-known to those skilled in the art. Thus, the method WO 00/36135 PCT/GB99/04 2 2 8 12 of the invention may be used to identify a compound that is able to modulate/inhibit a protein kinase derivable from a first tissue of an organism (ie the first source) to a different extent to an equivalent (non-identical) protein kinase derivable from a second tissue of the same organism (ie the second 5 source). Preferred embodiments of the invention include methods according to any of the above aspects of the invention wherein any of the appropriate said non-host yeast cell sources is a human (ie the protein kinase is derivable from a human). 10 It will be appreciated that if the protein kinase from the first and second sources are closely related then it may prove difficult to identify a compound that is able to inhibit the protein kinase from the first source but not the protein kinase from the second source, or vice versa. However, unless the said protein kinases are 15 identical it may be possible to identify such a compound. In these and other aspects of the invention described below, exemplary genera of yeast contemplated to be useful, either as a said host yeast cell or as a said source other than a said host yeast cell, in the practice of the present invention are 20 Pichia, Saccharomyces, Kluyveromyces, Candida, Torulopsis, Hansenula, Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis and the like. 25 By pathogenic yeast is included pathogenic yeast of any one of the genera Candida spp, Blastomyces spp, for example B. dermatitidis, Coccidioides spp, for example C. immitis, Histoplasma spp, for example H. capsulatum, Sporothrix spp, for example S. schenckii, Aspergillus spp, for example A. flumigatus, A. flavus, A. niger, Phialophora compacta (Fonsecaea compacta), P. pedrosoi (F.

WO 00/36135 PCT/GB99/04228 13 pedrosi), P. verrucosa, Cladosporium carrionii, Rhinocladiella aquaspersa, Cryptococcus spp, for example C. neoformans, Cephalosporium spp, Fusarium spp, Histoplasma spp, for example H. capsulatum, Pneumocystis carinii, Rhizopus spp, Rhizomucor spp, Madurella spp, for example M. mycetomatis, M. 5 grisea, Pseudallescheria boydii, Paracoccidioides spp, for example P. brasiliensis, Prototheca spp, for example P. wickerhamii, Epidermophyton spp, Microsporum spp, Trichophyton spp, and Malassezia spp, for example M. furfur (Pityrosporum orbiculare). 10 It will be appreciated that by "yeast" we include "fungi" and, in particular we include the pathogenic fungi of the genera Aspergillus, including Aspergillus fumigatus, Cryptococcus, including Cryptococcus neoformans, and Histoplasma, including Histoplasma capsulatum. 15 It will be appreciated that Saccharomyces spp, for example S. cerevisiae are not considered pathogenic yeasts. Schizosaccharomyces spp, for example S. pombe are not considered pathogenic yeasts. It will be appreciated that, in a simple embodiment, the host yeast cell is rendered 20 not capable of expressing the said host yeast cell protein kinase or kinases by virtue of a mutation or mutations in the gene or genes encoding the said host yeast cell protein kinase. Typically the mutation is any mutation that prevents expression of an active protein kinase; it is particularly preferred if the mutation is one which cannot be spontaneously reversed, for example a deletion of all or 25 part of the gene encoding the protein kinase. The host yeast cell which is capable of expressing the said equivalent protein kinase from a non host yeast cell source is most conveniently made by WO 00/36135 PCT/GB99/04228 14 introducing into a suitable yeast a genetic construct which encodes and is capable of expressing the said equivalent protein kinase. It will be appreciated that the host yeast cell parental strain may not be wild-type. 5 For example, particularly in relation to S. cerevisiae as the host yeast cell, mutant strains containing Ade- or Leu- or Ura- mutations may be used as the parental strain to allow selection of plasmid uptake. It will be appreciated that a constitutive or inducible promoter may be used in the 10 expression of the non-host yeast cell protein kinase. Examples of constitutive promoters well known in the art are the adh or the SV40 promoter. As discussed above and in Example 1, the GAL1 promoter may be used. Alternatively, the promoter from the gene encoding the host yeast cell protein kinase may be used to express the non-host yeast cell protein kinase. For example, the promoters 15 from the Pkhl or Pkh2 genes may be used, in a manner analogous to that described in Example 1. It is preferred that the exogenous polynucleotide is stably maintained in the yeast. It is further preferred, though not essential, that the exogenous polynucleotide is 20 stably integrated into the yeast genome. Transformation of cell hosts is accomplished by well known methods that typically depend on the type of vector used and host cell. Transformation of Saccharomyces and related cells is described in Sherman et al (1986) Methods in 25 Yeast Genetics, a Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 may also be useful. Schizosaccharomyces pombe may be transformed following LiCl treatment or by electroporation.

WO 00/36135 PCT/G B99/04228 15 Conveniently, a Bio-Rad Pulse Controller may be used for electroporation of S. pombe cells. The technique of electroporation of yeast is disclosed in Becker & Guarente (1990) Meth Enzymol 194, 182. 5 S. cerevisiae may be transformed by, for example, methods analogous to those described in Example 1. It is particularly preferred if, in relation to the first aspect of the invention, the host yeast cell is incapable of expressing the protein kinase Pkhl and/or Pkh2. 10 Thus, a further preferred embodiment of the invention is a method of the invention wherein the said host yeast cell protein kinase or kinases is Pkh1 and/or Pkh2, wherein Pkh1 is the polypeptide encoded by open reading frame YDR490c of S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae 15 and Pkh2 is the polypeptide encoded by open reading frame YOL100w of S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae. Pkh1 from S. cerevisiae may have the following amino acid sequence: 20 MGNRSLTEADHALLSKPLVPTSAEHTQTQEYPRPFVDGSNSQSGSELQAS PQGQFGEKALTSTNRFI PLANDDPGMQHEMGLDPSMRRRREEWAERGAAK IVKDVVDPATGELTKHVVKMGIKDFKFGEQLGDGSYSSVVLATARDSGKK YAVKVLSKEYLIRQKKVKYVTVEKLALQKLNGTKGIFKLFFTFQDEASLY FLLEYAPHGDFLGLIKKYGSLNETCARYYASQIIDAVDSLHNIGIIHRDI 25 KPENILLDKNMKVKLTDFGTAKILPEEPSNTADGKPYFDLYAKSKSFVGT AEYVSPELLNDNYTDSRCDIWAFGCILYQMLAGKPPFKAANEYLTFQKVM KIQYAFTAGFPQIVKDLVKKLLVRDPNDRLTIKQIKAHLFFHEVNFEDGS VWDDNPPE IQPYKINAEAMKPLQKVSESDTTVKMANLQLAGNGHADTPLQ

APAATSQEHSVISMTAATAAFNKDYTSQPKLGSKSSTSVRSASNNTDREV

WO 00/36135 PCT/GB99/04228 16 IQKKVSKNRASVSSPSISTTSRGKDNRSRSSDAFWSRYLQNMDERVLLMK EVALSTRNLEDS PVGLENVALDYKNPLDIEPPTDSAGKFYKKMFLITNLG RALVFVKRRSLSMWEEQEFELQFELELNDVEKIRFI SDQVLEIDGSRTIF IGCKERAVLMKLWKLIHNGMTAKPKVVSPKSDHKMFDKFILQKRQNTKKK 5 NQAPPVPQSNRLINGLPDRCILKTPEEGALHTKRPTSLQTRSSSNYSKLL ARSTQMRKNMTRTDEK (766 aa complete sequence) Pkhl from S. cerevisiae may be encoded by the following nucleotide sequence: 10 ATGGGAAATAGGTCTTTGACAGAGGCAGACCACGCCCTGCTGTCCAAGCC CTTGGTACCGACATCTGCGGAACATACACAAACGCAAGAGTATCCTCGTC CTTTCGTAGATGGCAGCAATTCTCAGAGCGGGTCTGAACTACAGGCTTCT CCACAAGGTCAGTTT GGAGAAAAGGCATT GACTAGTACTAATCGCTT CAT TCCCCTGGCAAATGATGACCCGGGTATGCAGCACGAGATGGGTCTTGATC 15 CCTCAATGAGGCGTAGAAGAGAAGAATGGGCAGAACGTGGTGCGGCAAAA ATCGTCAAAGATGTT GTCGACCCAGCTACGGGGGAGTTAACTAAGCATGT TGTCAAGAT GGGAATAAAGGACTT CAAGTTTGGAGAGCAACT CGGGGATG GATCATATTCTAGTGTTGTTCTGGCTACCGCCCGTGATTCGGGCAAGAAA TATGCAGTAAAAGT GTTGAGTAAAGAATATCT GATCCGT CAAAAAAAAGT 20 TAAATACGTCACAGTGGAGAAATTGGCTTTGCAGAAGCTGAATGGCACCA AGGGCATATTCAAGCTTTTCTTCACTTTCCAGGACGAGGCAAGCTTGTAT TTCCTTCTAGAATATGCCCCCCACGGTGATTTCTTGGGCTTGATTAAGAA ATATGGATCTTTAAACGAGACATGTGCACGCTATTATGCGTCGCAGATCA TCGATGCCGTTGACTCCTTGCATAATATCGGCATTATTCACAGGGATATC 25 AAGCCCGAAAACATATTGCTCGACAAAAATATGAAAGTGAAGTTGACGGA TTTTGGTACAGCCAAAATTTTACCGGAGGAACCTTCGAACACCGCAGATG GCAAGCCTTATTTCGATTTGTATGCTAAGTCGAAATCATTTGTTGGTACC GCAGAATAT GTTTCTCCTGAGCTACTGAATGATAATTATACAGATT CCCG TTGTGACATTTGGGCATTTGGTTGCATATTGTACCAAATGCTTGCGGGAA 30 AACCGCCTTTTAAAGCTGCCAATGAATATTTGACATTCCAAAAAGTAATG WO 00/36135 PCT/GB99/04228 17 AAGATTCAATATGCGTTTACTGCAGGTTTTCCGCAAATAGTAAAAGATTT AGTTAAAAAACTATTAGTTAGGGATCCAAATGATAGATTGACCATAAAGC AGATCAAAGCACACCTCTTTTTCCATGAAGTCAACTTTGAAGATGGTTCT GTTTGGGATGATAATCCACCGGAGATACAGCCATATAAAATAAATGCAGA 5 GGCGATGAAGCCCCTGCAAAAGGTTTCTGAATCTGATACCACTGTCAAAA TGGCCAACCTTCAGCTGGCTGGTAATGGACATGCAGATACTCCCCTGCAA GCACCAGCAGCGACCT CT CAAGAGCATT CTGTGAT CAGTATGACTGCAGC AACCGCCGCATTCAATAAAGATTATACAAGTCAACCCAAATT GGGGAGCA AGTCAAGCACATCTGTTAGATCTGCCTCCAACAACACAGATCGCGAGGTA 10 ATTCAAAAGAAGGTTTCAAAAAATCGCGCATCTGTATCATCTCCTTCAAT TTCTACTACATCACGGGGGAAAGATAATAGAAGTCGCTCTTCTGACGCCT TCTGGTCACGCTACTTGCAAAATATGGATGAACGTGTCTTGTTGATGAAG GAGGTAGCGCTTTCCACACGAAACTTAGAGGACTCACCTGTAGGTCTTGA GAACGTGGCTCTGGACTACAAGAACCCTCTTGATATCGAGCCTCCTACTG 15 ATAGTGCAGGCAAATTTTACAAGAAAATGTTTCTAATAACAAACCTAGGC AGAGCACTTGTTTTTGTCAAGAGAAGAAGT CT CAGCATGTGGGAAGAACA GGAATTTGAATTGCAATTCGAACTAGAGTTGAATGACGTTGAGAAGATAC GCTTTATAAGTGATCAAGTCCTTGAAATTGACGGTTCCAGGACGATTTTC ATAGGAT GCAAAGAGAGAGCAGTTTTAAT GAAACTATGGAAATTAATACA 20 TAATGGAATGACCGCCAAACCTAAAGTAGTATCGCCGAAGTCGGACCATA AAATGTTTGATAAGTTCATTCTTCAAAAAAGACAGAATACAAAAAAAAAG AATCAAGCTCCTCCCGTACCTCAATCGAATAGGCTAATAAATGGTTTACC GGACCGTTGCATCTTAAAGACACCCGAAGAAGGCGCACTTCACACAAAAC GTCCCACTTCGTTGCAGACCCGATCGTCATCTAATTACTCAAAATTGCTG 25 GCAAGATCGACACAAATGCGGAAAAACATGACACGGACAGATGAAAAATG A (Genbank accession number 927745; 2301 bp complete sequence). Pkh2 from S. cerevisiae may have the amino acid sequence: 30 MYFDKDNSMSPRPLLPSDEQKLNINLLTKKEKFSHLDPHYDAKATPQRST WO 00/36135 PCT/G B99/04228 18 SNRNVGDLLLEKRTAKPMIQKALTNTDNFIEMYHNQQRKNLDDDTIKEVM INDENGKTVASTNDGRYDNDYDNNDINDQKTLDNIAGS PHMEKNRNKVKI EHDSSSQKPIAKESSKAQKNI IKKGIKDFKFGSVIGDGAYSTVMLATSID TKKRYAAKVLNKEYLIRQKKVKYVS IEKTALQKLNNSPSVVRLFSTFQDE 5 SSLYFLLEYAPNGDFLSLMKKYGSLDETCARYYAAQIIDAIDYLHSNGII HRDIKPENILLDGEMKIKLTDFGTAKLLNPTNNSVSKPEYDLSTRSKSFV GTAEYVS PELLNDS FTDYRCDIWAFGCILFQMIAGKPPFKATNEYLTFQK VMKVQYAFTPGFPLIIRDLVKKILVKNLDRRLTISQIKEHHFFKDLNFKD GSVWSKTPPE IKPYKINAKSMQAMPSGS DRKLVKKSVNTLGKSHLVTQRS 10 ASSPSVEETTHSTLYNNNTHASTESEISIKKRPTDERTAQILENARKGIN NRKNQPGKRTPSGAASAALAASAALTKKTMQSYPTSSSKSSRSSS PATTS RPGTYKRTSSTESKPFAKSPPLSASVLSSKVPMPPYTPPMSPPMTPYDTY QMTPPYTTKQQDYSDTAIAAPKPCI SKQNVKNSTDS PLMNKQDIQWSFYL KNINEHVLRTEKLDFVTTNYDILEKKMLKLNGSLLDPQLFGKPRHTFLSQ 15 VARSGGEVTGFRNDPTMTAYSKTEDTYYSKNIIDLQLLEDDYRIEGGDLS ELLTNRSGEGYKCNQNSSPMKDDDKSESNNKGSSVFSGKIKKLFHPTSAA ETLSSS DEKTKYYKRTIVMTS FGRFLVFAKRRQPNPVTNLKYELEYDINL RQQGTKIKELIIPLEMGTNHIVVIQTPYKSFLLSTDKKTTSKLFTVLKKI LNSNTNKIEKELLQRNQKVIERRTSSSGRAI PKDLPTSKSPSPKPRTHSQ 20 SPSISKHNSFSESINSAKSNRSSRIFETFINAKEQNSKKHAAPVPLTSKL VNGLPKRQVTVGLGLNTGTNFKNSSAKSKRS (1081 aa complete sequence). Pkh2 from S. cerevisiae may be encoded by the nucleotide sequence: 25 ATGTATTTTGATAAGGATAATTCCATGAGCCCTAGGCCGTTATTGCCAAG TGATGAGCAGAAGCTAAACATTAATCTTCTAACGAAAAAGGAGAAATTCT CGCATTTAGACCCCCATTATGACGCAAAAGCCACTCCACAAAGAAGCACT TCGAATAGAAACGTTGGCGATTTACTTTTGGAAAAAAGAACCGCTAAGCC TATGATT CAAAAGGCCTTGACGAATACGGATAATTTCATTGAAATGTACC 30 ATAATCAGCAGAGAAAAAATCTTGATGATGACACTATTAAAGAAGTAATG WO 00/36135 PCT/GB99/04228 19 ATTAATGATGAAAACGGAAAAACTGTCGCTAGTACCAACGACGGCAGATA TGACAACGATTACGATAATAACGATATTAATGACCAAAAAACTTTGGATA ATATAGCGGGAAGTCCCCACATGGAAAAAAATCGAAACAAAGTAAAGATT GAACATGACTCTTCATCTCAAAAACCAATAGCTAAAGAGTCATCCAAAGC 5 CCAAAAAAATATAATCAAAAAGGGAATCAAGGACTTTAAATTTGGTAGTG TAATAGGTGATGGCGCGTATTCTACTGTAATGTTAGCGACGTCGATTGAT ACCAAAAAGAGGTACGCCGCAAAAGTACTAAACAAAGAATATTTAATACG CCAGAAGAAAGTCAAATACGTCAGCATAGAAAAAACCGCCCTTCAAAAGC TCAATAATTCTCCTAGTGTTGTGCGATTATTTTCCACTTTTCAGGATGAA 10 TCAAGCCTATACTTTCTCTTAGAGTATGCCCCCAATGGGGACTTTCTTTC TTTAATGAAAAAATACGGTTCATTAGACGAAACCTGCGCACGATATTATG CTGCGCAAATAATAGATGCCATAGACTACTTACATTCCAACGGTATTATT CATAGAGATATAAAACCAGAAAATATTCTTTTAGATGGAGAAATGAAGAT CAAACTGACTGATTTTGGTACTGCGAAGTTACTGAATCCTACAAATAATA 15 GCGTTTCGAAACCAGAATACGATTTATCAACAAGGTCGAAATCTTTCGTT GGAACTGCAGAATACGTATCTCCAGAACTTTTAAATGACAGTTTTACAGA CTATCGTTGCGATATTTGGGCCTTCGGATGTATACTTTTCCAGATGATTG CCGGAAAACCACCATTCAAAGCTACCAATGAATACTTGACTTTCCAAAAG GTAATGAAAGTTCAGTACGCCTTTACACCAGGTTTCCCACTTATTATCAG 20 AGATTTGGTTAAGAAAATCTTAGTAAAAAACTTAGACCGAAGATTGACGA TAAGCCAAATTAAGGAACATCATTTTTTCAAAGATTTGAATTTTAAAGAC GGCTCTGTTTGGTCAAAAACGCCTCCAGAGATCAAACCATATAAAATCAA CGCCAAATCCATGCAGGCAATGCCAAGCGGAAGCGATAGAAAACTGGTGA AGAAATCAGTCAACACACTTGGCAAATCGCATCTAGTGACTCAAAGGTCA 25 GCTTCAAGTCCCTCTGTTGAGGAAACTACTCATTCAACCCTATACAATAA CAATACTCACGCTTCTACTGAAAGTGAAATATCAATAAAGAAGAGACCCA CTGATGAAAGAACAGCGCAGATACTTGAAAATGCAAGAAAGGGTATAAAC AATAGGAAAAATCAACCAGGCAAGAGAACACCAAGTGGTGCAGCTTCTGC TGCCCTAGCAGCTTCTGCTGCTTTAACCAAGAAAACCATGCAAAGCTATC 30 CAACTTCTAGTTCGAAAAGTAGCAGGTCAAGCTCTCCTGCGACAACATCA WO 00/36135 PCT/GB99/04228 20 AGACCAGGAACTTATAAGCGTACTT CTTCTACAGAAAGTAAACCATTTGC CAAATCTCCACCTTTGTCAGCATCAGTTTTATCGTCAAAAGTCCCAATGC CTCCATACACACCTCCAATGTCGCCCCCTATGACACCATATGATACATAT CAAATGACACCTCCCTATACGACAAAACAGCAGGATTATTCTGATACCGC 5 AATTGCCGCACCTAAGCCTTGTATTAGTAAGCAAAATGTTAAAAATAGCA CAGATTCTCCCTTGATGAACAAGCAAGATATTCAATGGTCCTTTTACCTG AAAAACATCAACGAACATGTACTAAGGACGGAAAAACTGGATTTT GTTAC CACAAATTACGATATCTTAGAGAAGAAAATGCTTAAACTAAATGGTTCAT TGTTAGATCCTCAACTGTTTGGTAAGCCTAGACATACTTTTTTATCCCAA 10 GTAGCTAGGAGTGGGGGAGAGGTTACAGGTTTTCGAAATGATCCAACTAT GACTGCTTATTCCAAAACAGAAGATACGTACTATTCGAAAAATATTATCG ATTTGCAGCTCTTGGAAGATGATTATCGAATTGAAGGAGGTGACTTATCG GAGTTGCTTACTAACAGAAGCGGAGAAGGGTACAAATGCAATCAAAACAG CT CACCAAT GAAAGACGAT GATAAAT CCGAAT CTAACAATAAAGGAAGCT 15 CTGTTTTTTCTGGCAAGATTAAAAAATTATTTCACCCTACCTCAGCAGCT GAAACGCTCTCTTCCTCTGATGAAAAAACCAAGTACTATAAACGAACCAT TGTAATGACATCATTTGGAAGGTTTCTAGTATTTGCCAAGAGGAGGCAGC CAAATCCAGTTACAAATTTAAAGTATGAACTAGAATATGACATAAATTTG CGTCAACAGGGTACCAAAATAAAAGAACTAATCATTCCCTTGGAAATGGG 20 AACTAATCATATAGTTGTGATTCAGACACCTTACAAGTCATTTCTTTTGA GCACTGATAAAAAAACCACGAGCAAATTGTTTACTGTTCTCAAAAAAATT CTTAAT T CGAATACAAATAAAATAGAGAAAGAACT GTT GCAAAGAAACCA AAAGGTAAT T GAAAGAAGAACAT CAT CAT CCGGAAGAGCCATACCTAAAG ATCTTCCAACTTCCAAGTCTCCTTCGCCAAAACCCAGGACGCATAGCCAA 25 TCTCCATCAATTTCAAAGCACAATTCGTTTTCTGAATCGATTAATAGCGC TAAGAGCAACAGATCAAGCAGAATTTTTGAAACCTTTATCAATGCCAAGG AACAAAATT CAAAAAAACACGCTGCTCCAGTACCGTTAACCAGTAAATTA GTTAACGGATTGCCAAAAAGACAAGTTACCGT GGGATTAGGTCTAAACAC AGGAACAAATTT CAAAAACT CATCTGCAAAAT CGAAGAGGTCGTAAT 30 (Genbank accession no 1419952; 3247 bp complete sequence).

WO 00/36135 PCT/GB99/04228 21 Equivalent open reading frames in yeast other than S. cerevisiae may be identified as such by methods well known to those skilled in the art and as described below. 5 The KSG1 gene (PDK1-like) from Schizosaccharomyces pombe may encode a polypeptide which comprises the following amino acid sequence: MRNTHNPNETEASEDAENDTQSESDLSFDHGSSEKLNRASLPKTQNSAI PQSNALN TTPNESTSQIDSSPKI PSAVPHI STPNPSSGASTPNIKRVSDFKFGEILGEGSYST 10 VLTATENSTKREYAIKVLDKRHIIKEKKEKYVNIEKEALCILSKHPGFIKLFYTFQ DAHNLYFVLSLARNGELLDYINKLGRFNEICAQYYAALIVDSIDYMHGRGVIHRDL KPENILLDDNMRTKITDFGSAKILNSSHGSHEEDTHHADKPQAHSRSFVGTARYVS PEVLSDKIAGTASDIWAFGCILFQMLAGKPPFVAGNEYLTFQSILHLSYEIPPDIS DVASDLIKKLLVLDPKDRLTVDE IHQHPFFNGIKFDNTLWELPPPRLKPFGHTSVL 15 SLSVPNASNKHENGDLTSPLGVPSMVSASTNAAPSPVGTFNRGTLLPCQSNLEEEN KEWSSILQDDEKISKIGTLNVYSMSGINGNDAFRFFSSLFRKRKPRTFILTNFGRY LCVASDGEGRKTVKEEI PIKSVGMRCRMVKNNEHGWVVETPTKSWS FEDPNGPASA WVELLDKASS ISLPFGNHSVTSFSRS IARSAV (>gi|3341488|gn|PIDIe1312259 protein kinase. S. pombe). 20 The KSG1 gene from S. pombe may comprise the following nucleotide sequence: GGTATCTTCAGTTAAATCTTGCGTTGGTTGTCCTTTAATGAAGATTTGTTCGTAAG CAAAATATATGAATTCGCTAGCAAGATATTGCCATTCATACCAACCCTTTTCTTTA TGTTATATCTCCAAAATGAATTGTTTACAACACTAAATTGAAACAAATCAAACACA 25 CTTTGTTCCTATTAGGTGTAGTGGAAAATGCAATTCTAAACAATGGCACTTTATTT TTGATTCTGTGCTGAACAAATAATTCATATTTATAAACTGAATGGTGCATAATTTT CTCTCTCTGTAAAATTGATACGGTAAGTTGAAATTCAAGTAAAATATGTTTCCCGC ATATTCAGCTACGATTCTGGTATAAAAGCTTCTAATGCTCTTCGTATATAAACTGA GTTATTCATTAAAACCTAAGATCTTTTAAGTTATTGAAATTAAATCATATTGAATT 30 CCACACCCGAAGGTAATGCTATTCAACCAGCCAAGACGAAAATACTTGGTTTGACA WO 00/36135 PCT/GB99/04228 22 ATTTTTTTACTTTTATGTAATGATTTTCTCTACTCCATTAAGAATTTGCGTATGAC TGTGTATACTCAGCGCTGATTCACATAGTAATATAAATCATTACCTTGATGAACTA CTTCTCTAGGCTCCTTAAAAAAATAAATAAACTTATAGTTACTAAAAAAAGCATGA TCTAAATCAAATGAATGAATATTTAGTTTATCTAAAATGTCGAATTCACTATTAAT 5 TTTCGCACTCACAACGATGCAAATCATAACCCGGATAGCAAAATCTTATATAACAA TAGTACAATAGTTGTATACTTGCAGTTATAAGCACTAATTTTACCGAATATGTGCA ATTTTCTTTTGATGACATTGCCGTCAATTCGTAGTTTACTGACACTGATTCACCTA CCCAGTACTCTCTTCACACCAGATATTGGTTATGCGAAATACGCACAATCCGAATG AAACTGAAGCATCAGAAGATGCAGAAAATGATACTCAAAGCGAATCCGACCTTAGT 10 TTTGATCATGGATCAAGTGAAAAACTAAACCGAGCTTCATTACCAAAGACGCAAAA TAGTGCCATCCCGCAGTCTAATGCTTTAAATACTACACCAAATGAGTCAACGTCTC AGATCGATTCATCGCCGAAGATTCCCTCTGCAGTCCCACATATCTCTACTCCAAAT CCGTCTAGCGGTGCATCGACTCCTAACATAAAACGAGTTTCGGATTTTAAATTTGG CGAAATCCTTGGAGAAGGATCATACAGTACTGTATTAACAGCTACCGAAAACTCAA 15 CAAAAAGAGAATATGCTATTAAGGTGTTGGATAAAAGACATATTATCAAGGAAAAA AAGGAAAAATACGTCAACATTGAAAAAGAAGCTTTGTGCATCCTCTCTAAGCATCC TGGATTTATTAAGCTATTTTATACTTTCCAAGATGCTCACAATTTGTATTTTGTTT TGAGTCTTGCTCGAAATGGTGAACTGTTGGATTATATCAATAAGGTATGTATAAAT AATGTTCATGCAGCTTGATGATGGATAGCTTACTGATTATTACAATCTTGTCTTTT 20 TTCTTATTAAATGTTTTATTTTTTGCTTACACATAATTTAGTTGGGACGATTTAAC GAAATATGTGCACAATATTATGCTGCACTTATCGTTGACTCAATAGACTATATGCA TGGACGAGGAGTGATTCACCGTGACTTGAAGCCCGAAAATATTTTGCTGGATGATA ATATGCGTACAAAAATTACCGACTTTGGTTCAGCAAAAATATTAAACTCTTCACAT GGCAGTCATGAAGAAGACACACACCATGCTGACAAACCACAGGCTCATTCTCGATC 25 GTTTGTAGGAACTGCTCGTTATGTATCTCCTGAAGTTTTAAG TGATAAAATTGCTGGTACGGCTTCCGATATTTGGGCGTTTGGTTGCATTCTTTTCC AAATGCTAGCTGGCAAACCCCCTTTTGTAGCAGGAAATGAATATTTGACATTCCAA AGTATTCTTCATTTGAGTTATGAGATACCACCTGACATATCAGATGTCGCTTCTGA TTTGATTAAAAAACTGCTAGTTCTAGATCCTAAAGATCGCTTAACTGTTGATGAAA 30 TACACCAGCATCCGTTTTTTAATGGTATCAAATTTGATAATACCCTTTGGGAACTT WO 00/36135 PCT/GB99/04228 23 CCTCCTCCCCGTCTTAAACCTTTTGGTCATACTAGCGTCCTCAGCCTTTCCGTTCC TAATGCATCTAACAAACACGAAAATGGTGATTTGACCTCGCCTCTTGGTGTTCCAT CGATGGTTTCAGCATCCACCAATGCTGCACCCTCTCCGGTTGGTACTTTTAACCGA GGCACT CTATTGCCGT GTCAATCCAACCTTGAGGAGGAAAACAAGGAATGGTCGAG 5 TATTCTTCAAGACGATGAAAAAATCTCAAAAATTGGAACCCTCAATGTTTATAGTA TGTCTGGTATCAATGGAAATGATGCCTTTCGCTTTTTCTCCAGTCTTTTTCGAAAA AGGAAACCTCGGACCTTTATACTTACAAATTTTGGTCGATACCTGTGTGTTGCCTC GGACGGTGAAGGGCGAAAAACAGTTAAAGAAGAAATACCTATTAAGAGTGTAGGCA TGCGTTGTCGGATGGTAAAAAACAATGAACATGGCTGGGTAGTTGAGACTCCAACA 10 AAATCCTGGTCGTTTGAAGACCCGAATGGACCGGCTTCTGCTTGGGTTGAGCTTCT AGATAAGGCTAGCTCTATTTCTCTTCCATTCGGTAATCATTCTGTTACCAGCTTTT CAAGAAGCATTGCTAGAAGTGCTGTCTAATCTATCCTTTATGTCTATGAACACTTC CATCCATCCCCTCACTTTTTATTCTATCACTCTTTTTTCTGTTCTGTTCTGCATTA ATTTTCTTACGACCTTATTGATGTTTCAAGAAATTCTTTTTAAGTTTTTCTCTTAC 15 GTTCGTTGTTTAATTCGAAAAAAAATCTTTATATCCGTGATCCTTCAGTGTGATTA CAAAATTAGATTGCCGTATGATTTCTGTATTTTGTTATTAACGGTCAATTGATTTT ATTTGCTTATTCTTATTATGGTCTATTTATTTTATATTTTTTACCTTAGCCTTAAA ATGTTAAAATGGTAAAAGTTATCGATCCAAGAACATTTCACCGTTTACTAGTTAAT ATACCTCTACCTACGTAGTAAAAAAATTTAAAGAAATTATTCCCAAAAGCCAGTTG 20 TACATTCTCTCAATTTATGATTAATCTACAGATTAAGTTAATTATGCAAGTATGAC TTGTACATAAAAATTAGGTTTCGCTTAATATCAGAAAAAAAAACTTCAAGCATCAA TTTACTAGCATGATAAATAATGAATTACTTCTCCAAGTATGTTTTTGCGACACGAT C (>gil 33414871 emb I X992801 SPKSG1 S.pombe KSG1 gene (PDK1-like)) 25 A second PDK1-like gene (SPBC4C3. 11) of S. pombe may encode a polypeptide which includes the following amino acid sequence: MDLEHKRISRSTLPDYADPDYFEARGERNPVKPQSSNVVPGTSHIGSIKSPADYVF GDIIGDGSFSKVRRATDKKSWKEYAIKVLDKKYIVKENKVKYVNIERDSMMRLNGF 30 PGISRLFHTFQDDLKLYYVLELAPNGELLQYIKKYRFLDENCVRFYAAEILSSIEY WO 00/36135 PCT/G B99/04228 24 MHSCGIIHRDLKPENILFDGNMHVKITDFGTAKILPPKYVNSPDYTTFPSSFVGTA EYVAPELLSRQVVSKSSDLWAFACVVYQMIVGSPPFHGSNPNNIFKKIMSLEYELP KLLPPDIVPLFSHLFRIQPSDRSTTQQIKQFPFFATITWDNLWTQDPPPMQSFRPN YNIAIPNAPAYYRSNVTAAAAANAAAAFASASIVKHQETARRQELPTVNRFTAPTA 5 HYGYASLRSHQMPVDRLYYKLVPSSESI (>gi 128328921 gnl I PID I e1249768 hypothetical protein kinase phosphorylase). The second PDK1-like gene (SPBC4C3.11) of S. pombe may include the following nucleotide sequence: 10 acaacttcat ttttcatgaa aacctctttt ataaatgatg gatctggagc ataaacgcat tagccgaagt acattgccgg attatgcgga tcccgattac ttcgaggcta gaggtgaaag aaatccggta aaacctcagt cttccaacgt agtaccagga acaagtcata taggatcgat caaatctccg gcggattacg tttttggtga cattatagga gatggatcat tctcaaaggt aagttgatac 15 attgcttctc agttcagaag tttttcaacc gcaggtcgat taacctatgg accttcgatt actgacgaat aagttgtcta atattcgtta ggtgagaaga gcaactgata aaaagagttg gaaggagtac gctatcaaag tccttgataa aaaatatatt gtcaaggaaa ataaggttaa gtatgtgaat atagagagag attctatgat gagacttaat gggtttcctg gtatctctcg tcttttccat 20 acatttcagg atgatttaaa actttattat gtgcttgaac ttgcacccaa tggtgaactt ttgcaataca tcaaaaaggt atattttttc attagtctat tcatttttcc tttattaact aagctttggt agtatcgttt tcttgatgag aattgtgtgc gcttttatgc tgctgagatt ttatcaagta tcgagtatat gcactcctgc ggtataattc acagagatct caagccagaa aagtatgttt 25 gagtagtggt cattaaatgt tcgtttcctt ttcctaattc taacctattt tttagcattt tatttgatgg aaatatgcat gtaaaaatta ccgatttcgg cacagccaaa atcctacccc ctaaatatgt aaatagccct gattacacta cctttccaag ctcctttgtt ggcactgcgg aatatgttgc tcctgaacta ttgtctagac aagttgtttc aaaatcgtaa gaaaacctat tatcccagtc 30 tatttttttt ctgacaaata tttaagttcc gatttatggg cttttgcgtg tgttgtttat caaatgattg ttggttcccc tccttttcat ggcagcaatc ctaataatat tttcaaaaag ataatgagcc tggaatatga gcttccaaag ctcttaccac ctgatatcgt tcctttgttt agccatcttt tccgtattca gccatctgat cgatctacaa cccaacaaat aaaacaattt cctttttttg 35 ctactattac ttgggacaat ttatggactc aagatcctcc tcctatgcag tcattccggc ctaattataa catagccatt cctaatgctc ctgcttatta tcgctcaaat gtgacagccg cagctgctgc taatgctgcc gcggcatttg WO 00/36135 PCT/GB99/04228 25 cttctgcatc cattgtaaag catcaggaaa ctgctcgacg tcaggagctt cctacggtaa atcgtttcac tgctccaact gctcattatg gctatgcttc acttcgaagc catcagatgc ctgttgacag actttattac aagttggttc catcgtctga gtcgatc 5 The terms Pkhl and Pkh2 may represent PKB-activating Kinase Homologues 1 and 2, respectively. Yeast, for example S. cerevisiae, cells that are capable of expressing either Pkh1 or Pkh2 or both are capable of growing, but yeast, for example S. cerevisiae, cells that are not capable of expressing either Pkh1 or 10 Pkh2 are not capable of growing, as described in Example 1. A protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases, for example Pkh1 and/or Pkh2, may be PDK1, for example mammalian, preferably human PDK1. 15 As described in Example 1, yeast, for example S. cerevisiae, cells that are not capable of expressing either Pkh1 or Pkh2 may be capable of growing if they are capable of expressing human PDK1. The PDK1 may be plant PDK1. An example of plant PDK1 may be a 20 polypeptide which comprises the following amino acid sequence: MLAMEKEFDSKLVLQGNSSNGANVSRSKSFSFKAPQENFTSHDFEFGKIY GVGSYSKVVRAKKKETGTVYALKIMDKKFITKENKTAYVKLERIVLDQLE HPGI IKLYFTFQDTSSLYMALESCEGGELFDQITRKGRLSEDEARFYTAE 25 VVDALEYIHSMGLIHRDIKPENLLLTSDGHIKIADFGSVKPMQDSQITVL PNAASDDKACTFVGTAAYVPPEVLNSSPAT FGNDLWALGCTLYQMLSGTS PFKDASEWLIFQRIIARDIKFPNHFSEAARDLIDRLLDTEPSRRPGAGSE GYVALKRHPFFNGVDWKDLRSQTPPKLAPDPASQTASPERDDTHGSPWNL THIGDSLATQNEGHSAPPTSSESSGSITRLASIDSFDSRWQQFLEPGESV 30 LMISAVKKLQKITSKKVQLILTNKPKLIYVDPSKLVVKGNIIWSDNSNDL

NVVVTSPSHFKICTPKKVLSFEDAKQRASVWKKAIETLQNR*

WO 00/36135 PCT/G B99/04228 26 (492 aa complete sequence.) It will be appreciated that a further aspect of the invention is a substantially pure polypeptide comprising the above amino acid sequence or a variant, fragment, 5 fusion or derivative thereof, or a fusion of a said variant or fragment or derivative The said amino acid sequence may be encoded by the following nucleic acid sequence: 10 ATGTTGGCAATGGAGAAAGAATTTGATTCAAAGCTTGTTCTTCAAGGGAA CTCATCCAACGGTGCTAATGTTTCTAGAAGCAAAAGCTTCTCCTTTAAAG CTCCTCAAGAAAATTTCACCAGCCATGATTTCGAATTTGGCAAGATCTAT GGTGTTGGTTCTTACTCTAAGGTTGTTAGGGCAAAGAAGAAGGAAACTGG 15 AACTGTGTATGCTTTAAAGATTATGGACAAAAAGTTTATCACCAAGGAGA ATAAAACTGCTTATGTGAAATTGGAAAGGATTGTTCTTGATCAACTTGAA CATCCTGGGATCATTAAACTTTACTTCACGTTTCAAGACACATCCTCACT ATATATGGCACTTGAATCTTGTGAGGGTGGCGAGCTTTTCGACCAAATAA CCAGAAAAGGTCGGCTATCGGAGGATGAAGCTCGGTTCTACACTGCAGAA 20 GTTGTGGATGCTCTTGAGTATATACATAGTATGGGACTGATTCATCGAGA TATTAAGCCGGAGAATCTGTTGCTGACTTCAGATGGACACATTAAGATTG CGGATTTTGGAAGTGTAAAGCCGATGCAGGATAGCCAGATCACAGTTCTA CCTAATGCAGCTTCTGACGATAAGGCGTGCACTTTTGTCGGGACTGCTGC ATATGTTCCTCCAGAAGTTCTCAACTCCTCTCCCGCAACTTTCGGGAATG 25 ATCTTTGGGCTCTCGGCTGCACTCTCTATCAGATGCTTTCGGGGACTTCC CCATTTAAAGATGCAAGTGAATGGCTGATTTTCCAAAGAATTATAGCCAG AGATATAAAGTTCCCAAATCATTTTTCAGAAGCAGCAAGAGACCTCATCG ACCGGTTGCTGGATACCGAGCCAAGCAGAAGGCCAGGTGCTGGCTCAGAA GGTTATGTTGCTCTTAAGAGACATCCTTTCTTTAATGGAGTTGACTGGAA 30 GGATCTAAGGTCCCAGACTCCTCCAAAACTAGCTCCAGATCCTGCGTCTC WO 00/36135 PCT/GB99/04228 27 AGACAGCAT CTCCCGAGAGGGAT GACACACATGGTT CT CCAT GGAACCTG ACACATATTGGAGATTCTTTAGCCACACAGAACGAGGGGCACAGTGCTCC TCCTACATCTTCTGAATCATCGGGTTCCATAACTCGACTTGCTTCAATAG ACTCTTTTGATTCAAGATGGCAACAGTTTTTAGAGCCAGGAGAAT CGGTT 5 CTGATGATATCAGCGGTGAAGAAGCTTCAGAAAATAACGAGCAAGAAGGT GCAGCTAATACTCACCAACAAACCCAAGCTGATATATGTCGACCCGTCAA AACTAGT TGTGAAAGGAAACATTATATGGT CT GATAACTCGAATGACCTC AACGTTGTAGTCACTAGCCCTTCACATTTCAAGATTTGCACGCCAAAGAA GGTTTTAT CATTTGAAGACGCAAAACAGAGAGCTTCAGTGTGGAAAAAGG 10 CAATCGAGACTCTTCAGAACCGCTGA (1476 bp complete sequence) It will be appreciated that a further aspect of the invention is a recombinant polynucleotide comprising the above nucleotide sequence, or a recombinant 15 polynucleotide encoding or suitable for expressing the above polypeptide. A still further embodiment of the invention is a method of the invention wherein the said host yeast cell protein kinase or kinases is Ypkl and/or Ykr2. The identification of the Ypk1 and Ykr2 genes is described in 20 references 41 to 43. Ypkl from S. cerevisiae may comprise the following amino acid sequence: MYSWKSKFKFGKSKEEKEAKHSGFFHSSKKEEQQNNQATAGEHDASITRS SLDRKGTINPSNSSVVPVRVSYDASSSTSTVRDSNGGNSENTNSSQNLDE 25 TANIGSTGTPNDATSSSGMMTIKVYNGDDFILPFPITSSEQILNKLLASG VPPPHKE ISKEVDALIAQLSRVQIKNQGPADEDLISSESAAKFIPSTIML PGSSTLNPLLYFTIEFDNTVATIEAEYGTIAKPGFNKISTFDVTRKLPYL KIDVFARIPSILLPSKTWQQEMGLQDEKLQTIFDKINSNQDIHLDSFHLP INLSFDSAAS IRLYNHHWITLDNGLGKINISIDYKPSRNKPLSIDDFDLL 30 KVIGKGSFGKVMQVRKKDTQKVYALKAIRKSYIVSKSEVTHTLAERTVLA WO 00/36135 PCT/GB99/04228 28 RVDCPFIVPLKFS FQSPEKLYFVLAFINGGELFYHLQKEGRFDLSRARFY TAELLCALDNLHKLDVVYRDLKPENILLDYQGHIALCDFGLCKLNMKDDD KTDT FCGT PEYLAPELLLGLGYTKAVDWWTLGVLLYEMLTGLPPYYDEDV PKMYKKILQEPLVFPDGFDRDAKDLLIGLLSRDPTRRLGYNGADEIRNHP 5 FFSQLSWKRLLMKGYIPPYKPAVSNSMDTSNFDEEFTREKPIDSVVDEYL SESVQKQFGGWTYVGNEQLGSSMVQGRSIR (680 aa complete sequence). Ypk1 from S. cerevisiae may be encoded by the following nucleotide sequence: 10 ATGTATTCTTGGAAGTCAAAGTTTAAGTTTGGAAAATCTAAAGAAGAAAA AGAAGCCAAACATAGTGGGTTTTTTCACTCCTCTAAAAAGGAAGAACAGC AGAATAATCAAGCAACTGCGGGGGAACATGATGCTTCAATTACTCGTTCA T CTTTAGACAGGAAGGGCACGATAAAT CCAT CAAATT CATCAGTGGT CCC GGTTCGTGTTTCATACGATGCATCCTCATCGACCTCTACTGTACGAGATT 15 CAAATGGTGGTAATTCAGAAAACACGAACTCATCTCAAAATTTAGATGAA ACAGCAAATATTGGTTCTACAGGGACACCCAATGATGCCACCTCAAGTTC AGGAATGATGACTATCAAAGTATATAATGGTGATGACTTTATTTTACCTT TTCCGATTACCTCAAGCGAGCAAATATTGAATAAACTATTGGCCTCGGGT GTTCCACCGCCACATAAGGAGATTAGTAAAGAAGTAGATGCTTTAATTGC 20 ACAACTGTCCCGTGTTCAAATAAAGAACCAAGGTCCAGCTGATGAAGATT TGATCTCTAGCGAGTCAGCAGCAAAGTTCATCCCATCTACTATCATGTTA CCAGGGTCTTCGACCTTAAATCCCTTACTATATTTCACCATTGAATTTGA TAATACAGTTGCAACTATTGAAGCAGAGTATGGTACGAT CGCTAAACCTG GTTTCAATAAGATATCTACCTTTGATGTGACAAGGAAATTACCTTATTTA 25 AAGATTGATGTATTTGCAAGAATTCCGTCCATTCTTTTGCCCTCGAAGAC ATGGCAACAGGAGATGGGTTTGCAAGACGAGAAGCTACAGACCATTTTTG ATAAAATAAATTCCAATCAAGATATACATTTAGATTCTTTCCATTTGCCC ATCAACTTAAGTTTTGACTCTGCAGCTAGTATTAGACTATATAATCACCA CTGGATCACATTGGATAATGGCTTGGGTAAGATCAATATTAGTATTGATT 30 ATAAACCTTCCAGAAATAAACCTTTGTCCATCGATGATTTCGATCTTTTG WO 00/36135 PCT/GB99/04228 29 AAAGTTATCGGTAAGGGTTCGTTTGGTAAAGTGATGCAAGTCAGAAAGAA AGATACACAAAAAGTATACGCTT T GAAGGCAAT CAGAAAAT CATACATT G TCTCTAAATCCGAAGTCACGCATACTTTGGCAGAAAGAACCGTTCTAGCA CGTGTTGATTGTCCATTTATTGTACCTTTGAAGTTTTCTTTCCAATCACC 5 GGAAAAATTATACTTTGTTTTAGCGTTTATCAATGGTGGTGAGTTGTTTT ATCATCTACAAAAGGAAGGAAGGTTTGATTTATCACGT GCCAGATTTTAT ACCGCAGAATTGTTATGTGCGCTAGACAACTTGCATAAACTAGATGTTGT CTATCGTGATTTGAAACCAGAGAATATTTTATTGGATTATCAAGGCCACA TTGCCCTTTGTGATTTCGGGCTATGCAAATTGAATATGAAGGATGATGAT 10 AAGACAGATACTTTTTGTGGGACCCCAGAATACTTGGCACCAGAACTATT GCTAGGTTTAGGCTATACAAAGGCAGTAGATTGGTGGACATTGGGAGTCT TGTTATACGAGATGCTCACAGGTCTTCCTCCTTATTATGATGAAGATGTT CCAAAAATGTATAAGAAGATTCTACAGGAGCCACTAGT TTTCCCAGATGG ATTTGATAGAGATGCAAAGGATCTATTGATTGGATTATTGAGCCGTGATC 15 CGACAAGAAGATTGGGCTACAATGGTGCCGACGAAATTCGGAACCATCCT TTTTTCAGCCAATTATCATGGAAGCGCCTGTTGATGAAGGGTTATATTCC ACCATATAAACCAGCTGTTAGTAATTCCATGGATACTAGTAATTTCGATG AGGAAT TCACTAGGGAGAAGCCAATTGATAGT GTGGTAGATGAATACTTG AGTGAAAGTGTTCAAAAGCAATTTGGTGGCTGGACATACGTTGGAAATGA 20 ACAGCTAGGTAGCTCAATGGTGCAAGGTAGAAGCATTAGATAG (Genbank accession no 486213; 2043 bp complete sequence) Ykr2 (also known as Ypk2) from S. cerevisiae may have the following amino acid sequence: 25 MHSWRISKFKLGRSKEDDGSSEDENEKSWGNGLFHFHHGEKHHDGSPKNH NHE HEHHIRKINTNETLPSSLSSPKLRNDASFKNPSGIGNDNSKASERKA SQSSTETQGPSSESGLMTVKVYSGKDFTLPFPITSNSTILQKLLSSGILT SSSNDASEVAAIMRQLPRYKRVDQDSAGEGLIDRAFATKFIPSSILLPGS TNSSPLLYFTIEFDNSITTISPDMGTMEQPVFNKISTFDVTRKLRFLKID 30 VFARIPSLLLPSKNWQQEIGEQDEVLKEILKKINTNQDIHLDSFHLPLNL WO 00/36135 PCT/GB99/04228 30 KIDSAAQIRLYNHHWI SLERGYGKLNITVDYKPSKNKPLS I DDFDLLKVI GKGS FGKVMQVRKKDTQKIYALKALRKAYIVSKCEVTHTLAERTVLARVD CPFIVPLKFSFQSPEKLYLVLAFINGGELFYHLQHEGRFSLARSRFYIAE LLCALDSLHKLDVIYRDLKPENILLDYQGHIALCDFGLCKLNMKDNDKTD 5 TFCGTPEYLAPEILLGQGYTKTVDWWTLGILLYEMMTGLPPYYDENVPVM YKKILQQPLLFPDGFDPAAKDLLIGLLSRDPSRRLGVNGTDEIRNHPFFK DISWKKLLLKGYIPPYKPIVKSEIDTANFDQEFTKEKPIDSVVDEYLSAS IQKQFGGWTYIGDEQLGDSPSQGRSIS (677 aa complete sequence). 10 Ykr2 (also known as Ypk2) from S. cerevisiae may be encoded by the following nucleotide sequence: ATGCATTCCTGGCGAATATCCAAGTTTAAGTTAGGAAGGTCCAAAGAAGA TGATGGGAGTAGTGAAGATGAAAATGAAAAATCGTGGGGTAATGGCCTGT 15 TTCATTTCCACCATGGAGAAAAACATCACGATGGTAGCCCGAAGAATCAT AATCATGAACACGAACACCATATAAGAAAGAT CAATACAAATGAGACTCT CCCAAGTTCCTTAAGTTCTCCAAAATTACGTAATGATGCATCCTTCAAGA ATCCAT CGGGGATAGGAAATGACAATT CTAAGGCTTCCGAAAGGAAAGCT AGTCAGTCGTCTACTGAGACGCAGGGACCGAGTTCGGAATCCGGACTAAT 20 GACAGTGAAGGTGTATTCTGGTAAAGATTTTACTCTTCCCTTCCCTATCA CCTCTAACTCTACTATTTTACAAAAACTACTAAGTTCCGGCATCCTTACT TCATCATCCAATGACGCTTCCGAAGTTGCAGCCATAATGCGGCAGCTACC ACGATACAAGAGAGTGGATCAAGATTCAGCAGGGGAAGGCTTGATAGATA GAGCTTTTGCCACTAAATTCATTCCTTCCTCTATATTGTTACCTGGGTCA 25 ACAAATTCAAGCCCATTACTTTATTTTACAATTGAATTTGATAATTCTAT TACTACTATTAGTCCAGATATGGGAACGATGGAGCAACCAGTGTTTAACA AAATATCGACATTTGATGTAACAAGAAAATTACGATTTTTAAAAATCGAT GTCTTTGCAAGGATTCCATCCCTACTTTTACCCTCTAAAAACTGGCAACA GGAGATTGGCGAGCAGGACGAAGTACTGAAGGAGATTTTAAAAAAAATCA 30 ATACAAATCAGGATATCCATTTGGACTCCTTCCATTTACCTTTGAATTTA WO 00/36135 PCT/GB99/04228 31 AAAATCGATTCTGCAGCCCAAATAAGACTATACAATCACCATTGGATTTC TTTAGAAAGGGGATATGGTAAATTAAATATCACGGTGGACTACAAACCTT CTAAGAACAAGCCTCTCTCCATTGATGACTTTGATCTATTGAAGGTTATC GGGAAGGGTTCGTT CGGCAAAGT GATGCAAGTAAGGAAAAAAGATACCCA 5 AAAGATTTACGCTTTGAAGGCTCTGAGAAAAGCATATATTGTATCGAAAT GTGAAGTGACACATACTTTAGCGGAGAGGACTGTCCTAGCAAGAGTTGAC TGCCCCTTTATTGTTCCGTTGAAGTTCTCATTCCAATCTCCGGAGAAGTT GTACCTAGTATTAGCTTTCATTAATGGCGGTGAACTGTTCTACCATTTAC AACACGAGGGACGATTCAGTCTAGCACGCTCCCGTTTTTATATTGCAGAA 10 CTATTATGTGCTCTCGATTCATTACACAAACTTGACGTCATTTATCGTGA CCTAAAGCCTGAAAACATTCTATTGGATTACCAAGGACATATTGCACTGT GTGATTTTGGGCTTTGCAAGCT GAACATGAAGGATAAT GACAAAACAGAC ACTTTCTGTGGTACTCCCGAATATTTGGCACCAGAAATCTTGTTGGGGCA GGGCTATACTAAAACAGTTGACTGGTGGACATTAGGTAT CTTACTGTATG 15 AGATGATGACAGGGCTGCCACCATACTATGATGAGAACGTTCCTGTTATG TACAAGAAAATTCTGCAGCAACCGCTACTATTTCCTGATGGATTTGACCC TGCGGCAAAAGACCTATTAATT GGCCT CTTAAGCAGAGACCCAAGCAGAA GACTCGGCGTTAACGGTACAGATGAAATTCGTAACCATCCTTTCTTTAAA GACATCTCATGGAAAAAGCTACTTTTGAAGGGCTATATTCCGCCTTACAA 20 GCCAATTGTAAAGAGTGAAATAGATACTGCAAATTTTGATCAAGAGTTCA CTAAGGAAAAACCGAT CGATAGTGTAGT GGACGAGTACTTAAGTGCAAGT ATTCAAAAGCAGTTTGGTGGGTGGACGTACATTGGTGACGAACAGTTGGG T GATTCT CCTTCGCAGGGGAGAAGCATTAGTTAG (Genbank accession no. 817862; 2034 bp complete sequence) 25 Yeast, for example S. cerevisiae, cells that are capable of expressing either Ypk1 or Ykr2 or both are capable of growing, but yeast, for example S. cerevisiae, cells that are not capable of expressing either Ypkl or Ykr2 are not capable of growing under certain conditions, as described in Example 1. 30 WO 00/36135 PCT/GB99/04228 32 A protein kinase derivable from an source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases, for example Ypk1 or Ykr2, may be serum and glucocorticoid induced protein kinase (SGK), for example mammalian, preferably human SGK. As described in Example 1, 5 yeast, for example S. cerevisiae, cells that are not capable of expressing either Ypkl or Ykr2 may be capable of growing if they are capable of expressing human SGK . Rat SGK may have the nucleotide and amino acid sequences shown in Figures 12 10 and 13, respectively. The term SGK encompasses isoforms of SGK, for example SGK 1, 2 and 3 as described in Webster et al (1993) Mol. Cell. Biol. 13, 1031 2040 and Kobayashi et al (1999) Biochem J 344(Pt 1), 189-197 and in EMBL database records with Accession Nos AAD41091, AF169034 and AF169035. 15 PKB may also have some activity that is equivalent to Ypk1 or Ykr2 as yeast, for example S. cerevisiae, cells that are not capable of expressing either Ypk1 or Ykr2 may be capable of growing if they are capable of expressing high levels of PKB, ie if the cells have a high accumulation of a plasmid capable of expressing 20 PKB, as described in Example 1. Alternatively, the ability to grow of cells that are capable of expressing PKB at a lower level may be increased by growing the cells in the presence of an activator of PKB activity. A further aspect of the invention is a method of identifying a compound that 25 modulates (inhibits or increases) the activity of PDK1 derivable from a first source, wherein a compound is exposed to 1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing a yeast polypeptide that is a functional equivalent of human PDK1 (which may be WO 00/36135 PCT/GB99/04228 33 Pkh1 and Pkh2) and (b) is capable of expressing PDK1 derivable from the said first source and optionally 2) a second host yeast cell wherein the yeast cell is capable of expressing a yeast 5 polypeptide that is a functional equivalent of human PDK1 (which may be Pkh1 and/or Pkh2) and the effect of the compound on the viability of the said yeast cell or cells is measured, and a compound that affects the viability of the first said yeast cell, or optionally that affects the viability of the first said yeast cell and the said second 10 yeast cell differently, is identified. A further aspect of the invention is a method of identifying a compound that modulates (inhibits or increases) the activity of a functional equivalent of Ypk1 and/or Ykr2 derivable from a first source, for example SGK or PKB, preferably 15 human SGK or human PKB, wherein a compound is exposed to 1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing a yeast polypeptide that is a functional equivalent of Ypk1 and/or Yrk2 and (b) is capable of expressing a functional equivalent of Ypkl and/or Ykr2 (for example SGK) derivable from the said first source 20 and optionally 2) a second host yeast cell wherein the yeast cell is capable of expressing a yeast polypeptide (for example, an endogenous polypeptide) that is a functional equivalent of Ypkland/or Yrk2 and the effect of the compound on the viability of the said yeast cell or cells is 25 measured, and a compound that affects the viability of the first said yeast cell, or optionally that affects the viability of the first said yeast cell and the said second yeast cell differently, is identified.

WO 00/36135 PCT/GB99/04228 34 As in the first aspect of the invention, it will be appreciated that the said first and second host yeast cells differ substantially only in the features indicated such that apart from these features the cells are essentially the same. This ensures that there is a reasonable expectation that the effect of the compound on the phenotype 5 of the said yeast cells can be attributed to the compound's effect on the modulation of the activity to different extents of the protein kinases as said. Thus, the first and second host yeast cells are from the same species and have substantially the same genetic content with the exception of the features indicated ie the capability to express the said host yeast cell protein kinase or kinases and 10 the ability to express the said equivalent protein kinase derivable from a source other than the host yeast cell. It will be appreciated that the said first and second host yeast cells may differ in genetic content relating to the generation or selection of the said first or second host yeast cells (for example, in selectable marker genes used in recombinant techniques, as well known to those skilled in 15 the art). It will be appreciated that the PDK1 or SGK or PKB that the said first cell is capable of expressing may not be full-length PDK1 or SGK or PKB; for example, it may be a truncated PDK1 or SGK as appropriate. It will be appreciated that it 20 is preferred that any PDK1 or SGK or PKB (either activated or not activated) that is not full length PDK1 or SGK or PKB may have at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the enzymatic activity of full length PDK1 or SGK or PKB (either activated or not activated), as herein described. 25 However, it will be understood that in the methods of the invention described above it will be desirable to identify compounds that may modulate (including activate or inhibit), the activity of the relevant protein kinase or kinases, for example PDK1 or SGK, in vivo. Thus it will be understood that reagents and conditions used in the method may be chosen such that the interactions between WO 00/36135 PCT/GB99/04228 35 the said protein kinase and its substrate or substrates and any regulating protein/substance are substantially the same as in vivo. It will be appreciated that these methods of the invention may be used to identify 5 an activator of, for example, PDK1, SGK or PKB in a manner similar to that described above for the first and second aspects of the invention. Thus, the said first yeast cell may, under the growth conditions used in the method, express the said functional equivalent of Ypkl, Ykr2, Pkh1 and/or Pkh2 (for example, PDK1, SGK or PKB) at a level below that required for the said yeast cell to be 10 capable of growing at the level of the wild-type cell, preferably at a level below that required for the said yeast cell to be substantially capable of growing. In the presence of an activator of the said functional equivalent (for example, PDK1, SGK or PKB), the level of activity of the said functional equivalent may be sufficient for the said yeast cell to be more capable, preferentially substantially 15 capable of growing, under the same growth conditions. As discussed above and in Example 1, the said functional equivalent may expressed from a regulatable promoter, for example the GAL1 promoter that is repressed in the presence of glucose. 20 The optional said second yeast cell may similarly be substantially unable to grow unless an activator of the said yeast polypeptide (for example, an endogenous polypeptide) is present. It will be appreciated that the yeast polypeptide may be expressed from a heterologous promoter, for example the GAL1 promoter, as discussed above and in Example 1. 25 It will be appreciated that when identifying compounds on the basis of the ability to increase growth (as opposed to reducing growth) of the target cell that it may be less important to make use of the optional second yeast cell in the methods above as it may be less likely that a compound that is not acting on the "target" WO 00/36135 PCT/GB99/04228 36 polypeptide (for example, the said functional equivalent of Ypkl, Ykr2, Pkh1 and/or Pkh2, which may be PDK1, SGK or PKB) would have the desired effect of increasing cell growth. Compounds acting through a "non-specific" mechanism, as discussed above, more frequently act to reduce cell growth. 5 Preferences regarding the host yeast cell and sources of the equivalent protein kinases may be as given in regard to the earlier aspects of the invention. It will be appreciated that in the methods described herein, which may be drug 10 screening methods, a term well known to those skilled in the art, the compound may be a drug-like compound or lead compound for the development of a drug like compound. The term "drug-like compound" is well known to those skilled in the art, and 15 may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small 20 molecule, which may be of less than 5000 daltons and which may be water soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential. 25 The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise WO 00/36135 PCT/GB99/04228 37 or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics. The compound may be suitable for use as a plant protection product or may be a 5 lead compound for the development of a compound suitable for use as a plant protection product. It will be appreciated that such compound may have characteristics similar to those described above for drug like compounds. It will be appreciated that compounds that may be identified by methods or screens performed with cells may be capable of interacting with or entering cells. 10 Alternatively, the methods may be used as "library screening" methods, a term well known to those skilled in the art. Thus, for example, the methods of the invention may be used to detect (and optionally identify) a polynucleotide capable of expressing a polypeptide activator of a functional equivalent of Ypkl, Ykr2, 15 Pkh1 and/or Pkh2 for example, PDK1, SGK or PKB. Aliquots of an expression library in a suitable vector, as described below, may be tested for the ability to increase the growth of a first yeast cell that, under the growth conditions used in the method, expresses the said functional equivalent of Ypkl, Ykr2, Pkh1 and/or Pkh2 (for example, PDK1, SGK or PKB) at a level below that required for the 20 said yeast cell to be capable of growing at the level of the wild-type cell, preferably at a level below that required for the said yeast cell to be substantially capable of growing. In the presence of a polynucleotide expressing a polypeptide activator of the said functional equivalent (for example, PDK1, SGK or PKB), the level of activity of the said functional equivalent may be sufficient for the said 25 yeast cell to be more capable, preferentially substantially capable of growing, under the same growth conditions. As discussed above and in Example 1, the said functional equivalent may expressed from a regulatable promoter, for example the GAL1 promoter that is repressed in the presence of glucose.

WO 00/36135 PCT/GB99/04228 38 It will be appreciated that several cycles of identifying pools of polynucleotides comprising a polynucleotide having the required property and then rescreening those polynucleotides may be required in order to identify a single species of polynucleotide with the required property. 5 It will be appreciated that further tests and/or sequence analysis may be required in order to distinguish polynucleotides encoding a functional equivalent of Ypkl, Ykr2, Pkh1 and/or Pkh2 from polynucleotides encoding an activator of a said functional equivalent. 10 Methods of introducing a polynucleotide into a host yeast cell are well known to those skilled in the art. Methods of preparing a suitable expression library for screening are well known to those skilled in the art. The library may preferably be from the same source as the said functional equivalent that is expressed in the 15 said host yeast cell ie a human expression library may be screened for effects on yeast cells expressing human PDK1, SGK or PKB at levels below the threshold required for cell growth. The above compound/library screening methods may conveniently be carried out 20 in a 96-well microtitre plate format. A yeast cell, such as a Saccharomyces cerevisiae cell that has the properties of a host yeast cell for use in the method of the invention may be picked and put into liquid culture in minimal medium supplemented as necessary (as described, for example, in Example 1). The culture is grown up and may then be diluted to an optical density (OD) at 595 nm 25 of 0.01 to 0.1. The diluted cultures are then aliquoted into wells of a sterile 96 well microtitre plate containing individual test compounds. The growth of the cells is monitored over time until the ODs95 reached is about ~0.8 for control cultures (ie those cultured in the absence of test compound). The OD595 is assessed using a microtitre plate reader. It will be appreciated that a control WO 00/36135 PCT/GB99/04228 39 culture may contain an aliquot of the solvent used to dissolve the test compound. Thus, for example, the test compound may be dissolved in dimethylsulfoxide (DMSO) and both test and control cultures may therefore contain DMSO, for example at between 0.1 and 5%, preferably at about 1 % to 2% by volume. 5 Alternatively, the cells may be grown on solid medium and the presence, asbence, number and/or size (or other characteristic) of colonies detected. It will be appreciated that this may be performed in a high-throughput format, for example in microtitre plates. The presence, absence, number and/or size (or 10 other characteristic) of the colonies may be detected by visual inspection, either manually or by automated techniques, including computer image analysis. It will be appreciated that dose response measurements, as well known to those skilled in the art, may be made for selected compounds. 15 If appropriate, duplicate plates of compounds may be exposed to aliquots (including solid cultures) of a particular yeast culture, or may be exposed to aliquots of different yeast cultures. For example, one such plate may be exposed to wild-type (with respect to Pkhl, Pkh2, Ypkl, Yrkl as appropriate) yeast cells, 20 and a further such plate may be exposed to cells which are mutant with respect to Pkhl, Pkh2, Ypkl, Yrk2 as appropriate but which express, for example, human equivalents (PDK1 is a human equivalent of Pkh1 and Pkh2, and SGK and PKB are human equivalents of Ypkl and Yrk2), as described in the methods of the invention. 25 Yeast cells WO 00/36135 PCT/GB99/04228 40 A further aspect of the invention is a yeast cell that is not capable of expressing Pkh1 and Pkh2 or any functional equivalent thereof. It will be appreciated that such a cell may not be capable of growing ie may be of limited viability. 5 A further aspect of the invention is a yeast cell that is not capable of expressing endogenous Pkhl and/or Pkh2. The yeast cell may be capable of expressing a functional equivalent of Pkh1 and/or Pkh2 that is not the Pkh1 or Pkh2 endogenous to a wild-type yeast cell from which the said yeast cell is derived. The functional equivalent may be human PDK1 or a variant, fusion or derivative 10 thereof, for example PDK1-APH as described in Example 1. PDK1 or PDK1 APH may be capable of phosphorylating the same target substrates essential for the viability of yeast cells as Pkh1 and Pkh2, as discussed in Example 1. It will be appreciated that the cell may be capable under given conditions of expressing the said functional equivalent of Pkh1 and/or Pkh2 at a level below that necessary 15 full wild-type viability of the cell, for example at a level at which the cell is substantially unable to grow under given conditions. Thus, the yeast cell may be a S. cerevisiae cell that is not capable of expressing Pkhl from S. cerevisiae or a S. cerevisiae cell that is not capable of expressing 20 Pkh2 from S. cerevisiae, or a S. cerevisiae cell that is not capable of expressing Pkhl and Pkh2 from S. cerevisiae. Alternatively, the yeast cell may be a cell of a different yeast species that is not capable of expressing a Pkh1 that is expressed in wild type cells of that yeast species (which may be an endogenous functional equivalent of Pkh1 from S. cerevisiae), or that is not capable of expressing a 25 Pkh2 that is expressed in wild type cells of that yeast species (which may be an endogenous functional equivalent of Pkh2 from S. cerevisiae), or that is not capable of expressing any Pkhl and Pkh2 (ie not capable of expressing any endogenous functional equivalent of Pkhl and Pkh2 of S. cerevisiae) that are expressed in wild type cells of that yeast species.

WO 00/36135 PCT/GB99/04228 41 A yeast cell not capable of expressing Pkh1 or Pkh2 or endogenous functional equivalents may be made by methods known in the art. For example, the open reading frame encoding, for example, either Pkhl or Pkh2 may be disrupted by 5 insertion of a selectable marker, for example as described in Example 1. A further aspect of the invention is a yeast cell wherein one or more genes encoding a functional equivalent of PDK1, for example human PDK1, are mutated such that the yeast cell is not capable of expressing the said functional 10 equivalent of (human) PDK1. Each such gene encoding a functional equivalent of human PDK1 may be mutated such that the yeast cell is not capable of expressing a functional equivalent of (human) PDK1. Such a functional equivalent of (human) PDK1 may be Pkhl or Pkh2 in, for example, a S. cerevisiae cell. 15 It will be appreciated that cells of a yeast species other than S. cerevisiae may express one, two, three, four or more polypeptides that are at least partially functionally equivalent to Pkhl and/or Pkh2 from S. cerevisiae. It will further be appreciated that the yeast cell of the invention encompasses a cell of such a yeast 20 species that is not capable of expressing one, two, three, four or more or all of the said polypeptides. It will be appreciated that Pkhl, Pkh2 and any polypeptide that is a functional equivalent of Pkh1 and/or Pkh2 may be a functional equivalent of mammalian, 25 preferably human, PDK1. Thus, the invention encompasses a yeast cell wherein one or more gene that is present in a wild-type yeast cell that encodes a functional equivalent of human PDK1 is mutated such that the yeast cell is not capable of expressing the said functional equivalent of human PDK1. Each such gene present in a wild-type yeast cell that encodes a functional equivalent of human WO 00/36135 PCT/GB99/04228 42 PDK1 is mutated such that the yeast cell is not capable of expressing a functional equivalent of human PDK1. The yeast cell of this aspect of the invention encompasses a yeast cell that is not 5 capable of expressing a polypeptide with the sequence of Pkh1 or a functional equivalent thereof and/or Pkh2 or a functional equivalent thereof. It will be appreciated that a said yeast cell that is not capable of expressing any functional equivalent of Pkhl or Pkh2 may not be as capable of cell growth and/or division as a wild-type yeast cell or a yeast cell that is capable of expressing a functional 10 equivalent of Pkh1 and/or Pkh2. A functional equivalent of Pkh1 and/or Pkh2 may be capable of phosphorylating Ypkl and/or Ykr2. PDK1 or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment that is capable of phosphorylating Ypk1 and/or Ykr2 may be a functional equivalent of Pkhl and/or Pkh2. 15 A further aspect of the invention is a said yeast cell capable of expressing a functional equivalent of Pkh1 and/or Pkh2 that is not Pkhl or Pkh2 derivable from the wild-type yeast cell ie is an exogenous functional equivalent of Pkh1 and/or Pkh2. The said functional equivalent of Pkhl and/or Pkh2 may be 20 mammalian, preferably human, PDK1 or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment that may be capable of phosphorylating Ypk1 and/or Ykr2. Such a yeast cell is described in Example 1. The said functional equivalent of Pkh1 or Pkh2 may be Pkh1 or Pkh2 from a different yeast species, for example a pathogenic yeast species; thus 25 the yeast cell may be a S. cerevisiae cell that is (1) not capable of expressing S. cerevisiae Pkhl or S. cerevisiae Pkh2 and (2) is capable of expressing Candida Pkhl or Candida Pkh2.

WO 00/36135 PCT/GB99/04228 43 Thus, a yeast cell of the invention may be a yeast cell wherein the wild-type gene encoding a functional equivalent of Pkh1 or Pkh2 is altered such that it is not capable of expressing a functional equivalent of the polypeptide Pkhl or Pkh2. Thus, the yeast cell may be a S. cerevisiae cell wherein the polypeptide encoded 5 by open reading frame YDR490c in wt yeast cells is not capable of being expressed and/or the polypeptide encoded by open reading frame YOL100w in wt yeast is not capable of being expressed. The gene may be altered by the open reading frame of the gene being disrupted 10 by insertion of a selectable marker. The marker may be TRP1 or HIS3, as described in Example 1. A further aspect of the invention is a method of identifying a compound that modulates (including increases or inhibits) the activity of PDK1, for example 15 mammalian, preferably human, PDK1, wherein a yeast cell according to the invention is used. A further aspect of the invention is the use of a yeast cell of the invention in a method of identifying a compound that modulates (activates or inhibits) the 20 activity of PDK1. By PDK1 is included mammalian, preferably human, PDK1, and a functional equivalent of PDK1 derivable from another source, preferably a source that may be pathogenic (including an opportunistic pathogen) to a mammal, preferably a 25 human. Thus, a functional equivalent of PDK1 from a pathogenic yeast, for example Candida is included. Such a functional equivalent may be a polypeptide that is an equivalent of the Pkh1 or Pkh2 polypeptide identified in S. cerevisiae. Thus, such a functional equivalent may be the Pkh1 or Pkh2 identified in WO 00/36135 PCT/GB99/04228 44 Candida, as discussed in Example 4, or in the plant Arabidopsis thaliana, as discussed below. A further aspect of the invention is a yeast, for example S. cerevisiae or Candida, 5 or other yeast as listed above, cell wherein one or more endogenous gene(s) encoding a functional equivalent of human SGK or PKB is mutated such that the yeast cell is not capable of expressing the said functional equivalent of human SGK or PKB. The said gene may be the Ypkl or Yrk2 gene. Each such endogenous gene encoding a functional equivalent of human SGK or PKB may be 10 mutated such that the yeast cell is not capable of expressing an endogenous functional equivalent of, for example, human SGK or PKB. A further aspect of the invention is a yeast, for example S. cerevisiae or Candida, or other yeast/fungus, as listed above, cell wherein one or more endogenous 15 genes encoding a functional equivalent of human SGK is mutated such that the yeast cell is not capable of expressing the said functional equivalent of human SGK. As discussed in Example 4, the following nucleotide sequences may encode 20 polypeptides related to S. cerevisiae Ypkl and Yrk2 polypeptides: 384362E11.sl.seq and 384286E10.sl.seq. The said gene may be a gene from which Ypk1 may be expressed or a gene from which Ykr2 may be expressed. 25 A further aspect of the invention is a said yeast cell wherein each such endogenous gene encoding a functional equivalent of human SGK or Ypkl or Yrk2 is mutated such that the yeast cell is not capable of expressing an endogenous functional equivalent of, for example, human SGK or Ypkl or Yrk2. 30 WO 00/36135 PCT/GB99/04228 45 A further aspect of the invention is a method of identifying a compound (including a polypeptide or polynucleotide encoding a polypeptide) that modulates (activates or inhibits) the activity of SGK, for example fungal or mammalian, preferably human, SGK, wherein a yeast cell according to the above aspect of the 5 invention is used. A further aspect of the invention is the use of a yeast cell according to the above aspect of the invention in a method of identifying a compound that modulates (activates or inhibits) the activity of SGK. 10 Ypkl and Ykr2 A further aspect of the invention is a protein kinase derivable from yeast capable of phosphorylating a polypeptide comprising the consensus sequence Arg-Xaa 15 Arg-Xaa-Xaa-Ser/Thr-Hyd, for example the polypeptide Crosstide (which has the amino acid sequence GRPRTSSFAEG) or polypeptide 2, 8 or 11 shown in Table 2 of Example 1. A further aspect of the invention is a protein kinase derivable from yeast capable 20 of being phosphorylated by Pkh1 or Pkh2 or mammalian, preferably human, PDK1. The yeast may be S. cerevisiae. The protein kinase may share functional characteristics with mammalian PKBc and/or SGK. It was not previously known 25 that yeast, for example S. cerevisiae, may have a protein kinase with similar functional properties to mammalian PKBa or SGK. The protein kinase may be Ypkl from S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae, for example Candida spp or Ykr2 from S.

WO 00/36135 PCT/GB99/04228 46 cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae, for example Candida spp. Alternatively and less preferably, it may be a related polypeptide pkcl or sch9 or other AGC (protein kinase A/protein kinase G/protein kinase C) family member. 5 A further aspect of the invention is a variant, derivative, fragment or fusion thereof or a fusion of a variant, derivative or fragment of a protein kinase according to the previous aspect of the invention that is capable of being phosphorylated by Pkh1 or Pkh2 or mammalian, preferably human, PDK1 and/or 10 capable of phosphorylating a polypeptide comprising the consensus sequence Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr-Hyd. The protein kinase may be the polypeptide encoded by the YPK1 gene [41] or the YKR2/YPK2 gene [42]. 15 Pkhl and Pkh2 A further aspect of the invention is a substantially pure polypeptide encoded by open reading frame YDR490c of S. cerevisiae or equivalent open reading frame 20 in yeast other than S. cerevisiae or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative or a substantially pure polypeptide encoded by open reading frame YOL100w of S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae. 25 or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative wherein the polypeptide does not comprise the amino acid sequence of human PDK1 or Drosophila PDK1 (DSTPK61).

WO 00/36135 PCT/GB99/04228 47 The said polypeptides are considered to be protein kinases, in particular protein kinases that are capable of phosphorylating PKBx, particularly human PKBa, in particular on residue Thr308. They are further considered to be capable of phosphorylating the yeast polypeptides known as Ypk1 and Ykr2, as described 5 above, human SGK or a polypeptide with the consensus sequence Thr-Phe-Cys Gly-Thr-X-Glu-Tyr (which may be present with the "activation loop" of the catalytic domain between conserved subdomains VII and VIII of a protein kinase). 10 The polypeptides described above are herein referred to as Pkhl (PKB-activating kinase homologue 1; encoded by open reading frame YDR490c of S. cerevisiae, for example) or Pkh2 (PKB-activating kinase homologue 2; encoded by open reading frame YOL100w of S. cerevisiae, for example) [42, 43]. 15 Partial amino acid sequences of Pkh1 and Pkh2 are also shown in Figure 1. Example 4 describes the identification of Candida albicans nucleotide sequences that may encode polypeptides related to S. cerevisiae Pkhl and Pkh2. The method given in Example 4 may be applicable to identifying related open reading 20 frames in other organisms, for example other yeasts. The following nucleotide sequences may encode polypeptides related to S. cerevisiae Pkh1 and Pkh2: 384194F08.sl.seq and 396076E03.s2.seq 25 By "substantially pure" we mean that the said polypeptide is substantially free of other proteins. Thus, we include any composition that includes at least 30% of the protein content by weight as the said polypeptide, preferably at least 50%, more preferably at least 70%, still more preferably at least 90% and most preferably at least 95% of the protein content is the said polypeptide.

WO 00/36135 PCT/GB99/04228 48 Thus, the invention also includes compositions comprising the said polypeptide and a contaminant wherein the contaminant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more 5 preferably less than 30% of the composition, still more preferably less than 10% of the composition and most preferably less than 5% of the composition by weight. The invention also includes the substantially pure said polypeptide when 10 combined with other components ex vivo, said other components not being all of the components found in the cell in which said polypeptide is found. It will be appreciated that the said substantially pure polypeptide may be obtained by expression from a recombinant nucleic acid, for example in a prokaryotic or eukaryotic cell, as discussed further below. 15 Variants (whether naturally-occurring or otherwise) may be made using the methods of protein engineering and site-directed mutagenesis well known in the art using the recombinant polynucleotides described below. 20 By "fragment of said polypeptide" we include any fragment which retains activity, for example a protein kinase activity, for example as described above, or which is useful in some other way, for example, for use in raising antibodies or in a binding assay. 25 By "fusion of said polypeptide" we include said polypeptide fused to any other polypeptide. For example, the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions to GST are given in Example 1. Similarly, the said polypeptide may be fused to an oligo-histidine tag WO 00/36135 PCT/GB99/04228 49 such as His6 or to an epitope recognised by an antibody such as the well known Myc tag epitope. Fusions to any variant, fragment or derivative of said polypeptide are also included in the scope of the invention. 5 By "variants" of the polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. In particular we include variants of the polypeptide where such changes do not substantially alter the activity of the said polypeptide (for example, the ability to phosphorylate PKBa, particularly human PKBa, in particular on residue Thr308). Variants of Pkh1 or 10 Pkh2 do not include polypeptides which have the amino acid sequence of Schizosaccharomyces pombe, human PDK1 or Drosophila PDK1 (DSTPK61); see Figure 1. It will be appreciated that a variant that comprises substantially full-length Pkhl 15 or Pkh2 may be particularly useful. By "substantially full-length" is meant comprising at least 80%, preferably 90%, still more preferably 95%, 98% or 100% (ie all) of the sequence of the full length polypeptide. By "conservative substitutions" is intended combinations such as Gly, Ala; Val, 20 Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. It is particularly preferred if the polypeptide variant has an amino acid sequence which has at least 65 % identity with the amino acid sequence given above, more preferably at least 75 %, still more preferably at least 80%, yet more preferably at 25 least 90%, and most preferably at least 95% or 99% identity with the amino acid sequence given above. The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of WO 00/36135 PCT/GB99/04228 50 Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally. 5 The alignment may alternatively be carried out using the Clustal W program (Thompson et al (1994) Nucl Acid Res 22, 4673-4680). The parameters used may be as follows: Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. 10 Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM. Thus, using these parameters, the catalytic domain of Pkhl and Pkh2 may have 15 50% and 49% identity respectively with the catalytic domain of human PDK1 and 41% identity with the catalytic domain of Drosophila PDK1. There appears to be no significant identity between the non-catalytic domains of human or Drosophila PDK1 and either Pkhl or Pkh2. 20 A particular embodiment of the invention provides a substantially pure yeast Pkh1 polypeptide or naturally occurring allelic variants thereof. A partial amino acid sequence is also shown in Figure 1. A further particular embodiment of the invention provides a substantially pure 25 Pkh2 polypeptide or naturally occurring allelic variants thereof. It is particularly preferred, although not essential, that the variant or fragment or derivative or fusion of the said polypeptide, or the fusion of the variant or fragment or derivative has at least 30% of the enzyme activity of Pkh1 or Pkh2, WO 00/36135 PCT/GB99/04228 51 respectively, with respect to the phosphorylation (or activation) of PKBc (particularly human PKBa) or other polypeptide comprising the consensus sequence B-T-F-C-G-T-P/I-D/E-Y-L/I/M-A-P-E, for example a protein kinase belonging to the AGC subfamily (protein kinase A/G/C subfamily), in particular 5 SGK, p70 S6 Kinase or PKCg, as discussed in Example 1 and shown in Figure 6. It is more preferred if the variant or fragment or derivative or fusion of the said polypeptide, or the fusion of the variant or fragment or derivative has at least 50%, preferably at least 70% and more preferably at least 90% of the enzyme activity of Pkh1 or Pkh2 with respect to the phosphorylation of PKBa or any one 10 of the alternatives described above. However, it will be appreciated that variants or fusions or derivatives or fragments which are devoid of enzymatic activity may nevertheless be useful, for example by interacting with another polypeptide, or as antigens in raising antibodies. 15 A further aspect of the invention provides a recombinant polynucleotide encoding a said polypeptide of the invention (for example, Pkhl or Pkh2 from S. cerevisiae) or encoding a variant or fragment or derivative of fusion of said polypeptide or a fusion of a said variant or fragment or derivative. Preferences and exclusions for the said polynucleotide variant are the same as given above. 20 A further aspect of the invention provides a recombinant polynucleotide suitable for expressing a polypeptide of the invention or suitable for expressing a variant or fragment or derivative of fusion of said polypeptide or a fusion of a said variant or fragment or derivative. Preferences and exclusions for the said 25 polynucleotide variant are the same as in the first aspect of the invention. By "suitable for expressing" is mean that the polynucleotide is a polynucleotide that may be translated to form the polypeptide, for example RNA, or that the polynucleotide (which is preferably DNA) encoding the polypeptide of the WO 00/36135 PCT/GB99/04228 52 invention is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. The polynucleotide may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by any desired host; such controls may be 5 incorporated in the expression vector. Thus a further aspect of the invention is a replicable vector suitable for expressing a polypeptide of the invention or suitable for expressing a variant or fragment or derivative of fusion of said polypeptide or a fusion of a said variant 10 or fragment or derivative. Preferences and exclusions for the said polynucleotide variant are the same as for the polypeptide of the invention. For example, the replicable vector may be suitable for expressing a fusion of the polypeptide of the invention, in particular a GST fusion, for example as described in Example 1. It will be appreciated that a construct is not a recombinant polynucleotide as defined 15 above if it lacks sequences necessary for the translation and therefore expression of the encoded polypeptide of the invention. A further aspect of the invention is a polynucleotide encoding a fusion of the polypeptide of the invention, or a fusion of a variant or fragment or derivative, in 20 particular a GST fusion. A still further aspect is a vector suitable for replication in a mammalian/eukaryotic cell, comprising a polynucleotide encoding the polypeptide, or a variant or fragment or derivative or a fusion of the polypeptide, as defined in the first aspect of the invention, or a fusion of a variant or fragment or derivative, in particular a GST fusion. 25 Characteristics of vectors suitable for replication in mammalian/eukaryotic cells are well known to those skilled in the art, and examples are given below. It will be appreciated that a vector may be suitable for replication in both prokaryotic and eukaryotic cells.

WO 00/36135 PCT/GB99/04228 53 A polynucleotide comprising a fragment of the recombinant polynucleotide encoding a polypeptide of the invention or a variant, fragment, fusion or derivative may also be useful. Preferably, the polynucleotide comprises a 5 fragment which is at least 10 nucleotides in length, more preferably at least 14 nucleotides in length and still more preferably at least 18 nucleotides in length. Such polynucleotides are useful as PCR primers. A polynucleotide complementary to the polynucleotide (or a fragment thereof) encoding a polypeptide of the invention or a variant, fragment, fusion or derivative may also 10 be useful. Such complementary polynucleotides are well known to those skilled in the art as antisense polynucleotides. The polynucleotide or recombinant polynucleotide of the invention may be DNA or RNA, preferably DNA. The polynucleotide may or may not contain introns in 15 the coding sequence; preferably the polynucleotide is a cDNA. A "variation" of the polynucleotide includes one which is (i) usable to produce a protein or a fragment thereof which is in turn usable to prepare antibodies which specifically bind to the protein encoded by the said polynucleotide or (ii) an 20 antisense sequence corresponding to the gene or to a variation of type (i) as just defined. For example, different codons can be substituted which code for the same amino acid(s) as the original codons. Alternatively, the substitute codons may code for a different amino acid that will not affect the activity or immunogenicity of the protein or which may improve or otherwise modulate its 25 activity or immunogenicity. For example, site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro Mutagenesis" Science, 229: 193-210 (1985), which is incorporated herein by reference. Since such modified WO 00/36135 PCT/GB99/04228 54 polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are within the scope of the claimed invention. 5 Moreover, it will be recognised by those skilled in the art that the polynucleotide sequence (or fragments thereof) encoding a polypeptide of the invention can be used to obtain other polynucleotide sequences that hybridise with it under conditions of high stringency. Such polynucleotides includes any genomic DNA. Accordingly, the polynucleotide of the invention includes polynucleotide that 10 shows at least 60%, preferably 70%, and more preferably at least 80% and most preferably at least 90% homology with the polynucleotide identified in the method of the invention, provided that such homologous polynucleotide encodes a polypeptide which is usable in at least some of the methods described below or is otherwise useful. Such a polypeptide may be a functional homologue of the 15 polypeptide of the invention. The polypeptide may, for example, have a similar enzymatic activity to the polypeptide of the invention or may be able to substitute for the polypeptide of the invention in a cell, as discussed above. It will be appreciated that such a method may be used in the identification of a 20 functional homologue of Ypk1 or Ykr2, as discussed above. It is preferred that the polynucleotide is derivable from a yeast, for example a yeast other than S. cerevisiae or Candida. It is particularly preferred that the polynucleotide is derivable from a pathogenic yeast or a yeast that may be useful 25 as the host cell in a screening assay as described below. Per cent homology can be determined by, for example, the GAP program of the University of Wisconsin Genetic Computer Group.

WO 00/36135 PCT/GB99/04228 55 DNA-DNA, DNA-RNA and RNA-RNA hybridisation may be performed in aqueous solution containing between 0. 1XSSC and 6XSSC and at temperatures of between 55*C and 70*C. It is well known in the art that the higher the temperature or the lower the SSC concentration the more stringent the 5 hybridisation conditions. By "high stringency" we mean 2XSSC and 65"C. 1XSSC is 0.15M NaCI/0.015M sodium citrate. Polynucleotides which hybridise at high stringency are included within the scope of the claimed invention. The present invention also relates to a host cell transformed with a polynucleotide 10 vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, 15 USA (No ATCC 31343). Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include 20 Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors. 25 Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and WO 00/36135 PCT/GB99/04228 56 Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 5 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA. 10 Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect cells and vertebrate cells. For example, many bacterial species may be transformed by the methods 15 described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5X PEB using 6250V per cm at 25:FD. 20 Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182. Methods and vectors for transformation of Candida spp are described in, for example, Zhou P, et al "A system for gene cloning and manipulation in the yeast 25 Candida glabrata." Gene. 1994 May 3;142(1):135-40; Mehra RK, et al "Cloning system for Candida glabrata using elements from the metallothionein-Ia encoding gene that confer autonomous replication." Gene. 1992 Apr 1;113(1):119-24; Ohkuma M, et al "Cloning of the C-URA3 gene and construction of a triple auxotroph (his5, adel, ura3) as a useful host for the WO 00/36135 PCT/GB99/04228 57 genetic engineering of Candida maltosa." Curr Genet. 1993 Mar;23(3):205-10; Sakai Y, et al "Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5'-phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for 5 integrative transformation." J Bacteriol. 1991 Dec;173(23):7458-63; Haas LO, et al "Development of an integrative DNA transformation system for the yeast Candida tropicalis." J Bacteriol. 1990 Aug; 172(8):4571-7; Sakai Y, et al "High frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces 10 cerevisiae. " J Bacteriol. 1993 Jun;175(11):3556- 6 2; Kitada K, et al "Cloning of the Candida glabrata TRP1 and HIS3 genes, and construction of their disruptant strains by sequential integrative transformation." Gene. 1995 Nov 20; 165(2):203 6; Macreadie IG, et al "Heterologous gene expression and protein secretion from Candida glabrata." Biotechnol Appl Biochem. 1994 Jun;19 ( Pt 3):265-9. Hsu 15 WH, et al "Construction of a new yeast cloning vector containing autonomous replication sequences from Candida utilis." J Bacteriol. 1983 Jun;154(3):1033-9; Hanic-Joyce PJ, et al "A high-copy-number ADE2-bearing plasmid for transformation of Candida glabrata." Gene. 1998 May 12;211(2):395-400. 20 Successfully transformed cells, ie cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention. Cells can be harvested and lysed and their DNA content examined for the presence of the 25 DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described below.

WO 00/36135 PCT/GB99/04228 58 In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce 5 proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies. Thus, in addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally 10 homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. A further aspect of the invention provides a method of making the polypeptide of the invention or a variant, derivative, fragment or fusion thereof or a fusion of a 15 variant, fragment or derivative the method comprising culturing a host cell comprising a recombinant polynucleotide or a replicable vector which encodes said polypeptide, and isolating said polypeptide or a variant, derivative, fragment or fusion thereof or a fusion of a variant, fragment or derivative from said host cell. Methods of cultivating host cells and isolating recombinant proteins are well 20 known in the art. The invention also includes a polypeptide, or a variant, fragment, derivative or fusion thereof, or fusion of a said variant or fragment or derivative obtainable by the above method of the invention. 25 A still further aspect of the invention provides an antibody reactive towards a polypeptide of the invention. Examples of such antibodies and of methods of preparing such antibodies are given in Example 1.

WO 00/36135 PCT/GB99/04228 59 Antibodies reactive towards the said polypeptide of the invention may be made by methods well known in the art. In particular, the antibodies may be polyclonal or monoclonal. 5 Suitable monoclonal antibodies which are reactive towards the said polypeptide may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", SGR Hurrell (CRC Press, 1982). 10 In a preferred embodiment the antibody is raised using any suitable peptide sequence obtainable from the amino acid sequence of Pkh1 or Pkh2 as appropriate. It is preferred if polyclonal antipeptide antibodies are made. 15 It is particularly preferred if the antibody does not react substantially with another protein kinase, in particular another yeast protein kinase. Accordingly, it may be preferred if peptides based on the Pkhl or Pkh2 sequence are used which vary significantly from any peptides found in any other protein kinase, particularly a yeast protein kinase. It may also be preferred that an antibody reacts with Pkhl 20 but does not react substantially with Pkh2, and vice versa. Techniques for preparing antibodies are well known to those skilled in the art, for example as described in Harlow, ED & Lane, D "Antibodies: a laboratory manual" (1988) New York Cold Spring Harbor Laboratory. 25 Other methods A further aspect of the invention provides a method of identifying a drug-like compound or lead compound for the development of a drug-like compound that WO 00/36135 PCT/GB99/04228 60 modulates the activity of the polypeptide Pkhl, Pkh2, Ypk1 or Ykr2, the method comprising contacting a compound with the polypeptide or a suitable variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof and determining whether the protein kinase activity of the said 5 polypeptide is changed compared to the activity of the said polypeptide or said variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof in the absence of said compound. The terms "drug-like compound" and "lead compound" are well known to those 10 skilled in the art, as discussed above. The compound may act by interacting with the said polypeptide and modulating, for example inhibiting, its activation by an "upstream activator". The compound may act by interacting with Ypkl or Ykr2 and modulating, for example 15 inhibiting, its activation by an "upstream activator", for example Pkh1 or Pkh2. It will be understood that it will be desirable to identify compounds that may modulate the activity of the polypeptide in vivo. Thus it will be understood that reagents and conditions used in the method may be chosen such that the 20 interactions between the said polypeptide and its substrate are substantially the same as between yeast, for example S. cerevisiae Pkhl, Pkh2, Ypk1 or Ykr2 and their substrate or substrates in vivo. An example of a substrate of said Pkh1 or Pkh2 polypeptide is Ypkl or Ykr2. 25 In one embodiment, the compound decreases the activity of said polypeptide. For example, the compound may bind substantially reversibly or substantially irreversibly to the active site of said polypeptide. In a further example, the compound may bind to a portion of said polypeptide that is not the active site so as to interfere with the binding of the said polypeptide to its substrate. In a still WO 00/36135 PCT/GB99/04228 61 further example, the compound may bind to a portion of said polypeptide so as to decrease said polypeptide's activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said polypeptide's activity, for example in the activation of the said polypeptide by an 5 "upstream activator", such as Pkh1 or Pkh2 for Ypkl and Ykr2. In a further embodiment, the compound increases the activity of said polypeptide. For example, the compound may bind to a portion of said polypeptide that is not the active site so as to aid the binding of the said polypeptide to its substrate. In 10 a still further example, the compound may bind to a portion of said polypeptide so as to increase said polypeptide's activity by an allosteric effect. This allosteric effect may be an allosteric effect that is involved in the natural regulation of the said polypeptide's activity for example in the activation of the said polypeptide by an "upstream activator", such as Pkh1 or Pkh2 for Ypk1 and Ykr2. 15 Conveniently, the method makes use of the fact that Pkhl or Pkh2 phosphorylates, for example Ypk1 or Ykr2 as described in Example 1, or that Ypkl or Ykr2 phosphorylate peptides as shown in Table 2 and discussed in Example 1, but any suitable substrate may be used. Thus the phosphorylation of 20 Ypk1 or Ykr2 or peptide substrate of Ypkl or Ykr2 may be measured using techniques well known to those skilled in the art. Conveniently, the method makes use of an assay which may be substantially the same as that described in Example 1 or which may be adapted to render the assay more convenient for high-throughput screening, as well known to those skilled in the art. For 25 example, a scintillation proximity assay (SPA; Amersham) may be useful. It is preferred that the polypeptide (for example, Pkhl, Pkh2, Ypk1 or Ykr2) is recombinant. It is preferred that the substrate is recombinant or synthetic.

WO 00/36135 PCT/GB99/04228 62 Alternatively, a change in the activity of the substrate may be measured. For example, the protein kinase activity of Ypkl or Ykr2 or a substrate of Ypkl or Ykr2 may be measured, as described above. This may be done in a whole cell system or using purified or partially purified components. Expression of an 5 protein encoded by an RNA transcribed from a promoter regulated by a susbtrate of Ypkl or Ykr2 may be measured. The protein may be one that is physiologically regulated by a substrate of Ypkl or Ykr2 or may be a "reporter" protein, as well known to those skilled in the art (ie a recombinant construct may be used). A reporter protein may be one whose activity may easily be assayed, 10 for example p-galactosidase, chloramphenicol acetyltransferase or luciferase (see, for example, Tan et al (1996)). A further aspect of the invention is a method of identifying a compound which modulates, for example blocks, the activation of a polypeptide that is a functional 15 equivalent of Ypkl and/or Ykr2 that is not SGK, PKBa or p70S6 kinase(or is derivable from a non-mammalian source) by an interacting polypeptide, for example Pkhl, Pkh2 or PDK1, the method comprising determining whether a compound enhances or disrupts the interaction between (a) a polypeptide that is a functional equivalent of Ypk1 and/or Ykr2 that is not SGK, PKBc or p70S6 20 kinase (or is derivable from a non-mammalian source) or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative and (b) the interacting polypeptide, or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or determining whether the compound substantially blocks activation of 25 the said polypeptide that is a functional equivalent of Ypk1 and/or Ykr2 that is not SGK, PKBa or p70S6 kinase (or is derivable from a non-mammalian source) or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion by the interacting polypeptide, or a suitable variant, derivative, fragment or fusion thereof.

WO 00/36135 PCT/GB99/04228 63 It will be understood that it will be desirable to identify compounds that may modulate the activity of the polypeptide in vivo. Thus it will be understood that reagents and conditions used in the method may be chosen such that the 5 interactions between the said polypeptide that is a functional equivalent of Ypkl and/or Ykr2 that is not SGK, PKBa or p70S6 kinase (or is derivable from a non mammalian source) and the interacting polypeptide, for example Pkhl, Pkh2, PDK1 and/or a protein kinase with PDK2 activity are substantially the same as between a said naturally occuring polypeptide that is a functional equivalent of 10 Ypkl and/or Ykr2 and a naturally occuring interacting polypeptide in vivo. PDK1 may be a polypeptide with PDK2 activity, for example when bound to PIF or a related polypeptide, as discussed above. It will be appreciated that the said suitable variant, fragment, derivative or fusion 15 of a polypeptide that is a functional equivalent of Ypkl and/or Ykr2 that is not SGK, PKBa or p70S6 kinase (or is derivable from a non-mammalian source), or a fusion of a said fragment, derivative or fusion is not SGK, PKBx or p70S6 kinase. 20 A further aspect of the invention is the use of Pkhl or Pkh2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof that is not PDK1 to phosphorylate and/or activate a polypeptide that is Ypk1 and/or Ykr2 or SGK or PKBa or a functional equivalent thereof or suitable variant, fragment, derivative or fusion thereof, or a fusion of a said 25 fragment, derivative or fusion. A further aspect of the invention is the use of PDK1 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof to phosphorylate and/or activate a polypeptide that is Ypk1 WO 00/36135 PCT/GB99/04228 64 and/or Ykr2 or SGK or a functional equivalent thereof that is not PKBa or p70S6 kinase or suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion. 5 A further aspect of the invention is a method of identifying a compound which binds to Ypk1 or Ykr2 or SGK (or other substrate of Pkhl, Pkh2 or PDK1) and either enhances or prevents its activation by Pkhl, Pkh2 or PDK1, the method comprising determining whether a compound enhances or prevents the interaction of Ypk1 or Ykr2 or SGK (or other substrate of Pkhl, Pkh2 or PDK1) or a 10 suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative with Pkhl, Pkh2 or PDK1 or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative or determining whether the compound substantially blocks activation of Ypk1 or Ykr2 or SGK (or other substrate Pkhl, Pkh2 or PDK1) or a suitable 15 fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative by Pkhl, Pkh2 or PDK1. Suitable assays may be similar to those described above. 20 A further aspect of the invention is a method of identifying a polypeptide that interacts with Pkhl or Pkh2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof that is not PDK1, the method comprising 1) contacting a) the said polypeptide with b) a composition that may contain such an interacting polypeptide, 2) detecting the 25 presence of a complex containing the said polypeptide and an interacting polypeptide, and optionally 3) identifying any interacting polypeptide bound to the said polypeptide.

WO 00/36135 PCT/G B99/04228 65 In one embodiment, the composition may comprise material from cells. In particular, the cells may be selected from the following types: (1) cells which have Pkh1 or Pkh2 activity after exposure to a stimulus, but which have not been so exposed and (2) cells of type 1 after exposure to the stimulus. Polypeptides 5 that are found in one only of types 1 or 2 are of particular interest and may be characterised further. Such a peptide may be an activator of Pkhl or Pkh2. Alternatively, it may be an inactivator of Pkhl or Pkh2. It will be appreciated that the method may be performed within a cell, for 10 example using the yeast two hybrid system as is well known in the art. In this example, cDNAs copied from mRNA from the two cell types described above would be used. It will further be appreciated that a recombinant yeast cell in which a Pkh1 or 15 Pkh2 gene is altered and/or a recombinant polynucleotide capable of expressing Pkh1 or Pkh2 is present, may be useful in, for example, identifying a substrate of Pkh1 or Pkh2, as described in Example 1. A further aspect of the invention is a polypeptide identifiable by the said method. 20 A still further aspect of the invention provides a method of identifying a compound which modulates the activation of Pkh1 or Pkh2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof that is not PDK1 by an "upstream activator". By "upstream 25 activator" is meant a molecule that interacts with the polypeptide of the invention with the result that the protein kinase activity of the polypeptide of the invention is increased. It may be a polypeptide. Preferably, it is a physiological activator of native Pkh1 or Pkh2. Such an activator may be identified by the method given above.

WO 00/36135 PCT/GB99/04228 66 A further aspect of the invention is the use of Pkhl or Pkh2 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof for the activation of Ypk1 or Ykr2 or a suitable variant, 5 fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion that is not SGK or PKBa. A further aspect of the invention is a method of identifying a compound which blocks the activation of Pkhl or Pkh2 or a suitable variant, fragment, derivative 10 or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof by a said interacting polypeptide identifiable by the above method, the method comprising determining whether a compound enhances or disrupts the interaction between (a) the said Pkh1 or Pkh2 and (b) said interacting polypeptide or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a 15 variant, derivative or fragment, or determining whether the compound substantially blocks activation of the said polypeptide as defined in the first aspect of the invention by said interacting polypeptide or a suitable variant, derivative, fragment or fusion thereof. 20 It will be appreciated that screening assays which are capable of high throughput operation will be particularly preferred. Examples may include cell based assays and protein-protein binding assays. An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used. For example, beads comprising scintillant and a polypeptide that may be phosphorylated may be 25 prepared. The beads may be mixed with a sample comprising the protein kinase, as described above and 1 2 P-ATP or "P-ATP and with the test compound. Conveniently this is done in a 96-well format. The plate is then counted using a suitable scintillation counter, using known parameters for 1 2 P or "P SPA assays. Only 32 P or "P that is in proximity to the scintillant, i.e. only that bound to the WO 00/36135 PCT/GB99/04228 67 polypeptide, is detected. Variants of such an assay, for example in which the polypeptide is immobilised on the scintillant beads via binding to an antibody, may also be used. 5 Other methods of detecting polypeptide/polypeptide interactions include ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Fluorescence Energy Resonance Transfer (FRET) methods, for example, well known to those skilled in the art, may be used, in which binding of two fluorescent labelled entities may be measured by measuring 10 the interaction of the fluorescent labels when in close proximity to each other. Medical aspects A further aspect of the invention is a compound identified or identifiable by any 15 appropriate one of the above methods of the invention. A still further aspect of the invention is a said compound for use in medicine. The compound may be a compound that inhibits the activity mammalian, 20 preferably human PDK1 or mammalian, preferably human SGK. Alternatively, the compound may inhibit a functional homologue of PDK1 or SGK in a pathogen or opportunistic pathogen, for example of a mammal, preferably a human. For example, the compound may inhibit a functional homologue of PDK1 (which may be Pkhl or Pkh2) or SGK (which may be Ypk1 or Yrk2) in 25 Candida species or any other pathogenic yeast species as listed above. Such a compound may be useful as an antifungal agent. It will be appreciated that the pathogen or opportunistic pathogen may affect a plant, which may be a monocotyledenous plant or a dicotyledenous plant. For WO 00/36135 PCT/GB99/04228 68 example, the pathogen may be a member of the Uredinales group of fungi that may cause the plant disease rust, for example Puccinia graminis which may cause black rust of cereals, or may be Phtophthora infestans which may cause potato blight. Thus, a compound that may inhibit a functional homologue of PDK1 5 (which may be Pkh1 or Pkh2) or SGK (which may be Ypk1 or Yrk2) in a yeast that is pathogenic for a plant may be useful as an antifungal agent for use as a plant protection product ie for treating or preventing a fungal infection of a plant. It will be appreciated that a compound that inhibits mammalian, for example 10 human PDK1 may be useful in medicine. Thus, a further aspect of the invention is a method of modulating in a cell the activity of PDK1 (which may include its interactions with protein kinase B or SGK), the method comprising exposing the cell to a compound of the invention. It will be appreciated that the method may be used in vitro or in vivo. It is preferred that the compound of the invention is 15 capable of entering the cell and that it enters the cell. A still further aspect of the invention is a method of treating a patient in need of modulation, preferably inhibition, of the activity of PDK1 or its interactions with protein kinase B the method comprising administering to the patient an effective 20 amount of a compound of the invention that inhibits mammalian, for example human, PDK1. Compounds identifiable in the screening methods of the invention that inhibit PKB, PDK1 or the activation of PKB by PDK1 are believed to be useful in 25 treating cancer. PKB is the cellular homologue of v-akt which is involved in leukaemias. Two isoforms of PKB are overexpressed in ovarian, pancratic and breast cancers. It is believed that PKB mediates protection of cells to apoptosis mediated, for example, by IGF-1. Overexpression of PKB may allow cancer cells to proliferate by stopping apoptosis. Promotion of apoptosis may be WO 00/36135 PCT/GB99/04228 69 beneficial in the resolution of inflammation. A compound that results in activation of PKB or PDK1 may be useful in the treatment of diabetes or obesity. Such a compound may also be useful in the 5 treatment of patient before, after or during heart surgery. Such a compound may also be useful in reducing apoptosis; thus, such a compound may be useful in treating a patient in need of protection against apoptosis. Reducing apoptosis may be useful following ischaemic injury, for example stroke or myocardial infarction, and in tissue repair. 10 A compound of the invention that inhibits a fungal functional homologue of PDK1 (which may be Pkh1 or Pkh2) or SKG (which may be Ypkl or Yrk2) may be useful in medicine, for example as an antifungal agent. It may be useful in treating a Candida infection, for example thrush. Thus, a further aspect of the 15 invention is the use of a compound of the invention that inhibits a fungal functional homologue of PDK1 or SGK in the manufacture of a medicament for treating or preventing a fungal infection, for example thrush. Further examples of fungal infections that may be treated by a compound of the 20 invention include infections that may be caused by a pathogenic fungus listed above. Still further examples of infections or conditions that may be treated or prevented by a compound of the invention are listed below. It will be appreciated that it may be preferred that the compound of the invention has been selected, for example using a method of the invention as described above, for an effect on a 25 protein kinase of the yeast genus or species indicated. Thus, a compound of the invention may be used in the manufacture of a medicament for the treatment or prevention of an infections that may be caused by a pathogenic fungus listed above or an infection or condition listed below.

WO 00/36135 PCT/GB99/04228 70 Aspergillosis may be caused by fungi of the genus Aspergillus, usually A. fumigatus. Blastomycosis is caused by the fungus Blastomyces dermatitidis. Infection may take place through the lungs but the infection may become widely 5 disseminated, with the skin, skeleton and genito-urinary system becoming infected. Candidiasis is caused by Candida spp, particularly C. albicans, and usually requires a predisposing factor, such as antibacterial therapy, diabetes, pregnancy 10 or immunodeficiency. Mucocutaneous candidiasis is known as thrush. Chromoblasomycosis may be caused by opportunistic pathogens including Phialophohra compacta, P. pedrosoi, P. verrucosa, Cladosporium carrionii and Rhinocladiella aquaspersa following skin trauma, particularly in tropical and subtropical climates. 15 Coccidioidomycosis (valley fever, desert fever, desert rheumatism) may be caused by inhalation of spores of Coccidioides immitis and occurs particularly in arid and semi-desert areas of North, Central and South America. Cryptococcosis is caused by the fungus Cryptococcus neoformans. It is rare in normal 20 individuals, but important in immunocompromised patients, often occurring as cryptococcal meningitis. Endocarditis may be caused by fungi such as Aspergillus or Candida, as may eye infections. Cephalosporium, Fusarium, Blastomyces, Cryptococcus and 25 Sporothrix may also cause eye infections. Histoplasmosis may be caused by Histoplasma capsulatum, found in soil and in bird and bat excrement. It is a sytemic infection that is endemic in central USA and central Africa (mainly H. capsulatum var duboisii.

WO 00/36135 PCT/GB99/04228 71 Mucormycosis is caused by Rhizopus or Rhizomucor species and usually affects only immunocompromised individuals. Mycetoma is a tropical and subtropical disease caused by Madurella mycetomatis, M. grisea or Pseudallescheria boydii 5 which enter the tissue through skin trauma. Paracoccidioidomycosis is a disease principally of Central and South America caused by Paracoccidioides brasiliensis. Pneumocystis carinii pneumonia is caused by Pneumocystis carinii, which is 10 thought to be a fungus. It is of particular significance in immunocompromised patients, particularly those with AIDS. Skin infections include the dermatophytoses, pityriasis versicolor and candidiasis (discussed above), as well as forms of other fungal infections discussed above, 15 such as apergillosis. Dermatophytoses include ringworm and tinea (including athlete's foot), and are caused by Epidermophyton, Microsporum and Trichophyton spp. Pityriasis versicolor (tinea versicolor) is caused by Malassezia furfur and is more common in tropical than temperate climates and exposure to the sun may trigger the infection. 20 Sporotrichosis is caused by Sporothrix schenckii and is seen mainly in the Americas and Africa. It occurs in cutaneous and extracutaneous forms. Further information regarding fungal infections for which the compounds of the 25 invention may be useful may be found in, for example, the Martindale Pharmacopaeia, 32"' edition. It will be appreciated that fungal infections may be more serious in immunocompromised hosts, for example a human with AIDS (acquired WO 00/36135 PCT/GB99/04228 72 immunodeficiency syndrome); thus, yeast/fungi that are of low pathogenicity in otherwise healthy individuals may be capable of causing more severe disease in an immunocompromised individual. 5 Kit of parts A further aspect of the invention is a kit of parts comprising means useful for carrying out a screening method of the invention. 10 Thus, a kit of parts of the invention may comprise a first host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from a first source and 15 2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from an source other than the first source. 20 Figure Legends. Figure 1. Comparison of the primary structures of Pkhl and Pkh2 with PDK1. (a) Alignment of the deduced amino acid sequences of yeast Pkhl and Pkh2 with the catalytic domains of human PDK1 and its Drosophila homologue, DSTPK61 [27], carried out using the CLUSTAL W program. Identical residues are denoted 25 by white-on-black letters, and similar residues by grey boxes. In our work, five independent PKH1 clones generated by PCR all differed from the sequence deposited in the Saccharomyces Genome Database, and indicated that Phe187 (TTC) should actually be Ile (ATC). (b) Schematic diagrams of the structures of WO 00/36135 PCT/GB99/04228 73 PDK1-related proteins. Blue boxes indicate the catalytic domain in each protein kinase and green boxes indicate PH domains. Figure 2. Deletion of both PKH1 and PKH2 produces non-viable cells. 5 (a) Growth of spores from a representative tetratype tetrad from strain AC306 (MATa/MATa PKH1/pkh1A::HIS3 PKH2/pkh2i::TRPJ). The deduced pkhlA pkh2A spore is inviable. (b) Verification by PCR using the appropriate primers indicated the presence of the expected deletion-substitution alleles. Presence of the HIS3 and TRP1 markers inserted into PKH1 and PKH2, respectively, are 10 denoted by a shift of the size of the resulting product from about 0.5 kbp in the intact loci, to 1.2 kbp (pkhlA::HIS) or 1.3 kbp (pkh2z::TRP]). Figure 3. Genetic evidence that PKH1 and PKH2 are functional homologues. (a) Strain AC306 (MATa/MATa PKH]/pkhlzA::HIS3 PKH2Ipkh2A::TRPJ), 15 transformed with plasmid pYES2-PKH1, which is marked with URA3, was sporulated. Verification by PCR of the genotype of two derived haploids, PKH1 PKH2 [pYES2-PKHJ] (spore A) and pkhlA pkh2A [pYES2-PKHJ] (spore B). (b) Four spores derived from a tetratype ascus of the diploid described in (a), all carrying pYES2-PKHI, were plated either on medium lacking uracil (right) to 20 select for the presence of the plasmid, or on medium containing 5-FOA (left) to select for loss of the plasmid [40]. In the absence of the plasmid-borne copy of PKH1, pkh1A pkh2A cells are unable to continue vegetative proliferation. Figure 4. Intact human PDK1 or PDK1-APH rescue the inviability of pkhlA 25 pkh2A cells. (a) Derivatives of strain AC306 (MATa/MATa PKHlpkhlA::HIS3 PKH2/pkh2A::TRP1), transformed with either YEplacl95-PDK1, YEplacl95 PDK1-APH, or YEplac195-PKH1, all marked with URA3, were sporulated.

WO 00/36135 PCT/GB99/04228 74 Verification by PCR of the genotype of two derived double mutant spores maintained by expression of either authentic yeast PKH1 (spore A) or human PDK1 (spore B). As expected, only the haploid carrying YEplacl95-PDK1 possesses a PDK1-derived sequence. (b) Four spores derived from tetratype asci 5 of the diploids described in (a), carrying either YEplacl95-PDK1 or YEplacl95 PDK1-APH, as indicated, were plated either on medium lacking uracil (top left) to select for the presence of the plasmids (only one representative plate is shown), or on medium containing 5-FOA (top right and bottom right, respectively) to select for loss of the plasmids. The pkh1A pkh2A cells are able to grow only 10 when the plasmids expressing human PDK1 or PDK1-APH are present. Figure 5. Both yeast Pkh1 and human PDK1 phosphorylate and activate human PKB in a PtdIns[3,4,5]P3- or PtdIns(3,4)P2 -dependent manner. Purified GST-PKB was incubated for 30 min at 30'C with either GST-Pkhl (a) 15 or GST-PDK1 (c) in the presence of 100 p.M ATP and phospholipid vesicles containing 100 p.M PtdCho, 100 p.M PtdSer, and the various Ptdlns lipids indicated, all at a final concentration of 10 pM. Reactions were terminated by adjusting the mixtures to a final concentration of 1 % (by vol) Triton X-100 [17] to dissolve the lipid vesicles, and the resulting increase in specific activity (U/mg) 20 of GST-PKB was then determined in the presence of [y- 32 P]ATP, as described in Materials and Methods. The increase in specific activity given is that relative to control incubations in which GST-Pkhl or GST-PDK1 were omitted. To measure the amount of incorporation into GST-PKB catalysed by either GST Pkhl (b) or GST-PDK1 (d), reactions were conducted in the presence of 100 p.M 25 [y- 2 P]ATP and terminated by adjusting the solutions to a final concentration of 1% SDS. The resulting denatured samples were then subjected to SDS-PAGE. Extent of phosphorylation was assessed by autoradiography of the Coomassie blue-stained band corresponding to GST-PKBa. Abbreviations: SA-PI[3,4,5]P3 WO 00/36135 PCT/GB99/04228 75 is sn-1-stearoyl, 2-arachidonyl D-PtdIns[3,4,5]P3; DP-PI[3,4,5]P3 is sn-1,2 dipalmitoyl D-PtdIns [3,4,5]P3; DP-PI[3,4]P2 is sn-1,2-dipalmitoyl D PtdIns[3,4]P2; DP-PI[3]P is sn-1,2-dipalmitoyl D-PtdIns-3P. PI[4,5]P2 was purified from brain extract. 5 Figure 6. Consensus sites for PDK1- and PDK2-dependent phosphorylation, and compari-son of the primary structures of Ypkl and Ykr2 with their closest human homologues. (a) Conserved motifs predicted to be phosphorylated by PDK1-like and PDK2 10 like enzymes are indicated with the residue phosphorylated shown in boldface type. In all cases, the putative PDK2 site is located 157-166 residues C-terminal to the PDK1 site. GenBank accession numbers: .PKBa: X65687; p70S6Ka: M60725; SGK: Y10032; PKCg: L07032; Ypkl: P12688; Ykr2: P18961; Pkcl: M32491; and Sch9: U00029. (b) In the alignment of Ypk1 and Ykr2 to 15 each other and to mammalian SGK, PKBt, p70 S6 kinase and PARK, identities to Ypkl are indicated by white-on-black letters. Figure 7. Expression of either Ypkl or Ykr2 is required for viability. Strain YES7 (MATa/MATa YPK1/ypklA::HIS3 YKR2Iykr2A::TRPJ), transformed 20 with the LEU2-marked plasmid, pGAL-YKR2, were sporulated under conditions (galactose-containing media) that induce expression of YKR2 from the plasmid. The four spores of a tetratype ascus derived from this diploid were recovered on medium lacking leucine to demand the presence of the plasmid. When subsequently streaked out on the same medium containing glucose as the carbon 25 source, which represses expression of the plasmid-borne YKR2 gene, the ypklA ykr2z double mutant cells are unable to continue vegetative proliferation. In contrast, the cells expressing either YPK1 or YKR2 (or both) from their normal chromosomal loci remain viable.

WO 00/36135 PCT/GB99/04228 76 Figure 8. SGK, a PKB-related enzyme, rescues the inviability of ypklA ykr2A cells. (a) Strain YPT28 (MATa ypklA ykr2A) carrying pYKR2, a UR A3-marked plasmid that expresses YKR2 from its authentic promoter, was generated as described in 5 Materials and methods and then transformed with either empty LEU2-marked vectors (pAD4M or YEp351GAL) or the same vectors expressing from either the ADH1 or the GAL1 promoters, as indicated, either YPK1, YKR2, SGK, PKBa, p70 S6 kinase or PARK. When streaked onto medium selective for the presence of both plasmids (left), all of the transformants grew well. However, when 10 plated on the same medium containing 5-FOA (right), to demand loss of the URA3-marked plasmid expressing YKR2, only cells expressing SGK were able to sustain normal growth of the ypk1A ypk1A cells, and did so as efficiently as authentic YPK1 or YKR2. Survival of a few colonies was reproducibly observed for cells expressing PKBa, suggesting that only very high levels of PKBc 15 expression can support the continued growth of Ypkl- and Ykr2-deficient cells. (b) The temperature-conditional strain YPT40 (MA Ta ypk1-14 ykr2) was constructed as described in Materials and methods and then transformed with the indicated plasmids, described in (a). The transformants isolated at permissive temperature (left) were then tested for their ability to continue to grow at the 20 restrictive temperature (right). Again, SGK was able to substitute for Ypkl and Ykr2 function and permit growth just as well as YPK1 itself. Weak complementation by PKB was again observed, in that microcolonies indicative of very slow growth were reproducibly recovered at the non-permissive temperature. 25 Figure 9. Pkhl phosphorylates and activates yeast Ypkl and human SGK in a 3 phospho-inositide-independent manner. Either GST-Ypkl (a) or GST-SGK (c) were incubated for 30 min at 30"C with GST-Pkhl or GST-PDK1, as indicated, in the presence of 100 pM ATP, with WO 00/36135 PCT/GB99/04228 77 and without phospholipid vesicles containing 100 p.M PtdCho, 100 p.M PtdSer and 10 pM of the D-enantiomer of sn-l-stearoyl, 2-arachidonyl D PtdIns[3,4,5]P3 ("PIP3"). Reactions were terminated and the degree of activation assessed as indicated in the legend to Fig. 5. To measure the amount 5 of incorporation into GST-Ypk1 (b) or GST-SGK (d) catalysed by either GST Pkhl or GST-PDK1, as indicated, reactions were carried out in the presence of 100 M [y- 32 P]ATP and the products were analysed by SDS-PAGE followed by autoradiography, as described in the legend to Fig. 5. 10 Figure 10. Phosphorylation of Thr504 in Ypk1 is essential for its activation by Pkhl. (a) GST-Ypkl, GST-Ypkl(T504D), GST-Ypkl(T662D) or GST-Ypkl(T504D T662D) were incubated with either GST-Pkhl or a catalytically-inactive derivative, GST-Pkh1(KD), as indicated, for 30 min at 30 0 C with ATP (100 15 pM). Reactions were terminated and the degree of activation assessed as indicated in the legend to Fig. 5. To follow the amount of incorporation into GST-Ypkl and its various derivatives (b), reactions were carried out in the presence of 100 pM [y- 32 P]ATP and the products were analysed by SDS-PAGE followed by autoradiography, as described in the legend to Fig. 5. 20 Figure 11. Parallels between PDK1-dependent signaling pathways in animal cells and Pkhl- and Pkh2-dependent signaling in yeast. See text for further details. 25 Figure 12. Nucleotide sequence of rat SGK. Figure 13. Amino acid sequence of rat SGK (gi| 4770981 pir |A48094 serum and glucocorticoid-regulated kinase - rat).

WO 00/36135 PCT/GB99/04228 78 Example 1: Functional homologues of mammalian 3-phosphoinositide dependent protein kinase-1 (PDK1) and protein kinase B (PKB/c-Akt) are components of a signaling pathway in the yeast Saccharomyces cerevisiae 5 Background: In animal cells, recruitment and activation of phosphatidylinositol 3-kinase by the binding of growth factors to their cognate receptors generates 3 phosphoinositides. These lipids induce 3-phosphoinositide-dependent protein 10 kinase-1 (PDK1) to phosphorylate and activate a variety of target protein kinases, including protein kinase B (PKB/c-Akt) and p70 S6 kinase, that subsequently mediate appropriate physiological responses. Results: The Saccharomyces cerevisiae genome encodes two protein kinases 15 (PKH1 and PKH2 gene products) whose catalytic domains are 72 % identical to each other and 50% identical to human or Drosophila PDK 1. Both pkh1A and pkh2z single mutants are viable, but a pkh1A pkh2z double mutant is inviable, indicating that Pkhl and Pkh2 have essential, but overlapping, functions. Expression of human PDK1 permits growth of pkh1A pkh2z cells. The yeast 20 genome also encodes two other protein kinases (YPK1 and YKR2 gene products) whose catalytic domains are 88% identical to each other and 58% identical to serum- and glucocorticoid-induced protein kinase (SGK), an enzyme closely related to PKB (56% identity). Either a ypk1A or a ykr2A single mutant is viable, but a ypk1A ykr2A double mutant is inviable, indicating that Ypk1 and 25 Ykr2 also have essential, but overlapping, functions. Growth of ypk1z ykr2A cells is fully rescued by expression of rat SGK, weakly by mouse PKB, and not at all by rat p70 S6 kinase. Pkhl, expressed in 293 cells as a fusion to glutathione S-transferase (GST), phosphorylates and activates mammalian PKB and SGK. Likewise, in vitro Pkh1 activates Ypk1 by phosphorylating the residue WO 00/36135 PCT/GB99/04228 79 (T504) in the kinase domain equivalent to those in PKB and SGK that are phosphorylated by PDK1. Activation of Ypk1 by Pkhl does not require phosphatidylinositol-3,4,5-trisphosphate (PtdIns[3,4,5]P3), consistent with the lack of pleckstrin homology (PH) domains in these yeast proteins, whereas 5 activation of human PKB by Pkh1 is dependent on PtdIns[3,4,5]P3. The minimum consensus sequence for phosphorylation by Ypk1 is Arg-X-Arg-X-X Ser-Hyd (where Hyd is a bulky hydrophobic residue), as found for PKB. Conclusions: These results demonstrate that Pkhl and Pkh2 are functional 10 homologues of PDK1, that Ypkl and Ykr2 are functional homologues of SGK (and perhaps PKB), and that, as in animal cells, these enzymes are components of a protein kinase cascade that is required for cell growth and viability. Background 15 Protein kinase B (PKB) [1], also called RAC (for "Related to PKA and PKC") kinase [2], is the mammalian homologue of a retroviral oncogene product, v-Akt [3], and is, therefore, also designated c-Akt. There are several reasons for the current interest in this enzyme and its function. First, PKB is activated within minutes in response to insulin and other growth factors, and activation is 20 prevented by inhibitors of phosphatidylinositol (PtdIns) 3-kinase [4-6]. Second, increasing evidence indicates that PKB mediates a number of the actions of insulin, including stimulation of glucose and amino acid uptake, glycogen and protein synthesis and, in cardiac muscle, glycolysis (reviewed in [7, 8]), as well as induction of the transcription of specific genes [9, 10]. Third, the PKB 25 isoform is overexpressed in a significant percentage of ovarian and pancreatic cancers [11, 12], and the PKB isoform is elevated in some breast cancers [13]. Fourth, PKB action provides a survival signal that protects cells from apoptosis induced in a variety of ways [14, 15]. Hence, activation of PKB by gene amplification and other mechanisms may contribute to the generation of WO 00/36135 PCT/GB99/04228 80 malignant cells that are able to proliferate independently of extracellular growth and survival signals. PKB phosphorylates proteins and peptides at serine or threonine residues that lie 5 in an -Arg-X-Arg-X-X-Ser/Thr- motif [16]. In insulin signal transduction, two physiological substrates of PKB appear to be the protein kinase, glycogen synthase kinase-3 (GSK3) [17, 18], and the cardiac isoform of PFK2 [7, 19]. Phosphorylation by PKB inhibits GSK3 activity, leading to dephosphorylation and activation of glycogen synthase and protein synthesis initiation factor eIF-2B 10 [20]. These events presumably contribute to the insulin-induced stimulation of glycogen synthesis and protein synthesis, respectively. PKB activates cardiac PFK2, which seems to underlie the insulin-induced stimulation of glycolysis observed in the heart. In the protection of cells against apoptosis, BAD appears to be one of the physiological substrates of PKB [14, 15]. In its 15 dephosphorylated form, BAD interacts with the Bcl family member, BclXL, thereby inducing apoptosis in some cells. However, when phosphorylated by PKB at Ser136, BAD dissociates from BclXL, interacts with 14-3-3 proteins instead, and apoptosis is prevented [21]. 20 Activation of PKB in response to insulin or growth factors requires phosphorylation [22, 23], which occurs at two sites [23]. These are Thr308, located in a -Thr-Phe-Cys-Gly-Thr-X-Glu-Tyr- motif within the "activation loop" of the catalytic domain between conserved subdomains VII and VIII, and Ser473, situated in the hydrophobic motif -Phe-X-X-Phe-Ser-Phe-, close to the C 25 terminus. Both sequence motifs are present in a number of protein kinases that play important roles in signal transduction [7, 24, 25]. Phosphorylation of both sites in PKB is prevented by inhibitors of PtdIns 3-kinase [22]. Thr308 is phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) [26, WO 00/36135 PCT/GB99/04228 81 27] and Ser473 by a distinct protein kinase activity, termed PDK2 activity, which may be possessed by PDK1 (Balendran et al (1999) Current Biology 9, 393-404). PKB can only be activated by PDK1 in vitro in the presence of lipid vesicles 5 containing PtdIns[3,4,5]P3 or PtdIns[3,4]P2 [26]. The former lipid is generated from PtdIns[4,5]P2 by PtdIns 3-kinase, and can be converted to the latter by one of several different classes of 5-phosphoinositide 5-phosphatases [28, 29]. The 3 phosphoinositides bind to a pleckstrin homology (PH) domain at the N-terminus of PKB [30, 31], presumably inducing thereby a conformational change that 10 makes Thr308 accessible to PDK1. PDK1 also possesses a PH domain, C terminal to the catalytic domain [27], that binds Ptdlns[3,4,5]P3 and PtdIns[3,4]P2 even more tightly than does the PH domain of PKB [32, 33]. The interaction of PDK1 with 3-phosphoinositides enhances activation of PKB in vitro, probably by facilitating encounter of PDK1 and PKB via their mutual binding to lipid vesicles 15 [33]. There is increasing evidence that, in addition to PKB, PDK1 activates a number of other protein kinases in vivo, such as p70 S6 kinase [25, 34], certain PKC isoforms [35] and, more recently, SGK [36], by phosphorylating a threonine residue that lie at an equivalent position and within the same sequence motif as Thr308 of PKB. Like PK3, these enzymes also contain the second, 20 hydrophobic, phosphorylation consensus sequence, situated 160-165 residues C terminal to the PDK1-dependent phosphorylation site [7] and, hence, are likely to be substrates for PDK2 protein kinase activity. If enzymes of the PDK1 and PKB class perform functions vital to signaling in all 25 eukaryotic cells, then these molecules should be evolutionarily conserved. We have shown previously that this pathway is apparently present in the fruit fly, Drosophila melanogaster [27]. Here we demonstrate both genetically in vivo and biochemically in vitro that PDK1-like and PKB-like protein kinases exist even in the unicellular eukaryotic microbe, Saccharomyces cerevisiae (baker's yeast).

WO 00/36135 PCT/G B99/04228 82 Moreover, we show that these enzymes are indispensible for cell growth and viability. Additional findings suggests that, as in animal cells, the PDK1-like gene products are likely to play a role in activating other classes of protein kinases, in addition to the PKB-like enzymes. 5 Results PKH1 and PKH2 encode homologues of mammalian PDK1 The genome of the yeast Saccharomyces cerevisiae contains two, previously uncharacterised open reading frames (YDR490c and YOL100w) that encode 10 protein kinases whose catalytic domains share 50% amino acid sequence identity with either human or Drosophila PDK1 and are 72% identical to each other (Fig. 1A). Hence, these loci were designated PKH1 and PKH2, respectively (for "PKB-activating Kinase Homologues 1 and 2"). PKH1 is located on the right arm of chromosome IV [37] and encodes a 766-residue protein (86 kDa); PKH2 15 is situated on the left arm of chromosome XV [38] and encodes a 1,081-residue protein (121 kDa). The Pkh1 and Pkh2 polypeptides also contain N-terminal extensions and long C-terminal extensions (Fig. 1B) and are much less similar to each another in these regions (27% identity). In these domains, there is no significiant homology with the non-catalytic regions of PDK1 and no significant 20 homology with other known proteins. In particular Pkhl and Pkh2 lack the PH domain that is found near the C-termini of both the human and Drosophila PDK1. PKH1 and PKH2 are essential genes that are functionally redundant 25 As a means to determine if PKH1 and PKH2 are expressed genes, the phenotypic effect of loss-of-function mutations in these loci was examined. For this purpose, each open reading frame was deleted and replaced with a selectable marker. The resulting alleles, pkhlz::TRP1 and pkh2A::HIS3, were used to replace the normal chromosomal loci by homologous recombination. Both a haploid pkhl3::TRP1 WO 00/36135 PCT/GB99/04228 83 mutant (AC301) and a haploid pkh2A:.HIS3 mutant (AC303) grew normally and indistinguishably from congenic PKH1 + and PKH2 + haploids isolated from the same tetrad. In addition, the pkhll and pkh2i single mutants displayed no apparent phenotype when the cells were challenged under various conditions, 5 including high concentrations of salt or caffeine, different carbon sources, and different temperatures, or when subjected to heat shock. To determine if PKH1 and PKH2 might share a common function, strain AC301 (MAT pkhlA::TRPJ) was crossed with strain AC303 (MATa pkh2A::HIS3). 10 Upon sporulation of the resulting doubly-heterozygous diploid (AC306), the majority of the 30 tetrads dissected, yielded three viable and one non-viable spore (Fig. 2A). The viable spores were analysed both by plating on appropriate selective media and by PCR (Fig 2B). None of the viable haploid cells were Trp + and His +, and none carried both the pkh1A and the pkh2A mutations. 15 Microscopic observation of the non-viable spores revealed that most germinated and underwent two or three cycles of cell division before ceasing to grow. Hence, pkh1l pkh2A double mutants are inviable, indicating that PKH1 and PKH2 encode genes that are functionally redundant and that share some role that is essential for cell growth and survival. 20 To confirm that the lethality of pkh1l pkh2i cells was due solely to the absence of PKH1 or PKH2 function, AC306 was transformed with either YEplacl95 PKH1, a URA3-marked plasmid expressing PKH1 from its endogenous promoter, or the empty vector (YEplac195), and the resulting Ura+ transformants were 25 subjected to sporulation and tetrad dissection. It was possible to identify many Trp 1+ His + and Ura + spore clones from the diploid transformed with YEp 195 PKH1, but not from that transformed with the empty vector (data not shown). Thus, the pkh1A pkh2A double mutant was able to survive if it expressed PKH1 from a plasmid. Likewise, when AC306 was transformed with a URA3-marked WO 00/36135 PCT/GB99/04228 84 plasmid (pYES2) expressing either PKH1 (Fig. 3A) or PKH2 (data not shown) from the GAL1 promoter, it was possible to obtain viable pkh1A pkh2A spores, even when the cells were propagated on glucose (presumably because the particular constructs used are not efficiently repressed on this carbon source 5 [39]). Moreover, when the pkh1A pkh2A cells harboring either pYES2-PKH1 (Fig. 3B) or pYES2-PKH2 (data not shown) were plated on medium containing 5-fluoro-orotic acid (5-FOA), which selects for cells that lack a functional URA3 gene [40] and, hence, for loss of the URA3-marked pYES2 plasmid, the pkh1l pkh2A cells were no longer capable of growing (Fig 3B). 10 Human PDK1 is a functional homologue of Pkh1 and Pkh2 To determine if Pkhl and Pkh2 are homologous in function, as well as in sequence, to PDK1, AC306 was transformed with YEplac195 expressing either full-length human PDK1 or human PDK1 lacking its C-terminal PH domain 15 (PDK1-APH), under control of the authentic PKH1 promoter. Expression of either PDK or PDK1-APH from this vector permitted the recovery of viable pkh1A pkh2A spores (Fig. 4B). Moreover, selection against these URA3-marked plasmids by plating on 5-FOA medium prevented the growth of the phklz pkh2z double mutants, but not a pkhlA or a pkh2A single mutant carrying the same 20 plasmids (Fig. 4B). Viable phk1A pkh2A double mutant spores could also be recovered when AC306 was transformed with a vector (pYES2) expressing either PDK1 or PDK1-APH from the GAL] promoter, even when the cells were germinated and grown on glucose-containing medium, presumably because the particular constructs used are not efficiently repressed on this carbon source (data 25 not shown). These results demonstrate that the catalytic domain of PDK1 is sufficient to substitute for the function of Pkhl and Pkh2 and suggests that PDK1 is able to phosphorylate the same essential target substrates in yeast cells as Pkhl and Pkh2.

WO 00/36135 PCT/GB99/04228 85 Purification and characterisation of Pkh1 kinase activity The PKH1 coding region was expressed, as a fusion to GST, in human 293 cells and purified from cell lysates by affinity chromatography on gluthatione 5 Sepharose. The material obtained showed a single Coomassie blue-staining band on SDS-PAGE that migrated as a 112 kDa species, in agreement with the molecular mass expected for the GST-Pkh1 fusion. A catalytically-inactive ("kinase-dead") mutant altered in a conserved residue known to be critical for binding the Mg 2 "-ATP substrate, Pkhl(D267A), was also expressed and purified. 10 This GST-KD-Pkhl construct served as a control. The activity of GST-Pkhl was assessed via its ability to phosphorylate and activate GST-PKB, a known substrate of human PDK1. Phosphorylation of PKB was assessed by the incorporation of label in reactions containing [y- 2 P]ATP. 15 Activity of PKB was measured via its ability, after its incubation with GST Pkhl, to phosphorylate a specific peptide substrate, Crosstide [17]. Yeast Pkh1 was able to activate human PKB (Fig. 5A), provided that lipid vesicles containing 3-phosphoinositides that are known to interact with the PH domain of PKB were present. There was an excellent correlation between the Pkhl 20 dependent phosphorylation of PKB (Fig. 5B) and the degree of activation observed (Fig. 5A). No activation or phosphorylation of PKB was observed if Ptdlns[3,4,5]P3 or PtdIns[3,4]P2 were omitted, or replaced by Ptdlns[4,5]P2 or PtdIns-3P. As observed with human PDK1 (Fig. 5C and 5D), Pkh1 phosphorylated and activated PKB more efficiently in the presence of the 25 naturally-occurring stearoyl-arachidonyl derivative of Ptdlns[3,4,5]P3 than in the presence of the dipalmitoyl derivative. GST-KD-Pkhl did not activate or phosphorylate PKB under any condition tested (data not shown).

WO 00/36135 PCT/GB99/04228 86 To determine what residue in PKB was phosphorylated by Pkhl, GST-PKB was phosphorylated to completion by prolonged incubation in the presence of [y "P]ATP, cleaved with trypsin, and the resulting digest resolved by HPLC on an RP-Cis column. Only one major labelled phosphopeptide was observed (data not 5 shown). The elution position of this species was congruent with that of the phosphopeptide (residues 308-328) obtained by tryptic digestion of PKB phosphorylated by human PDK1. No "P-labelled material eluted at the position corresponding to the PKB peptide that contains Ser473. The peptide labelled by Pkh1 contained phosphothreonine (and no other phosphoamino acid) and all the 10 radioactivity was released after one cycle of Edman degradation (data not shown). This analysis establishes that yeast Pkhl phosphorylates PKB at Thr308, the same residue phosphorylated by human PDK1. Hence, the yeast and human enzymes display the same specificity in vitro using this substrate. 15 YPK1 and YKR2 encode homologues of mammalian PKB PKB is one of the founding members of the so-called AGC sub-family of protein kinases. Moreover, PDK1 is known to phosphorylate PKB and several other members of this sub-family at a specific motif that lies in the so-called "activation loop" situated between conserved elements VII and VIII in the catalytic domains 20 of these enzymes (Fig. 6A). In addition, all of these targets also share a different consensus sequence at a C-terminal phosphorylation site for another protein kinase activity (PDK2). The S. cerevisiae genome encodes four, previously characterised protein kinases that possess both of these same motifs (Fig. 6A). The YPK1 gene was originally identified by screening a yeast genomic library 25 using a cDNA encoding the catalytic subunit of mammalian cyclic-dependent protein kinase as the probe [41]. Similarly, the highly-related YKR2 gene, also called YPK2 [42], was first identified by library screening with a mammalian PKC cDNA probe [43]. The SCH9 gene was identified by its ability, when overexpressed, to rescue the lethality of cells defective in the activation of Ras WO 00/36135 PCT/GB99/04228 87 [44]. Finally, the PKC1 gene was identified as a bona fide yeast homologue of major mammalian PKC isotypes [45]. However, among these four gene products, the catalytic domains of Ypk1 and Ykr2 share 88% identity, as well as extensive similarities across their N- and C-terminal extensions. Most 5 significantly, among all other protein kinases in available databases, the catalytic domains of Ypkl and Ykr2 share greatest similarity to members of the AGC sub family (Fig. 6B), including mammalian SGK (58% identity) [36, 46], PKB (54% identity) and p70 S6 kinase (52% identity). ARK (50% identity) [47] is another AGC subfamily member but does not have consensus phosphorylation PDK1 10 motifs. This sequence homology raised the possibility that Ypk1 and Ykr2 may be physiological substrates for Pkh1 and Pkh2. YPK1 and YKR2 are essential genes that are functionally redundant It has been reported previously that cells lacking either Ypkl or Ykr2 are viable, 15 whereas cells lacking both Ypkl and Ykr2 are inviable [42]. To confirm this observation, and to generate a strain in which the ability of potential mammalian homologues to rescue this inviability could be readily tested, otherwise isogenic ypklA:.HIS3 and ykr2A::TRPJ haploid strains (YES5 and YES1, respectively) were constructed and crossed to form a doubly heterozygous diploid (YES7). 20 This diploid was transformed with a LEU2-marked plasmid expressing the YKR2 gene under the tight control of the GAL1 promoter, and a Leu+ transformant was subjected to sporulation and tetrad dissection on galactose-containing medium. Under these conditions, most of the tetrads yielded four viable spores, and His+ Trp+ Leu+ isolates were readily recovered; whereas, when the Leu+ 25 transformant was sporulated on glucose, no His+ Trp+ Leu+ spores could be recovered (data not shown). Moreover, as expected if the absence of both YPK1 and YKR2 function is lethal, when the ypklA ykr2A double mutant carrying pGAL-YKR2, previously maintained on galactose medium, was streaked on glucose medium, the cells failed to grow, whereas otherwise isogenic wild-type WO 00/36135 PCT/GB99/04228 88 cells or ypklA and ykr2A single mutants carrying the same plasmid grew well on glucose (Fig. 7). These results demonstrate that YPK1 and YKR2 encode genes that are functionally redundant and that share some role that is essential for cell growth and survival. In our hands, however, a ypklA mutant grows detectably 5 more slowly that otherwise isogenic wild-type cells or a ykr2z mutant, either in liquid culture or on solid agar medium (data not shown). PKB-related enzyme, SGK, is a functional homologue of Ypkl and Ykr2 To test the ability of mammalian protein kinases to rescue the lethality of the 10 ypklA ykr2A double mutant, diploid YES7 was transformed with a low-copy number (CEN) plasmid marked with URA3 and expressing YKR2 from its endogenous promoter. A Ura + transformant was sporulated, dissected, and a His + Trp + Ura + spore, representing a ypklA ykr2z haploid maintained by expression of YKR2 from the plasmid (strain YPT28), was recovered. YPT28 15 was then transformed with either empty LEU2-marked, high-copy-number (2 pLm DNA) vectors (pAD4M or YEp351GAL) or the same vectors expressing (from either the GAL] promoter or the constitutive ADH1 promoter [48]) YPK1, YKR2, or cDNAs encoding rat SGK, mouse PKB/c-AKT, rat p70 S6 kinase, or bovine PARK. The nucleotide and amino acid sequences of rat SGK are shown in 20 Figures 12 and 13, respectively. All of these strains are able to grow on galactose medium due to the presence of the plasmid expressing YKR2, which also demonstrated that expression of none of the heterologous protein kinases tested was deleterious to yeast cell growth (Fig. 8A). In contrast, when plated on the same medium containing 5-FOA, thereby demanding loss of the 25 pYKR2(URA3) plasmid, the ypklA ykr2A cells carrying the empty LEU2-marked vectors were unable to grow, whereas those harboring plasmids expressing either YPK1 or YKR2 remained viable, as expected. Likewise, any other protein kinase that is able to perform the essential function of Ypk1 and Ykr2 should also permit growth on 5-FOA. Of the four mammalian cDNAs tested, only SGK displayed WO 00/36135 PCT/GB99/04228 89 efficient complementation of the ypk1A ypk1 double mutant (Fig. 8A). However, we also reproducibly observed weak complementation by PKB, in that a small percentage of the colonies were able to survive (presumably representing cells expressing exceedingly high levels of PKB resulting from accumulation of 5 the 2 pm DNA plasmid due to its notoriously poor segregation efficiency [49]). All of the mammalian protein kinases were produced at high levels in yeast (grown on SCGal-Leu), as judged by immunoblotting with appropriate antibodies, and were active, as judged by assaying extracts of the yeast cells with appropriate specific substrates (data not shown); hence, the failure of p70 S6 10 kinase and PARK to complement was not due to their lack of expression. Because p70 S6 kinase, in particular, is under such complex regulation in animal cells [50], truncations of the N-, C-, and both N- and C-termini [51] were also tested in the same way; although all were expressed, none was able to rescue the lethality of the ypkl ykr2A cells (data not shown). 15 To confirm these results by an independent method for assessing the ability of the mammalian protein kinases to complement, the same plasmids described above were introduced into a yeast strain (YPT40) that displays temperature-conditional growth because it carries a null mutation (ykr2i) in YKR2 and a temperature 20 sensitive (ts) mutation (ypk1-1") in YPK1, the latter of which was generated as described in detail in Materials and Methods. As expected, all of the transformants were able to grow at the permissive temperature (26'C), whereas the strain carrying an empty vector (YEp351GAL) was unable to survive at the restrictive temperature (35"C), but the strain expressing YPK1 from the same 25 vector grew well (Fig. 8B). As observed before, the cells expressing SGK also were able to grow well at 35'C, and the cells expressing PKB were able to grow weakly, in that microcolonies were observed outside of the heavy initial streak, whereas cells expressing p70 S6 kinase or PARK did not grow (Fig. 8B).

WO 00/36135 PCT/GB99/04228 90 Ypkl and mammalian SGK are efficient substrates for Pkh1 Based on the observations described in the preceding sections, Ypk1 (and/or Ykr2) should be physiological substrates of Pkhl (and/or Pkh2). In addition, yeast Pkhl (and/or Pkh2) should be able to phosphorylate and activate 5 mammalian SGK in vitro, as it has previously been shown that PDK1 is capable of phosphorylating and activating SGK [36]. To test these hypotheses, Ypk1 and SGK lacking the N-Terminal 60 amino acids [36] were expressed as GST fusion proteins in 293 cells and purified. Each purified protein yielded a single Coomassie blue-stained band on SDS-PAGE with an apparent mobility in good 10 agreement with its expected molecular mass (data not shown). In the absence of any other factor, purified GST-Ypkl displayed no detectable activity toward the peptide substrate (Crosstide); however, after pre-incubation with purified GST Pkhl, GST-Ypkl was activated and catalysed a readily detectable level of incorporation into the substrate in the presence or absence of lipid vesicles 15 containing Ptdlns[3,4,5]P3 (Fig. 9A). Consistent with activation resulting from Pkhl-dependent phosphorylation, when [y-"P]ATP was included in the reaction, incorporation of label into GST-Ypkl was readily detected (Fig. 9B) and was present exclusively as phosphothreonine (data not shown). A "kinase-dead" derivative, GST-KD-Ypkl(D488A) (see Materials and methods), was 20 phosphorylated by GST-Pkhl but, as expected, was not catalytically-active (data not shown). Also, as predicted, GST-SGK was both phosphorylated and activated upon incubation in vitro with GST-Pkhl, even more efficiently than by GST-PDK1 (Fig. 9C and 9D). 25 As one means to map the Pkhl phosphorylation site in Ypkl, a GST Ypkl(T504D) mutant was expressed in 293 cells and purified. The altered residue is equivalent to the PDK1 target residue (Thr308) in PKBC. As expected, GST-Ypkl(T504D) was not detectably phosphorylated by Pkhl (Fig. 10), consistent with the conclusion that Pkh1 phosphorylates Ypkl at this residue.

WO 00/36135 PCT/G B99/04228 91 Since GST-Ypkl(T504D) was neither constitutively active nor activated by Pkhl, Asp at this position cannot substitute for phosphothreonine to activate this protein kinase. Ypkl also contains a putative consensus site (Thr662) for PDK2 phosphorylation (Fig. 6A), equivalent to Ser473 in PKBa. GST-Ypkl(T662D) 5 expressed and purified from 293 cells was also inactive; however, this protein was phosphorylated and activated by Pkhl in a manner identical to GST-Ypkl itself (Fig. 10). A form of Ypk1 in which both Thr504 and Thr662 were mutated to Asp was also generated. This GST-Ypkl(T504D T662D) mutant also displayed no detectable activity, before or after incubation with Mg 2 '-ATP and 10 GST-Pkhl, demonstrating that, unlike PKBc, mutation of these residues to Asp cannot substitute for phosphothreonine to produce a constitutively-active enzyme. Substrate specificity of Ypkl is similar to PKB PKBa phosphorylates its substrates at the minimal consensus sequence Arg-X 15 Arg-X-X-Ser-Hyd, where Hyd is a bulky hydrophobic residue [16]. Using several series of peptide substrates of related sequence, efficiency of phosphorylation by Pkhl-activated Ypk1 was observed to be quite similar to that observed for PDK1-activated PKB (Table 2). Moreover, the effect of alterations on the rate of phosphorylation showed nearly identical trends. For example, as 20 seen for PKBa, changing either Arg to Lys, or absence of the P-5 Arg, or absence of the P+1 hydrophobic residue, all drastically reduced or abolished phosphorylation by Ypkl. Discussion 25 Here we have shown that S. cerevisiae contains protein kinases that are homologues of mammalian PDK1 and PKB family members, in terms of sequence, physiological function in vivo, and biochemical specificity in vitro. Specifically, we demonstrated, first, that two previously uncharacterised open reading frames, now designated the PKH1 and PKH2 genes, encode PDK1-like WO 00/36135 PCT/GB99/04228 92 protein kinases. The function(s) of Pkhl and Pkh2 appear to overlap because loss of either enzyme produces no obvious phenotype, whereas a cell deficient in both enzymes is inviable. Expression of the N-terminal catalytic domain of mammalian PDK1 alone is sufficient to overcome the lethality of pkh1A pkh2A 5 cells. Consistent with this finding, purified Pkhl phosphorylates and activates known substrates of mammalian PDK1, including PKB and SGK and, where analysed, phosphorylates the same residue (Thr308 in PKBct) as PDK1. Next, we demonstrated that the YPK1 and YKR2 gene products are PKB-like 10 protein kinases. In agreement with a previous report [42], we found that loss of Ykr2 produces no discernable phenotype, that absence of Ypkl results in slower cell growth, and that a ypk1z ykr2i double mutant is inviable, indicating that these protein kinases also have some functional redundancy. Expression of SGK, a close relative of PKBa, that lacks an obvious N-terminal PH domain, rescues 15 efficiently the inviability of ypklA ykr2z cells. We observed that PKB itself was able to sustain only weakly the growth of the ypkli ykr2A double mutant. This latter result is consistent with the prediction that PKB will exist in yeast largely in the inactive state because Saccharomyces cerevisiae lacks any enzymic machinery capable of generating either Ptdlns[3,4,5]P3 or PtdIns[3,4]P2 [52-54]. Binding of 20 these phospholipids is apparently necessary to relieve conformational constraints in PKB and thereby allow it to be an efficient substrate either for mammalian PDK1 or, as we have shown, for yeast Pkhl. This situation probably also explains why all four of the yeast proteins (Pkhl, Pkh2, Ypk1 and Ykr2) lack discernible PH domains and why Pkhl-dependent activation of Ypkl is not 25 influenced by the presence or absence of such 3-phosphoinositides. Indeed, Pkhl does not bind PtdIns[3,4,5]P3 under conditions where this phospholipid binds tightly to PDK1 (results not shown). Therefore, the fact that Pkhl-mediated activation of PKB only occurs efficiently in the presence of lipid vesicles containing PtdIns[3,4,5]P3 or Ptdlns[3,4]P2 confirms unequivocally that these 3- WO 00/36135 PCT/GB99/04228 93 phosphoinositides exert their effects solely by interacting with the PH domain of PKBct. The signals that lead to activation of Pkhl and Pkh2, or mobilize recruitment of these enzymes to particular subcellular locations, is not yet known. Likewise, the signals that promote encounter of Ypk1 and Ykr2 with Pkhl and 5 Pkh2 are also unknown. Despite the fact that the catalytic domains of Ypk1 and Ykr2 are also rather similar to other AGC sub-family protein kinases from animal cells, including p70 S6 kinase and PARK, these enzymes, although expressed in active form in yeast, 10 were unable to support any detectable growth of cells deficient in Ypkl and Ykr2. In addition to functional complementation in vivo, other results also indicate that Ypk1 and Ykr2 represent PKB-like enzymes. First, like its ability to phosphorylate and activate PKB and SGK, yeast Pkhl is able to phosphorylate and activate Ypk1 in vitro. Second, mutagenesis studies indicated that Pkhl 15 phosphorylates Ypk1 at a residue (Thr504) that is equivalent to the position in PKB (Thr308) that is phosphorylated by PDK1 and by Pkhl. Finally, using a series of synthetic peptide substrates, we found that Pkhl-activated Ypk1 has a substrate specificity in vitro nearly identical to that displayed by PDK1-activated PKB [16]. 20 Specifically, Ypkl was able to phosphorylate Ser and Thr residues that lie in an Arg-X-Arg-X-X-Ser/Thr-Hyd motif. Thus, some of the physiological substrates of Ypkl and Ykr2 may contain this consensus sequence. However, the yeast homologues of some of the known substrates of mammalian PKBa do not 25 contain any perfect matches to this canonical site. For example, none of the four GSK3 homologues encoded in the S. cerevisiae genome [55] possess the Arg-X Arg-X-X-Ser motif that is present at the N-terminus of the GSK3 isoforms targeted by mammalian PKBs. Also, yeast PFK2 lacks the two-C-terminal Arg- WO 00/36135 PCT/GB99/04228 94 X-Arg-X-X-Ser motifs found in the cardiac isoform of PFK2. Hence, the physiologically relevant substrates of Ypk1 and Ykr2 remain to be determined. Taken together, these results indicate that Ypk1 (and, most likely, Ykr2) lie 5 down-stream of Pkhl (and/or Phk2) in a protein kinase cascade that is essential for both the growth and survival of yeast cells. In mammalian cells, PDK1 appears to be responsible for the activation of a number of different protein kinases of the AGC sub-family, in addition to PKB , including p70 S6 kinase, certain PKC isotypes and, most recently SGK [36], in agreement with results 10 presented here. Are Ypk1 and Ykr2 the only substrates of Pkhl and Pkh2 that are essential for yeast cell viability? Several observations indicate that, as for PDK1 in animal cells, Pkhl and Pkh2 have multiple targets. First, human PKB can be partially activated by a T308D mutation (in the PDK1 site) or by a S473D mutation (in the PDK2 site), and almost fully activated by the simultaneous 15 presence of both mutations [22]; nonetheless, expression of such a PKB (T308D S473D) mutant is unable to support vegetative growth of a pkh1 pkh2A double mutant (although, following germination of a pkh1l pkh2A spore, expression of such a construct does allow many more cycles of cell division before cessation of growth ensues than expression of PKB itself, yielding a microcolony visible 20 under the microscope(results not shown)). Second, if Ypk1 or Ykr2 are the sole essential targets of Pkhl and Pkh2, and possess any basal activity in the absence of Pkhl- and/or Pkh2-dependent phosphorylation, then rampant overexpression of these enzymes might bypass the need for Pkhl and Pkh2 function; however, gross overproduction (approximately 50-fold) of either Ypk1 or Ypk2 from the 25 strong GAL] promoter on multi-copy plasmids also does not rescue the inviability of pkh1 pkh2A cells (results not shown). In contrast to mammalian PKBa, it has not yet proved possible to generate constitutively-active variants of yeast Ypkl or human SGK [36] by mutating to Asp the residues equivalent to Thr308 and Ser473 in PKBca. Hence, we have been unable to test whether constitutively- WO 00/36135 PCT/GB99/04228 95 active forms of Ypkl (or SGK) would be able to rescue the lethality of a pkh1A pkh2z double mutant. Third, one other potential Pkh1 and Pkh2 substrate, Pkcl, is known to be essential for yeast cell viability under standard growth conditions [45] because it sits at the head of a protein kinase cascade that controls a MAP 5 kinase (Slt2/Mpkl) required for proper cell wall biosynthesis and survival under hypertonic stress (reviewed in [56]). However, again as expected if Pkh1 and Pkh2 have multiple essential targets, attempts to rescue the lethality of pkhli pkh2A cells by overexpression of PKC1 were also unsuccessful (results not shown). Yet another potential Pkh1 and Pkh2 target, Sch9, is thought, on the 10 basis of genetic results, to serve as an effector enzyme in a pathway that is parallel to, and largely redundant in function with, the three yeast cyclic-AMP dependent protein kinase catalytic subunits [44]. Indeed, additional genetic results suggest that activation of the a subunit of a heterotrimeric G protein, Gpa2, but not activation of Ras2, leads to activation of Sch9 [57]. Like the 15 absence of Ypkl alone, loss of Sch9 causes slow growth, apparently due to prolongation of the G1 phase of the cell cycle [44]. Intriguingly, like mammalian PKBa, p70 S6 kinase, and SGK, the known and suspected substrates for yeast Pkh1 and Pkh2, including Ypkl, Ykr2, Pkcl and 20 Sch9, all also contain the consensus sequence (Phe-X-X-Phe/Tyr-Ser/Thr Phe/Tyr) for phosphorylation by PDK2 protein kinase activity and at the same position (160-165 residues C-terminal) relative to the Thr known (or suspected) to be phosphorylated by Pkhl (and, presumbly, Pkh2). This conservation strongly suggests, first, that the S. cerevisiae genome encodes one (or more) 25 protein kinases with a PDK2-like function and, second, that this enzyme(s) is likely to be or act coordinately with Pkhl and/or Pkh2 to control activation of a number of target protein kinases, as observed for PDK1 and PDK2 activity in animal cells (Fig. 11).

WO 00/36135 PCT/GB99/04228 96 Conclusions We have demonstrated here that the yeast, S. cerevisiae, possesses functional homologues of two effector enzymes, PDK1 and PKB, which are vital to the proper metabolic regulation and survival of mammalian cells in response to a 5 variety of extracellular stimuli. This conservation suggests that the physiological roles of these two classes of protein kinase must be evolutionarily quite ancient and central to the growth and viability of all eukaryotic cells. The presence of these proteins in yeast should allow the application of genetic approaches to identify both upstream activators of the PDK1-like enzymes, Pkhl and Pkh2, and 10 downstream substrates for the PKB-like enzymes, Ypk1 and Ykr2. Such information may, in turn, shed light on previously unsuspected modes of regulation of mammalian PDK1 and previously unknown potential targets of PKB. In addition, analysis in yeast may assist in revealing the nature of the as yet uncharacterised PDK2 enzyme that is required, along with PDK1, for full 15 activation of PKB in animal cells, and also presumably required for full activation of Ypkl and Ykr2 in yeast. Materials and methods Cell culture 20 S. cerevisiae strains used in this study are described in Table 1, and were isogenic derivatives of either AYS927 (W303 background) or YPH499 (S288C background). Yeast cells were grown at 30'C in a rich medium (YPD) containing 1% yeast extract, 2% peptone (Difco) and 2% glucose, or in a synthetic minimal medium (S), containing either galactose (Gal) or glucose (Glc, 25 or D, for dextrose) as the carbon source and supplemented with nutrients appropriate for maintaining selection for markers and plasmids [58]. Standard methods [58, 59] for genetic manipulation of yeast were used. E. coli cells (typically DH5 ) were grown at 37 0 C in Luria-Bertani medium [60] containing (when needed) 50 pg/ml ampicillin for plasmid selection.

WO 00/36135 PCT/GB99/04228 97 Recombinant DNA techniques Restriction enzyme digests, DNA ligations and other recombinant DNA procedures were performed as described [60]. Transformation of bacterial cells 5 was achieved using electroporation. Transformation of yeast cells was accomplished by minor modifications of the lithium acetate method [61, 62]. The PKH1 and PKH2 genes used in this study, and the YPK1 gene used in some constructions, were recovered from genomic DNA of strain AYS927 using PCR amplification with the Expand High Fidelity PCR System (Boehringer 10 Mannheim), gel purified using the Kristal gelex kit (Cambridge Molecular Technologies, Cambridge, UK), and cloned first into the pCR2.1-TOPO vector using the TOPO system (Invitrogen). Site-directed mutagenesis was performed using the QuikChange Kit (Strategene) following instructions provided by the manufacturer. All DNA constructs were verified by automatic DNA sequencing 15 using an automated DNA Sequencer (Model 373; Applied Biosystems). PCR amplification of yeast genomic DNA The primers used to amplify the PKH2 coding region were 5'-CG GGA TCC GCC ACC ATG GAG CAG AAG CTG ATC TCT GAA GAG GAC TTG TAT 20 TTG ATA AGG ATA ATT CCA TG-3' (forward) and 5'-ATA AGA AT GCG GCC GC TTA CGA CCT CTT CGA TTT TGC AG-3' (reverse), and incorporated BamHI and NotI sites, respectively (indicated by italics). The primers used to amplify the PKH1 coding region were 5'-ATA AGA AT GCG GCC GC TGC CAC C ATG GAG CAG AAC CTG TCT CTG AAG AGG ACT 25 TG GGA AAT AGG TCT TGA CAG AGG-3' (forward) and 5'-ATA AGA AT GCG GCC GC TCA TTT TTC ATC TGT CCG TGT C-3' (reverse), and incorporated NotI sites (indicated in italics). Both 5'-primers also contained a sequence encoding a 10-residue c-Myc epitope tag (underlined). The primers used to amplify the YPK1 gene were: 5'-GGA TCC GCC ACC ATG TAC CCA WO 00/36135 PCT/GB99/04228 98 TAC GAT GTG CCA GAT TAC GCC TAT TCT TGG AAG TTT AAG-3' (forward) and 5'-GGT ACC CTA TCT AAT GCT TCT ACC TTG C-3' (reverse), and incorporated BamHI and KpnI restriction sites, respectively (italics). The initiator or termination codons in all these primers are also 5 indicated (boldface type). Gene disruptions and strain constructions A PCR-based method [63] was used for disruption of the PKH1 and PKH2 genes. To generate the pkh2::.HIS3 mutation, HIS3 was amplified from pRS313 [64] 10 using as primers 5'-AAG TAA CAT CTT GAT GAA CCG AGA AGC CAC TAA CTA GTT TT GTG CAC CAT AAT TTT CCG-3' (forward), where the underlined sequence corresponds to nucleotides -93 to 53 from the PKH2 initiator codon and the remainder of the primer corresponds to nucleotides -326 to -311 from the initiator codon of HIS3, and 5'-TAA GTA GCT TGA TGA 15 AAA CAT TAG ATA AAA TTA CTA A TTA CCG TCG AGT TCA AGA G 3', where the underlined sequence corresponds to the nucleotides immediately after the PKH2 stop codon (in boldface type) and the remainder corresponds to nucleotides 204 to 189 after the HIS3 stop codon. The resulting 3.3 kb product was used for DNA-mediated transformation of a diploid strain (AYS927). 20 Transformants were selected on SD-His plates, and disruption was verified by PCR analysis of from one of the His+ isolates using appropriate primers. This heterozygous PKH2/pkh2A::HIS3 diploid (AC200) was sporulated, and the resulting tetrads were dissected. Spore clones were analysed by plating on selective medium and confirmed by PCR to identify a haploid containing the 25 pkh2A::HIS3 disruption (AC303). To generate the pkhlA::TRP1 mutation, the TRP1 marker in pRS314 [64] was amplified using 5'-GCA CGT GTA CTT GCT TGA ATA CTG CTA CTA TAT CAT TAA T ATG GTA CTG AGA GTG CAC C-3' (forward), where the underlined sequence corresponds to nucleotides immediately upstream of the initiator codon (boldface type) and the remainder WO 00/36135 PCT/GB99/04228 99 corresponds to nucleotides situated -300 to 285 nucleotides from the initiator codon of TRP1, and 5'-TAT TAT GCA TTA CAC TTT CCC CTT CAC CAT GTC TTA CAT ATG CAT CCG CAG GCA AGT GCA C-3' (reverse), where the underlined nucleotides correspond to positions +69 to 25 after the PKH1 5 stop codon and the remainder of the primer corresponds to the region situated +51 to 36 nucleotides after the TRP1 stop codon. The resulting 2.4 kb product was used for transformation of AYS927, and transformants were selected on SD Trp plates. Disruption was verified by PCR analysis from one of the Trp+ isolates using appropriate primers. This heterozygous diploid 10 PKH1/pkh1A::TRP1 (AC201) was sporulated, dissected, analysed, and a haploid spore containing the pkh1A::TRP1 mutation (AC301) was identified. In both the ypk1-A1::.HIS3 allele and the ykr2-A1::TRPI alleles [65], the coding sequences for the entire catalytic domains of both enzymes have been deleted and 15 replaced with the indicated markers. To generate a ypklA ykr2A double mutant, a MATa ypklzA strain (YES5) was mated to a MATa ykr2A strain (YES 1) carrying pYKR2(URA3) (see below). The resulting diploid was subjected to sporulation and tetrad dissection, and a His+ Trp+ Ura+ spore, representing a MATa ypk1i ykr2i cell kept alive with the plasmid-borne YKR2 gene, was designated 20 YPT28. To create the temperature-conditional ypk1-1" ykr2A strain (YPT40), a temperature-sensitive allele of YPK1 was first generated, as follows. A genomic insert containing the YPK1 gene [41], cloned into the XbaI site in the vector, pGEM31 (Promega), and generously provided by Richard A. Maurer (then at the University of Iowa, Iowa City, IA), was excised as a 4.1 kb XbaI-Sall fragment 25 and inserted into the LEU2-containing vector, pRS315 [64], yielding pRS315 YPK1. The sequence encoding the catalytic domain of YPK1 was then amplified under moderately error-prone conditions using AmpliTaq" DNA polymerase (Perkin Elmer Cetus), with pRS315-YPK1 as the template and a 5'-primer (P1), 5'-TGC CCT CGA AGA CAT GGC-3', corresponding to a sequence beginning WO 00/36135 PCT/GB99/04228 100 at nucleotide 788 (where the first base of the ATG start codon is +1) and a 3' primer (P2), 5'-CTT GAA CAC AGT AAG TAA CGG-3', corresponding to the flanking genomic sequence commencing 68-bp downstream of the stop codon. The resulting 1350-bp linear PCR product was gel purified and co-transformed 5 into YPT28 along with a ~ 9 kb linear fragment of pRS315-YPK1, that had been generated by digestion with PstI and NcoI, and gel purified. Transformants were selected on SCD-Leu plates at 26"C. This procedure allows for replacement of the corresponding sequence in the parent vector with potentially mutant sequences, and regeneration of circular plasmids, via in vivo repair of the gapped 10 plasmid by recombination with homologous sequences present at each end of the PCR product [66, 67]. To determine which LEU2-containing plasmids expressed functional YPK1 at 26 0 C, the Leu + transformants were subsequently replica plated onto -Leu plates containing 5-fluoro-orotic acid (5-FOA) [39], which selects for loss of the pYKR2(URA3) plasmid initially present in the recipient 15 ypklA ykr2A strain. To determine which of the YPK]- and LEU2-containing plasmids harbored a temperature-sensitive allele of YPK1, the Leu + Ura- cells were tested by replica-plating for their ability to grow on SCD-Leu plates at 37 0 C. One transformant was identified that reproducibly failed to grow at this temperature. The LEU2-containing plasmid carried by this strain (pypk1TS ) was 20 recovered [68], and direct nucleotide sequence analysis of the YPK1 open reading frame in the plasmid revealed the presence of two amino acid substitutions (1484T and Y536C). Sub-cloning and re-transformation confirmed that these mutations were sufficient to confer the temperature-sensitive (ts) phenotype, and this allele was designated ypk1-1". The ypk1-P' allele was used to transplace the normal 25 YPK1 chromosomal locus in the ykr2A strain (YES1), as follows. First, PCR was used to generate a customized DNA fragment containing BamHI and SmaI restriction sites 96 and 108 bp, respectively, downstream from the stop codon of the YPK1 coding sequence in pypk1TS plasmid, and this fragment was substituted for the corresponding segment of the 3'-flanking region, yielding pINT. A 2.6 WO 00/36135 PCT/G B99/04228 101 kb ScaI-BamHI fragment containing the HIS3 gene, excised from vector, pRS303 [64], was gel-purified and inserted into pINT that had been digested with BamHI and SmaI, yielding pINT-HIS, which was able to confer both leucine and histidine prototrophy to a leu2 his3 strain, YPH499 [64]. Finally, a 4.6 kb ClaI 5 XhoI fragment from pINT-HIS, containing the ypk1-Ps allele, the HIS3 gene, and additional genomic DNA from the YPK1 locus flanking the HIS3 gene to its 3' side, was gel purified and used for transformation of YES 1. His+ transformants were selected at 26*C, and then the presence of the integrated ypk-1-' allele (and the absence of the normal YPK1 locus) was confirmed by PCR analysis of DNA 10 isolated from the transformants and by demonstrating that such cells were unable to grow at 37 0 C. One such isolate that met all of these criteria was designated strain YPT40. Plasmids 15 For expression of PKH1, YPK1 as GST fusions in mammalian 293 cells, the corresponding coding sequences were excised from the appropriate pCR2. 1 TOPO derivative and inserted into the mammalian expression vector, pEBG-2T [69]. PKB [26], PDK1 [27] and SGK lacking the N-terminal 60 residues [36] were subcloned into the pEBG-2T vector as described elsewhere. For expression 20 of PKH1 in yeast, a 2.1 kb fragment, from an internal SmaI site (situated about 250 bp downstream from the initiator codon) to an EcoRI site in the pCR2. 1 TOPO vector, was first inserted into the 2 ptm DNA vector, YEplac195 [70], that had been digested with SmaI and EcoRI, yielding YEplacl95-2.1PKH1. To restore the 5'-end, a 1 kb fragment was amplified by PCR using the primers 5' 25 GCT TGA CTC AAT TAA GGC GAC-3' (forward), corresponding to nucleotides 628-634 upstream of the initiator codon, and 5'-ACA TGC TTA GTT AAC TCC-3' (reverse), corresponding to the region located 350 bp downstream of the initiator codon. The resulting product was first cloned into pCR2.1 TOPO, which was then digested with SmaI and SphI to liberate a 0.9 kb fragment WO 00/36135 PCT/GB99/04228 102 that was inserted into the YEplacl95-2.1PKH1 construct that had been digested with SphI and SnaI, yielding YEplacl95-PKH1, which contains the complete coding region of the PKH1 gene plus 0.5 kb of its promoter region and carries the URA3 gene as the selectable marker. To express PKH1 under control of the 5 GAL1 promoter, a 2.3 kb NotI-NotI fragment containing the myc-tagged version of the entire PKH1 coding sequence was inserted into the URA3-marked, 2 pm DNA-containing vector, pYES2 [71], yielding pYES2-PKH1. Likewise, to express PKH2 under GAL] promoter control, a 3.3 kb BamHlI-NotI fragment containing the entire PKH2 open reading frame was inserted into pYES2, 10 yielding pYES2-PKH2. To express mammalian PDK1 in yeast under control of the PKH1 promoter, first, a 0.7 kb fragment corresponding to the PKH1 promoter region was amplified by PCR from the YEplacl95-PKH1 construct using the primers 5'-GG 15 GGT ACC GCT TGA CTC AAT TAA GGC GAC-3' (forward) and 5'-CTT CAG AGA TCA GCT TCT GCT CCA T ATT AAT GAT ATA GTA-3' (reverse), corresponding to the start of the PDK1 coding sequence (underlined). Second, a 1.4 kb fragment comprising the N-terminal sequence of PDK1 was amplified from a human PDK1 cDNA using as primers the 0.7 PCR 20 amplification product (forward) and 5'-ACA CGA TCT CAG CCG TGT AAA A-3'(reverse), corresponding to residues 190-184 of PDK1. The 1.4 kb product was cleaved at the KpnI site (italics) and also with HindIII, which cleaves at an internal site in the PDK1 coding sequence. The resulting KpnI-HindIII fragment was used to replace a 0.5 kb segment encoding the N-terminal end of the PDK1 25 protein either in a construct containing either the complete PDK1 coding sequence, yielding YEplacl95-PDK1, or in a construct containing just the first 404 residues of the PDK1, corresponding to its catalytic domain and lacking its C-terminal PH domain, generating YEplacl95-PDK1-APH. To express human PDK1 under control of the GAL1 promoter, a 2.0 kb BglII-XbaI fragment WO 00/36135 PCT/G B99/04228 103 containing the complete PDK1 coding sequence was inserted into pYES2, yielding pYES2-PDK1. Similarly, a 1.4 kb BglII-XbaI fragment containing the kinase domain of PDK1, but lacking the PH domain, was inserted into pYES2, creating pYES2- PDK1-APH. 5 To express YKR2 in yeast from a LEU2-marked, high-copy-number (2 pLm DNA based) plasmid under control of the GAL1 promoter, a 2.4 kb XhoI-HindIII fragment of genomic DNA containing the entire YKR2 open reading frame [43] was excised from an insert in pUC18 (generously provided by Shigeo Ohno, 10 Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) and ligated into the vector, YEp351GAL [72], that had been linearized by digestion with SalI and HindIII, yielding pGAL-YKR2. An essentially identical approach was used to express YPK1, excised from a genomic DNA fragment (see above), yielding pGAL-YPK1. Alternatively, a 2.1 kb 15 BamHI-NotI fragment encoding an HA-tagged version of the YPK1 coding sequence, generated by PCR, was inserted into pYES2, yielding pYES2-YPK1. To place the YKR2 gene under control of its endogenous promoter on a low-copy number (CEN) plasmid carrying the URA3 gene, a 2.5 kb EcoRI-EcoRI fragment of the original YKR2-containing insert in pUC18 was ligated into the vector, 20 pRS316 [64], that had been linearized with EcoRI, generating pYKR2(URA3). To generate a version of Ypk1 tagged at its C-terminal end with the c-Myc epitope recognized by the monoclonal antibody 9E10 [73], a PCR-based method for precise gene fusion [74] was performed using the YPK1 sequence cloned in pGEM3 as one template and, as the other template, a sequence encoding the 16 25 residue version of the Myc epitope followed by a (His)6 tag cloned in pBluescript (Stratagene), and three appropriate synthetic oligonucleotide primers: P1; T3 (Stratagene), 5'-AAT TAA CCC TCA CTA AAG GG-3', corresponding to sequences in the pBluescript vector; and, a "joiner" primer (P3), 5'-TTC AGA AAT CAA CTT TTG TTC TCT AAT GCT TCT ACC TTG C-3', WO 00/36135 PCT/GB99/04228 104 corresponding to the 3'-end of the YPK1 coding sequence and the first several residues of the c-Myc epitope. A 2 kb ClaI-Sall fragment of the resulting product was used to replace the corresponding segment in the original YPK1 containing pGEM3 vector, yielding pYPKlmyc. A 1.2 kb NcoI-HindIII fragment 5 of pYPKlmyc was used to replace the corresponding segment of p GAL-YPK1, yielding pGAL-YPKlmyc. To express SGK in yeast, a 1.3 kb NcoI-EcoRI fragment encoding a rat SGK cDNA (Webster et al (1993) Mol Cell Biol 13(4), 2031-2040) was converted to 10 blunt ends by treatment with the Klenow fragment of E. coli DNA polymerase I in the presence of dNTPs and inserted behind the ADH1 promoter in the vector pAD4M [75] that had been linearized with SmaI. Correct orientation of the fragment was confirmed by appropriate restriction enzyme digests. An essentially identical approach was used to express bovine PARK, yielding pADH 15 8ARK, which was constructed by Henrik Dohlman (Thorner laboratory). To express PKB in yeast, a 1.5 kb BamHI-BamHI fragment encoding mouse c-Akt was excised from an insert in a two-hybrid bait vector, pASIIA (supplied by Zhou Songyang, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA), and ligated into YEp351GAL that had been linearized by 20 digestion with BamHI, yielding pGAL-PKB. Alternatively, a 2.5 kb EcoRI-XbaI fragment encoding a human PKB cDNA [1] was inserted into pYES2, generating pYES2-PKBa. In addition, a 1.5 kb EcoRI-XbaI insert expressing a constitutively-active mutant version of PKBa [22], in which Thr308 and Ser473 have been replaced by Asp, was inserted into pYES2, creating pYES2-DD 25 PKBc. To express p70 S6 kinase in yeast, a 1.6 kb XbaI-Sall fragment encoding rat p70 S6 kinase [76] was excised from p2B4 (provided by George Thomas, Friedrich Miescher Institute, Basel, Switzerland) and inserted into YEp351GAL that had been linearized by digestion with XbaI-SalI, yielding pGAL-S6K. In addition, an N-terminal truncation, a C-terminal truncation, and a double N- and WO 00/36135 PCT/GB99/04228 105 C-terminal truncation of p70 S6 kinase (generously provided by John Blenis, Department of Cell Biology, Harvard Medical School, Boston, MA), whose constructions are described in detail elsewhere [51], were also inserted into a yeast expression vector, YEp352 [78], each under control of the methionine 5 repressible MET3 promoter [78], using essentially identical methods. Site-directed mutagenesis To generate a catalytically-inactive ("kinase-dead") version of Pkh1 (KD-Pkhl), Asp276 (GAT) was changed to Ala (GCT). This position corresponds to a 10 conserved residue critical for recognition of the Mg 2 -ATP substrate in all protein kinases [79]. Likewise, a catalytically-inactive Ypk1 derivative (KD Ypkl) was generated by changing Asp488 (GAT) to Ala (GCT). To attempt to generate a constitutively-active YPK1 derivative, the Phkl-dependent phosphorylation site (Thr504) and another presumptive phosphorylation site 15 (Thr662) that matches the consensus for phosphorylation by PDK2 protein kinase activity were both replaced by Asp codons. Expression and purification of GST-Pkhl, GST-Ypkl and GST-SGK The human embryonic kidney cell line, 293, was cultured on forty 10-cm dishes, 20 and each dish was transfected with 20 ptg of the appropriate expression construct using a modified calcium phosphate method [60]. At 24 h after transfection, each dish of cells was harvested and the cells ruptured in 1 ml of ice-cold lysis buffer, which contained 50 mM Tris/HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 % (by vol) Triton X-100, 1 mM sodium orthovanadate, 10 mM sodium p-glycerol 25 phosphate, 50 mM NaF, 5 mM sodium pyrophosphate, 1 iM microcystin-LR, 1 mM benzamidine, 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 10 ptg/ml leupeptin and 0.1 % (by vol) 2-mercaptoethanol. The forty lysates were pooled, clarified by centrifugation at 4"C for 10 min at 13,000 x g, and the GST-fusion proteins were purified by affinity chromatography on glutathione-Sepharose [26].

WO 00/36135 PCT/GB99/04228 106 Approximately 0.5 mg of each purified GST-fusion protein was obtained, snap frozen in aliquots in liquid N2, and stored at -80"C. Measurement of Ypkl, SGK and PKB activities 5 Assay of Ypk1 was carried out in two stages. First (Stage 1), GST-Ypk1 was activated by incubation with GST-Pkh1 and Mg 2 '-ATP, as follows. A reaction mixture (18 pl) containing 2.5 p.M PKI, 1 pM microcystin-LR, 10 mM Mg acetate, 100 p.M unlabelled ATP and 0.6 p.M GST-Ypkl was prepared in Buffer A [50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, and 0.1 % (by vol) 2 10 mercaptoethanol]. Reaction was initiated by addition of 2 pl of 50 nM GST-Pkh1 in Buffer A containing 1 mg/ml bovine serum albumin, and was incubated at 30"C for 30 min. Second (Stage 2), activated Ypk1 was assayed by adding 30 pl of a mixture in Buffer A containing 2.5 p.M PKI, 1 pM microcystin-LR, 10 mM Mg-acetate, 100 pM [y"P]ATP (200-400 cpm/pmol) and 100 p.M Crosstide 15 (GRPRTSSFAEG) [17], a peptide phosphoacceptor substrate. After incubation for 15 min at 30"C, reaction was terminated by spotting a portion (45 p) of each reaction mixture onto small squares of phosphocellulose paper (Whatman P81), which were washed and analysed as described [80]. Control reactions omitted either GST-Ypk1 or GST-Pkh1 and resulted in incorporation of less than 5% of 20 the radioactivity measured in the presence of both of these proteins. One unit of GST-Ypk1 activity was defined as that amount required to catalyse phosphorylation of 1 nmole of Crosstide in 1 min. Assay of SGK and PKB activities were carried out in identical manner, except that GST-SGK and GST PKBa replaced GST-Ypk1 in the first stage of the assay. 25 Phosphorylation of GST-Ypkl, GST-PKB and GST-SGK by Pkh1 Incubations were identical to Stage 1 of the Ypk1 assay described above, except that [y- 32 P]ATP (500-1000 cpm/pmol) was used instead of unlabelled ATP, and reactions were terminated by adding SDS to a final concentration of 1 %. The WO 00/36135 PCT/GB99/04228 107 resulting samples were resolved on 7.5% SDS-polyacrylamide gels and, after staining with Coomassie blue, analysed by autoradiography. Also, the stained band corresponding to the GST-fusion protein of interest was excised and the amount of radioactivity incorporated was quantified by liquid scintillation 5 counting. Ability of GST-Pkhl to phosphorylate and activate human PKBa was examined using methods identical to those described immediately above for the phosphorylation and activation of yeast Ypkl by GST-Pkhl, except that reactions were performed in the presence of lipid vesicles containing various 3 phosphoinositides [26], as described in Results. 10 Determination of Ypkl substrate specificity GST-Ypkl was activated with Pkhl in vitro and incubated under standard assays conditions, as described above, except that Crosstide was replaced by 100 pM of the peptides discussed in detail in Results. GST-PKB, derived from transfected 15 IGF1-stimulated 293 cells [26], was assayed in parallel. References 20 1. Coffer & Woodgett Eur J Biochem. 1991, 201:475-481. 2. Jones et al (1991) Proc Natl Acad Sci. USA 88:4171-4175. 3. Belacossa et al (1991) Science 254:244-247. 4. Franke et al (1995) Cell 81:727-736. 5. Burgering et al (1995) Nature 376:599-602. 25 6. Kohn et al (1995) EMBO J 14:4288-4295. 7. Alessi & Cohen (1998) Curr Opin Genet Dev 8:55-62. 8. Downward (1998) Curr Opin Cell Biol 10:262-267. 9. Barthel et al (1997) Endocrinol 183:3559-3562. 10. Cichy et al (1998) J Biol Chem 273:6482-6487.

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WO 00/36135 PCT/GB99/04228 110 64. Sikorski & Hieter (1989) Genetics 122:19-27. 65. Schnieders, EA: Ypkl and Ykr2. Appendix, Doctoral Dissertation, University of California, Berkeley, CA, 1996, pp.

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Strain Genotype Source 25 AYS927 MATa/MATa ade2-1/ade2-1 his3-11,15/his3-11,15 M.J.R. Stark leu2-3,112/leu2-3,112 trpl-1/trpl-1 ura3-1/ura3-1 cani-100/can1-100 ssdl-d2/ssdl-d2 AC200 AYS927 PKH2/pkh2A::HIS3 This study PCT/GB99/0 4228 -WO 00/36135 111 AC201 AYS927 pKHlpkhlA::TRPJ This study AC303 MATa pkh2A:.HIS3 (derived from AC200) This study AC301 MAT pkhlA::TRP1 (derived from AC201) This study AC306 MATa/MATa PKHJIpkhlA::TRP1 PKH2/pkh2A::HIS3 This study 5 (AC303 X AC301) YPH499 MATa ade2-1010c his3-A200 leu2-AJ lys2- 8 01' [64] trpl-Al ura3-5 2 YPH500 MATa otherwise isogenic to YPH499 [64] YES1 YPH499 ykr2-A1::TRP1 This study 10 YES5 YPH500ypkl-Al::HIS3 This study YES7 MATa/MATa YPKllypklA::HIS3 YKR2/ykr2A::TRP1 This study (YES 1 X YES5) YPT28 MATa ypklI::HIS3 ykr2A::TRPl [pYKR2(URA3)] This study YPT40 MATa ypkl-l" ykr2A This study 15 PCT/GB99/0 4228 WO 00/36135 112 Table 2. Substrate selectivity of yeast Ypk1 and mammalian PKBa. Peptide Relative Rate of Phosphorylation (at 0 .1 mM) GST-Ypkl GST-PKBa 5 1 GRPRTSSFAEG [100] [100] 2 RPRTSSF 92 139 3 KPRTSSF 11 27 4 RPKTSSF 5 30 10 5 RPRTSAF 0 0* 6 PRTSSF 0 0* 7 RPRTSS 0 2* 8 KKRNRTLSVA 133 157 15 9 KKKNRTLSVA 28 13 10 KKRNKTLSVA 14 25 11 RPRTSSF 92 139 12 RPRTSSV 24 108 13 RPRTSSL 31 130 20 14 RPRTSSA 9 70 15 RPRTSSK 14 67* 16 RPRTSSE 6 63 25 *Data taken from Ref. 16. Example 2: screening assay for inhibitors of human PDK1 The effect of compounds on parental (wild-type with respect to Pkhl and/or Pkh2) S. cerevisiae cells and S. cerevisiae cells in which the endogenous Pkhl 30 and Pkh2 genes have been inactivated and in which human PDK1 is expressed WOO00/36135 PCT/GB99/04 2 2 8 113 under control of the Pkhl promoter, are compared. Compounds that affect the growth of the PDK1-expressing cells but not the Pkhl/Pkh2 expressing (wild type) cells are selected and may be used to design further compounds for manufacture and test, in order to develop a structure-activity relationship (SAR). 5 The compounds for test may be selected on the basis of known kinase inhibitory activity or other known property, or may be part of a library of synthetic or natural molecules that may be screened in a "lead generation" screening project. 10 Example 3: screening assay for inhibitors of PDK1 from a pathogenic source The effect of compounds on parental (wild-type with respect to Pkhl and/or Pkh2) S. cerevisiae cells and S. cerevisiae cells in which the endogenous Pkhl and Pkh2 genes have been inactivated and in which Candida Pkh1 or Pkh2 is 15 expressed under control of the Pkhl promoter, are compared. Compounds that affect the growth of the Candida Pkhl or Pkh2-expressing cells but not the wild type Pkhl/Pkh2 expressing cells are selected and may be used to design further compounds for manufacture and test, in order to develop a structure-activity relationship (SAR). 20 The compounds for test may be selected on the basis of known kinase inhibitory activity or other known property, or may be part of a library of synthetic or natural molecules that may be screened in a "lead generation" screening project. 25 Example 4: Identification of Candida genes related to S. cerevisiae Pkhl, Pkh2, Ypk1 and Ykr2 genes. The Candida sequence database only allows DNA searches of the Candida data. Therefore, for each of the 4 S. cerevisiae sequences identified and characterised, PCT/GB99/0 4 2 2 8 WO 00/36135 114 as described in example 1 (Pkhl, Pkh2, Ypkl, Ykr2), the coding sequence for each S. cerevisiae gene was used to search the Candida database. The best matches in each case were selected and compared with proteins in the 5 Genbank database. With one exception, this comparison identified the two related S. cerevisiae genes (ie Pkhl/Pkh2 or Ypkl/Tkr2) as the most closely related proteins. The Candida albicans database entries selected for further study on the basis of 10 the four coding sequence searches were: 384230A10.s4.seq (search 1) 384362E11.sl.seq (search 1 and search 2) 384286E10.s1.seq (search 1 and search 2) 396076E03.s2.seq (search 3 and search 4) 15 384194F08.sl.seq (search 3 and search 4) There appear to be fragments of three Candida genes in the database related very closely to S. cerevisiae Pkhl, Pkh2, Ypk1 and Ykr2. 20 384194F08.s1.seq and 396076E03.s2.seq appear to be closely related to Pkhl and Pkh2. 384362E11.sl.seq and 384286E10.sl.seq appear to be closely related to Ypkl and Ykr2/Ypk2. 25 Full-length coding sequences may be determined for the genes from which the above nucleotide sequences are derived by well known techniques, which may include further database interrogation and/or techniques of molecular biology, 30 which may include PCR or library-based cloning techniques.

WO 00/36135 PCT/GB99/04 2 2 8 115 Search 1 Blast Results 5 Smallest Sum 10 High Probability Sequences producing High-scoring Segment Pairs: Score P(N) N 384230A10.s4.seq 635 3.2e-43 1 15 384362E11.sl.seq 595 3.8e-40 1 CAPKC1 C.albicans gene for protein kinase C. 490 5.2e-37 2 384286E10.sl.seq 411 1.e-24 1 384201A03.s2.seq 363 1.4e-20 1 385002F05.xl.seq 350 1.9e-19 1 20 396143F08.s3.seq 307 7.1e-16 1 CAU73457 Candida albicans Cst20p (CST20) gene, complete ... 240 4.4e-10 1 YSASTPK Candida albicans serine/threonine protein kinase... 240 4.4e-10 1 265251G7.xl . seq 233 1.4e-09 1 265251H07.xl.seq 233 1.4e-09 1 25 385040C4.xl.seq 229 2.9e-09 1 386003B12.xl.seq 218 2.5e-08 1 396076E03.s2.seq 189 7.le-06 1 384281A03.sl.seq 174 0.00012 1 384194F08.sl.seq 172 0.00017 1 30 384253B09.s1.seq 165 0.00069 1 384329A05.sl.seq 164 0.00084 1 265251H06.x1.seq 162 0.0012 1 265273H11.yl.seq 157 0.0032 1 265266H04.xl.seq 155 0.0049 1 35 384068G08.sl.seq 152 0.0069 1 396170G09.sl.seq 151 0.0096 1 Search 2 Blast Results 40 Smallest 45 Sum High Probability Sequences producing High-scoring Segment Pairs: Score P(N) N 50 384362E11.sl.seq 586 2.2e-49 2 384286E10.sl.seq 497 8.7e-32 1 CAU73457 Candida albicans Cst20p (CST20) gene, complete ... 287 5.3e-14 1 YSASTPK Candida albicans serine/threonine protein kinase... 287 5.3e-14 1 265251G07.xl.seq 264 3.6e-12 1 55 265251H07.xl.seq 262 5.3e-12 1 265251H06.xl.seq 242 2.4e-10 1 265266H04.xl.seq 226 5.6e-09 1 385040C04.xl.seq 219 2.0e-08 1 YSASTKIN Candida albicans (clone pKB66) serine/threonine ... 185 1.6e-05 1 WO 00/36135 PCT/G B99/04228 116 CAU72980 Candida albicans Map kinase kinase (STE7) gene,... 185 1.7e-05 1 386003B12.x1.seq 181 3.2e-05 1 385081C04.xl.seq 164 0.00079 1 396039B07.s1.seq 159 0.0021 1 5 384281A03.sl.seq 156 0.0039 1 Search 3 Blast Results 10 Smallest Sum 15 High Probability Sequences producing High-scoring Segment Pairs: Score P(N) N 396076E03.s2.seq 636 6.9e-85 2 20 384194F08.s1.seq 674 1.5e-46 1 384254G10.sl.seq 266 3.8e-12 1 385051D09.xl.seq 244 2.le-10 1 384022B04.sl.seq BCK1 244 2.6e-10 1 CAU73457 Candida albicans Cst20p (CST20) gene, complete ... 229 5.9e-09 1 25 YSASTPK Candida albicans serine/threonine protein kinase... 229 5.9e-09 1 384201A03.s2.seq 220 2.5e-08 1 384168E05.s2.seq 216 3.le-08 1 CAU87996 Candida albicans CLA4 protein kinase homolog ge... 218 4.8e-08 1 396039B07.s1.seq 195 3.3e-06 1 30 384282H11.sl.seq 187 1.5e-05 1 385066D07.xl.seq 171 0.00033 1 265235H07.yl.seq 169 0.00047 1 396039H02.si.seq 162 0.0012 1 396256F10.sl.seq 164 0.0013 1 35 384102B12.sl.seq 163 0.0016 1 265091D04.xl.seq PAK1 159 0.0035 1 Unp-F1-F17E2-Reverse (-) Assignment:KIN3 156 0.0061 1 385009G05.xl.seq 154 0.0091 1 384179F07.s1.seq 153 0.011 1 40 265001E11.r2.seq STE11 150 0.020 1 Search 4 Blast Results 45 Smallest Sum 50 High Probability Sequences producing High-scoring Segment Pairs: Score P(N) N 396076E03.s2.seq 636 6.9e-85 2 55 384194F08.sl.seq 674 1.5e-46 1 384254G10.sl.seq 266 3.8e-12 1 385051D09.xl.seq 244 2.1e-10 1 384022B04.si.seq BCK1 244 2.6e-10 1 CAU73457 Candida albicans Cst2Op (CST20) gene, complete ... 229 5.9e-09 1 WO 00/36135 PCT/GB99/04228 117 YSASTPK Candida albicans serine/threonine protein kinase... 229 5.9e-09 1 384201A03.s2.seq 220 2.5e-08 1 384168E05.s2.seq 216 3.1e-08 1 CAU87996 Candida albicans CLA4 protein kinase homolog ge... 218 4.8e-08 1 5 396039B07.sl.seq 195 3.3e-06 1 384282H11.s1.seq 187 1.5e-05 1 385066D07.xl.seq 171 0.00033 1 265235H07.yl.seq 169 0.00047 1 396039H02.sl.seq 162 0.0012 1 10 396256F10.sl.seq 164 0.0013 1 384102B12.sl.seq 163 0.0016 1 265091D04.xl.seq PAK1 159 0.0035 1 Unp-F1-F17E2-Reverse (-) Assignment:KIN3 156 0.0061 1 385009G05.xl.seq 154 0.0091 1 15 384179F07.sl.seq 153 0.011 1 265001E11.r2.seq STE11 150 0.020 1 Genbank search results using 384194F08.sl.seq (search 3 and search 4) 20 Blast Results Smallest Sum 25 Reading High Probability Sequences producing High-scoring Segment Pairs: Frame Score P(N) N gij663254 (Z48149) probable protein kinase [Saccharom... -3 452 4.0e-55 1 gil1419952 (Z74842) ORF YOLlO0w [Saccharomyces cerevis... -3 452 4.0e-55 1 30 gil1431588 (X99280) protein kinase [Schizosaccharomyce... -3 441 1.7e-54 1 gil927745 (U33050) Ydr49Ocp; CAI: 0.11 [Saccharomyces... -3 446 2.le-54 1 gil2407613 (AF017995) 3-phosphoinositide dependent pro... -3 232 4.0e-48 2 giJ2505936 (Y15056) PkB kinase [Homo sapiens] -3 232 4.0e-48 2 gil2665356 (Y15748) PkB kinase [Rattus norvegicus] -3 232 4.1e-48 2 35 gi12832892 (AL021730) hypothetical protein kinase phos... -3 393 1.8e-47 1 gil927730 (U33050) Ydr466wp; CAI: 0.11 [Saccharomyces... -3 250 3.5e-47 3 gi|2980851 (Y07908) serine/threonine protein kinase (D... -3 351 3.3e-41 1 giJ1000069 (U15210) rac-alpha serine/threonine kinase ... -3 184 2.0e-31 2 giJ1673493 (Z81140) WlOGG.2 [Caenorhabditis elegant) -3 277 4.0e-31 1 40 gil294637 (L01G24) serine/threonine protein kinase (R... -3 277 4.le-31 1 gil1834511 (Y10032) serine/threonine protein kinase [H... -3 276 5.6e-31 1 gil2463201 (AJOOO5l2) serine/threonine protein kinase ... -3 276 5.6e-31 1 gil189967 (M80335) protein kinase A-alpha [Homo sapiens] -3 156 5.9e-29 2 giJ2632252 (Y12464) serine/threonine kinase [Sorghum b... -3 161 2.3e-28 2 45 gil1401040 (D49836) RAC-PK gamma [Rattus norvegicus] -3 167 2.5e-28 2 giJ1890142 (D83380) catalytic subunit of cAMP-dependen... -3 167 4.1e-28 2 gi|773642 (U23730) vinclozolin resistance protein [Us... -3 172 6.5e-28 2 gil2213667 (U83459) Riml~p [Saccharomyces cerevisiae] -3 255 8.5e-28 1 gi|836721 (D50617) YFLO33C [Saccharomyces cerevisiae) -3 255 8.5e-28 1 50 gil465398 (D28577) Protein kinase C [Mus musculus] -3 253 1.2e-27 1 gi|432274 (118964) protein kinase C iota [Homo sapiens] -3 253 1.2e-27 1 giJ598225 (L33881) protein kinase C iota (Homo sapiens] -3 253 1.2e-27 1 giJ507141 (M81709) cAMP-dependent protein kinase cata... -3 166 1.2e-27 2 gil2253148 (Z82096) ZK909.2a (Caenorhabditis elegant] -3 162 1.2e-27 2 55 gi304273 (L17008) cAMP-dependent protein kinase [Bla... -3 166 1.4e-27 2 gi|2253149 (Z82096) ZK909.2b [Caenorhabditis elegant] -3 162 1.5e-27 2 giJ853791 (Z34989) Ndr protein kinase [Caenorhabditis... -3 178 1.7e-27 2 giJ2113796 (X94399) cAMP-dependent protein kinase cata... -3 159 1.9e-27 2 giJ56916 (X68400) protein kinase [Rattus norvegicus] -3 251 2.4e-27 1 60 giJ220527 (D90242) nPKC-eta [Mus musculus] -3 251 2.4e-27 1 WO 00/36135 PCT/GB99/04228 118 gil665540 (U15983) cAMP-dependent protein kinase cata... -3 160 3.1e-27 2 gi|189989 (M55284) protein kinase C-L [Homo sapiens] -3 250 3.3e-27 1 giJ806542 (Z49233) calcium-stimulated protein kinase ... -3 249 4.2e-27 1 gij473894 (U08622) cAMP-dependent protein kinase [Sch... -3 169 4.3e-27 2 5 gil484305 (D23667) catalytic subunit of the cAMP-depe..- -3 169 4.3e-27 2 gi|853820 (Z35103) Ndr protein kinase [Drosophila mel... -3 164 4.5e-27 2 gif2632254 (Y12465) serine/threonine kinase [Sorghum b... -3 163 5.6e-27 2 gil1911 (X05998) C-beta subunit (338 AM [Sus scrofa] -3 155 6.5e-27 2 gil516040 (U12335) cAMP-dependent protein kinase cata... -3 159 6.6e-27 2 10 giJ35479 (X07767) protein kinase catalytic subunit t... -3 156 7.5e-27 2 gi|1487920 (Z75953) F57F5.5 [Caenorhabditis elegant] -3 247 7.6e-27 1 giJ3005054 (AF041843) protein kinase Ukclp [Ustilago m... -3 177 8.4e-27 2 gi|392435 (U00181) protein kinase C [Caenorhabditis e... -3 247 8.7e-27 1 giJ2304746 (A52140) HUMAN NDR [unidentified] -3 172 8.9e-27 2 15 gi|854170 (Z35102) Ndr protein kinase [Homo sapiens] -3 172 8.9e-27 2 gi|173011 (M17073) cAMP-dependent protein kinase subu... -3 160 9.7e-27 2 gil4625 (Y00694) put. kinase (AA 1-380) [Saccharomy... -3 160 9.7e-27 2 gil1370422 (Z73559) ORF YPL2O3w [Saccharomyces cerevis... -3 160 9.7e-27 2 gil191175 (M63311) cAMP-dependent protein kinase aiph... -3 156 1.0e-26 2 20 gi156233 (M37120) cAMP-dependent protein kinase cata... -3 156 1.3e-26 2 gil1086654 (U41016) coded for by C. elegant cDNA CEESC... -3 178 1.6e-26 2 gi|288120 (X53261) cAMP-dependent protein kinase cata... -3 153 1.7e-26 2 gil2760821 (LO6196) serine/threonine protein kinase [E... -3 174 1.7e-26 2 gil1050791 (X92517) N1727 gene product [Saccharomyces ... -3 175 1.7e-26 2 25 gil1302128 (Z71437) ORF YNLl61w [Saccharomyces cerevis... -3 175 1.7e-26 2 giJ3043598 (ABO11109) KIAA0537 protein [Homo sapiens] -3 196 1.9e-26 2 gil1853976 (D31773) protein kinase [Schizosaccharomyce... -3 245 2.0e-26 1 gil200367 (M12303) cAMP-dependent protein kinase cata... -3 158 2.0e-26 2 gil387513 (M19960) cAMP-dependent protein kinase aiph... -3 158 2.0e-26 2 30 gil155792 (M94884) protein kinase C [Aplysia californ... -3 244 2.3e-26 1 gi1178326 (M95936) protein serine/threonine kinase [H... -3 160 2.5e-26 2 gil191177 (M63312) cAMP-dependent protein kinase beta... -3 152 2.7e-26 2 giJ200387 (J02626) cAMP-dependent protein kinase beta... -3 152 2.7e-26 2 gil220704 (D10770) cAMP-dependent protein kinase cata... -3 152 2.7e-26 2 35 gi1163532 (J02647) protein kinase beta-catalytic subu... -3 151 2.7e-26 2 giJ189983 (M34181) cAMP-dependent protein kinase cata... -3 151 2.7e-26 2 gil337491 (M77198) rac protein kinase-beta [Homo sapi... -3 160 3.0e-26 2 gil2911458 (AF046921) cAMP-dependent protein kinase ca... -3 164 3.1e-26 2 giJ2244814 (Z97336) protein kinase [Arabidopsis thaliana] -3 242 3.le-26 1 40 gil56912 (X57986) cAMP-dependent protein kinase subu... -3 156 3.8e-26 2 gij162787 (M60482) cAMP-dependent protein kinase Il-b... -3 151 4.0e-26 2 gi1165563 (M20014) protein kinase C [Oryctolagus cuni... -3 242 4.4e-26 1 giJ35495 (X65293) protein kinase C epsilon [Homo sap... -3 242 4.4e-26 1 gij2605804 (AF028009) protein kinase C epsilon [Mus mu... -3 242 4.4e-26 1 45 giJ633 (X67154) protein kinase [Bos taurus] -3 156 5.2e-26 2 gi|942578 (U22445) serine/threonine kinase [Mus muscu... -3 157 6.5e-26 2 gi(157052 (M18655) cAMP-dependent protein kinase cata... -3 155 7.3e-26 2 giJ7807 (X16969) catalytic subunit [Drosophila mela... -3 155 7.3e-26 2 50 Genbank results using 38423oAi.S4.seq (search 1) 55 Blast Results Smallest Sum 60 Reading High Probability Sequences producing High-scoring Segment Pairs: Frame Score P(N) N WO 00/36135 PCT/GB99/04228 119 giJ1050791 (X925l7) N1727 gene product [Saccharomyces ... +3 921 8.3e-137 3 gif1302128 (Z71437) ORF YNLl6lw [Saccharomyces cerevis... +3 921 8.3e-137 3 giJ3005054 (AF041843) protein kinase Ukclp [Ustilago m... +3 817 3.0e-117 2 5 giJ2443511 (AF009512) protein kinase Orb6p [Schizosacc... +3 769 8.7e-115 2 gi|1870020 (X97657) serine/threonine kinase [Neurospor... +3 725 1.2e-102 2 gij641979 (U14989) kinase [Colletotrichum trifoii] +3 733 4.5e-100 2 giJ1870019 (X97657) serine/threonine kinase [Neurospor... +3 725 1.3e-99 2 gil2304746 (A52140) HUMAN NDR [unidentified] +3 647 1.5e-93 2 10 giJ854170 (Z35102) Ndr protein kinase [Homo sapiens] +3 647 1.5e-93 2 gi|853820 (Z35103) Ndr protein kinase [Drosophila mel... +3 629 2.7e-92 2 giJ2304742 (A52135) D. MELANOGASTER NDR [unidentified] +3 372 1.8e-89 3 gil1086654 (U41016) coded for by C. elegant cDNA CEESC... +3 628 2.0e-85 2 gi|853791 (Z34989) Ndr protein kinase [Caenorhabditis... +3 618 l.1e-80 1 15 giJ506534 (X71057) protein kinase [Nicotiana tabacum] +3 435 1.1e-74 3 gi|3135270 (AC003058) putative protein kinase [Arabido... +3 533 5.8e-74 2 giJ457709 (Z30330) protein kinase [Spinacia oleracea] +3 533 6.3e-74 2 gi|1914507 (Z81594) T20F10.l [Caenorhabditis elegant] +3 575 1.9e-72 1 giJ903942 (U29608) LATS [Drosophila melanogaster] +3 466 2.0e-71 3 20 giJ755008 (L39837) tumor suppressor [Drosophila melan... +3 466 2.0e-71 3 gi|457689 (Z30329) protein kinase [Mesembryanthemum c... +3 548 1.4e-69 1 gil1200533 (M28500) protein kinase [Euplotes crassusl +3 469 5.0e-60 1 gi|1200509 (U47679) protein kinase [Euplotes crassus] +3 469 3.5e-58 1 gi11276901 (U43195) Rho-associated, coiled-coil contai... +3 300 3.7e-54 2 25 gil1514696 (U58512) Rho-associated, coiled-coil contai... +3 300 3.7e-54 2 giJ1438567 (U61266) Rho-associated kinase beta [Rattus... +3 300 3.8e-54 2 gil2736153 (AF021936) myotonic dystrophy kinase-relate... +3 280 1.6e-51 2 gi|181605 (L08835) myotonic dystrophy kinase [Homo sa... +3 271 2.2e-51 2 giJ181606 (L08835) myotonic dystrophy kinase [Homo sa... +3 271 2.2e-51 2 30 gii976145 (L00727) myotonin-protein kinase, Form VIII... +3 271 2.4e-51 2 giJ106l299 (ZX7757) unknown [Schizosaccharomyces pombe. +3 422 2.6e-51 1 giJ186756 (M94203) protein kinase [Homo sapiens] +3 271 3.0e-51 2 giJ976143 (L00727) myotonin-protein kinase, Form V [H... +3 271 3.2e-51 2 giJ 976146 (L00727) myotonin-protein kinase, Form VII ... +3 271 3.5e-51 2 35 giJ307177 (L19268) protein kinase [Homo sapiens] +3 271 3.8e-51 2 giJ181603 (LU8835) myotonic dystrophy kinase [Homo sa.t.r. +3 271 3.8e-51 2 giJ633865 (S72883) myotonin protein kinase, MtPK=thy ... +3 271 3.8e-51 2 gij181604 (L08835) myotonic dystrophy kinase [Homo sa... +3 271 3.9e-51 2 giJ97G147 (L00727) myotonin-protein kinase, Form VI [... +3 271 4.0e-51 2 40 giJ976144 (L00727) myotonin-protein kinase, Form I [H... +3 271 4.1e-51 2 gi11384133 (U38481) ROK-alpha [Rattus norvegicus] +3 285 5.7e-51 2 gi11326078 (U36909) Rho-associated kinase [Bos taurus] +3 285 5.7e-51 2 giJl5l4698 (U58513) Rho-associated, coiled-coil contai... +3 285 5.7e-51 2 giJ556903 (Z21503) DM protein kinase [Mus musculus] +3 268 6.8e-51 2 45 giJ1695873 (U59305) ser-thr protein kinase PK428 [Homo... +3 266 8.2e-51 2 giJ563526 (Z38015) myotonic dystrophy protein kinase ... +3 268 1.0e-50 2 giJ171380 (MU2506) putative [Saccharomyces cerevisiae] +3 402 1.4e-50 2 gi1938422 ((397001) Similar to serine/threonine-protei... +3 253 2.6e-50 3 giJ2982220 (AF037073) Rho-associated kinase alpha eXen... +3 283 3.8e-50 2 50 giJ1323137 (Z72877) ORF YGRO92w [Saccharomyces cerevis... +3 413 4.4e-50 1 Genbank search results using 384286E10.sl.seq (search 1 and search 2) 55 Blast Results 60 WARNING: -hspmax 100 was exceeded with 1 of the database sequences, with as many as 139 HSPs being found at one time.

WO 00/36135 PCT/GB99/04228 120 Smallest Sum Reading High Probability 5 Sequences producing High-scoring Segment Pairs: Frame Score P(N) N gi|295681 (M24929) protein kinase [Saccharomyces cere... +3 284 5.0e-32 1 gij817862 (Z49702) Ypk2p [Saccharomyces cerevisiae] +3 284 5.0e-32 1 giJ172181 (M21307) protein kinase [Saccharomyces cere... +3 280 1.8e-31 1 10 giJ486213 (Z28126) ORF YKL126w [Saccharomyces cerevis... +3 280 1.8e-31 1 giJ2911462 (AF046923) serine/threonine protein kinase ... +3 194 1.7e-20 2 gil458284 (U05811) serine/threonine protein kinase [T... +3 193 5.0e-20 2 giJ206181 (M18330) protein kinase C delta subspecies +3 175 2.6e-15 1 gil189510 (M60725) p70 ribosomal S6 kinase alpha-I [ +3 174 3.3e-15 1 15 gil206840 (M57428) S6 kinase [Rattus norvegicus] +3 174 3.3e-15 1 gif1562 (X54415) G3 serine/threonine kinase [Orycto... +3 174 3.4e-15 1 gi|189508 (MG0724) p70 ribosomal 56 kinase alpha-I [H... +3 174 3.4e-15 1 gil2222775 (Y12002) protein kinase C homologue [Neuros... +3 173 5.7e-15 1 gi|200381 (M69042) [mouse protein kinase C delta mRNA... +3 172 7.2e-15 1 20 giJ53437 (X60304) protein kinase [Mus musculus] +3 172 7.2e-15 1 giJ501075 (U10016) protein kinase C [Trichoderma reesei] +3 172 8.0e-15 1 giJ2687849 (Y5839) protein kinase C [Cochliobolus het.... +3 169 2.2e-14 1 gi1507900 (U10549) protein kinase C [Aspergillus niger] +3 168 3.le-14 1 giJ206842 (M58340) S6 protein kinase [Rattus norvegicus] +3 165 7.3e-14 1 25 giJll363o2 (D38108) protein kinase [Schizosaccharomyce... +3 162 1.1e-13 2 giJl65563 (M20014) protein kinase C [Oryctolagus cuni... +3 161 3.1e-13 1 giJ35495 (X65293) protein kinase C epsilon (Homo sap... +3 161 3.1e-13 1 giJ206183 (M18331) protein kinase C epsilon subspecie ... +3 160 4.3e-13 1 giJ2605804 (AF028009) protein kinase C epsilon [Mus mu... +3 159 6.0e-13 1 30 giJ832908 (X81142) protein kinase C [Candida albicans] +3 159 6.5e-13 1 giJSS8lOO (L07032) protein kinase C-theta [Homo sapiens] +3 156 1.6e-12 1 giJ558099 (L1087) protein kinase C-theta [Homo sapiens] +3 156 1.6e-12 1 giJ458923 (U0029) Sch9p: cAMP-dependent protein kina... +3 156 1.7e-12 1 gi14426 (X12560) SCH9 protein (AA 1-824) [Saccharom... +3 156 1.7e-12 1 35 giJ5279 (X57629) Sch9 [Saccharomyces cerevisiae] +3 156 1.7e-12 1 giJ1778590 (U82935) protein kinase C2 A isoform [Caeno... +3 150 2.5e-12 2 gi1000069 (U15210) rac-alpha serine/threonine kinase ... +3 151 2.6e-12 2 giJ214789 (M20188) S6 kinase II beta [Xenopus laevisl +3 154 3.1e-12 1 gi3l114960 (Y13104) Protein kinase C-related kinase (P... +3 149 3.4e-12 2 40 giJ173427 (L07637) protein kinase C [Schizosaccharomy... +3 154 3.4e-12 1 giJ303941 (D14338) protein kinase [Schizosaccharomyce... +3 154 3.4e-12 1 giJ401772 (L07598) ribosomal protein S6 kinase 2 [Hom.. . +3 152 5.9e-12 1 giJ220527 (D90242) nPKC-eta (Mus musculus] +3 152 6.3e-12 1 gij1033033 (X85106) ribosomal S6 kinase [Homo sapiens] +3 152 6.3e-12 1 45 (gi|551556 (M28488) ribosomal protein S6 kinase [Gallu... +3 152 6.4e-12 1 giJ56916 (X68400) protein kinase [Rattus norvegicus] +3 151 8.7e-12 1 50 Genbank search results using 384362E11.sl.seq (search 1 and search 2) Blast Results Smallest 55 Sum Reading High Probability Sequences producing High-scoring Segment Pairs: Frame Score P(N) N giJ4862l3 (Z28126) ORF YKLl26w [Saccharomyces cerevis... +2 391 6.6e-65 3 60 gi|172181 (M21307) protein kinase [Saccharomyces cere... +2 387 2.4e-64 3 giJ295681 (M24929) protein kinase [Saccharomyces cere... +2 380 5.8e-63 3 WO 00/36135 PCT/GB99/04228 121 giJ817862 (Z49702) Ypk2p [Saccharomyces cerevisiae] +2 380 5.8e-63 3 gil2911462 (AF046923) serine/threonine protein kinase ... +2 355 1.6e-58 3 gi|458284 (U05811) serine/threonine protein kinase fT... +2 353 7.5e-58 3 gil1000069 (U15210) rac-alpha serine/threonine kinase ... +2 267 2.le-52 3 5 giJ558100 (L07032) protein kinase C-theta [Homo sapiens] +2 274 3.1e-43 2 giJ558099 (L01087) protein kinase C-theta [Homo sapiens] +2 274 3.le-43 2 giJ220574 (D11091) 'Protein Kinase' [Mus musculus] +2 271 1.1e-42 2 gi|183451 (Y10032) serine/threonine protein kinase [H... +2 270 1.3e-42 2 gil2463201 (AJ000512) serine/threonine protein kinase ... +2 270 1.3e-42 2 10 gil1673493 (Z81140) WlOG6.2 [Caenorhabditis elegant] +2 263 1.6e-42 2 giJ294637 (L01624) serine/threonine protein kinase [R... +2 269 1.7e-42 2 gil1136302 (D38108) protein kinase [Schizosaccharomyce... +2 144 3.2e-42 3 gi|206191 (M15522) protein kinase [Rattus norvegicus] +2 237 1.5e-40 2 gil56920 (X04139) protein kinase C C-terminal region... +2 237 1.5e-40 2 15 giJ2996092 (U63742) rac serine-threonine kinase homolo... +2 189 1.9e-40 3 gi|210068 (M80675) gag:akt fusion protein [AKT8 retro... +2 222 2.5e-39 4 giJ3116064 (AJ223715) s-sgkl [Squalus acanthias] +2 264 1.1e-38 2 gi|3116066 (AJ223716) s-sgk2 [Squalus acanthias] +2 264 2.8e-38 2 giJ942578 (U22445) serine/threonine kinase [Mus muscu... +2 225 3.4e-38 3 20 gi12586064 (AF027183) protein kinase C-related kinase ... +2 244 3.8e-38 2 gil2652950 (Z50028) F46F6.2 tCaenorhabditis elegant] +2 256 4.3e-38 3 giJ1667370 (Y07611) protein kinase [Mug musculus] +2 250 6.0e-38 2 gi|1220289 (Z70043) unknown [Schizosaccharomyces pombe] +2 135 6.4e-38 3 giJ206181 (M18330) protein kinase C delta subspecies ... +2 235 7.1e-38 2 25 giJ167718 (M59744) protein kinase 2 [Dictyostelium di... +2 232 8.7e-38 3 gi|1778590 (U82935) protein kinase C2 A isoform [Caeno... +2 247 1.0e-37 2 gi|1000125 (U33052) PRK2 [Homo sapiens) +2 245 l.le-37 2 giJ914100 (S75548) protein kinase PRK2 [human, DX3 B-... +2 245 1.1e-37 2 gi|200642 (M94335) protein kinase [Mus musculus] +2 224 1.2e-37 3 30 gi12o62375 (U75358) myeloma protein kinase [Rattus nor... +2 245 1.5e-37 2 giJ189510 (M60725) p70 ribosomal S kinase alpha-II [... +2 242 1.7e-37 2 giJ206840 (M57428) S6 kinase [Rattus norvegicus], +2 242 1.7e-37 2 giJl562 (X54415) G3 serine/threonine kinase [Orycto... +2 242 2.0e-37 2 giJ189508 (M60724) p70 ribosomal S6 kinase alpha-I [H... +2 242 2.0e-37 2 35 giJ287807 (X65687) serine/threonine protein kinase M... +2 222 2.3e-37 3 giJ48S403 (D30040) RAC protein kinase alpha [Rattus n... +2 222 2.3e-37 3 giJ3548l (X61037) human protein kinase B [Homo sapiens] +2 222 2.8e-37 3 giJ520587 (D10495) protein kinase C delta-type [Homo ... +2 238 4.le-37 2 giJ3114960 (Y13104) Protein kinase C-related kinase (P... +2 236 4.5e-37 2 40 giJ200381 (M69042) [Mouse protein kinase C delta mRNA. .. +2 235 4.9e-37 2 giJ189985 (L07861) protein kinase C-delta 13 [Homo sa... +2 238 4.9e-37 2 giJ189680 (L07860) protein kinase C-delta 13 Homo sa... +2 238 4.9e-37 2 giJ173427 (L07637) protein kinase C [Schizosaccharomy... +2 252 5.4e-37 2 giJ303941 (D14338) protein kinase [Schizosaccharomyce... +2 252 5.4e-37 2 45 giJl90828 (M63167) rac protein kinase-alpha (Homo sap... +2 222 6.le-37 3 giI485405 (D30041) RAC protein kinase beta [Rattus no... +2 215 8.4e-37 3 giJ4l4286 (U02967) protein kinase C [Lytechinus pictus] +2 246 8.7e-37 2 giJ53437 (X60304) protein kinase [Mus musculus] +2 233 9.2e-37 2 giJ206169 (K03486) protein kinase C type III [Rattus ... +2 237 1.2e-36 2 50 giJ8255l0 (D26180) PK [Rattus norvegicus] +2 246 1.7e-36 2 giJ1778592 (U82936) protein kinase C2 B isoform [Caeno... +2 247 1.8e-36 2 giJ1213263 (Z69795) unknown [Schizosaccharomyces pombe] +2 244 2.2e-36 2 giJ631 (X61036) bovine protein kinase B [Bos taurus] +2 222 2.2e-36 3 giJ303939 (D14337) protein kinase [Schizosaccharomyce... +2 244 2.5e-36 2 55 giJ868172 (U29376) similar to protein kinase C [Caeno... +2 247 2.8e-36 2 giJ158129 (J04848) protein kinase C [Drosophila melan... +2 238 2.9e-36 2 giJl041l83 (D43890) PKN' [Xenopus dp.] +2 248 3.0e-36 2 giJ206842 (M58340) S6 protein kinase [Rattus norvegicus] +2 242 3.5e-36 2 gi8353 (XU5076) protein kinase C (AA 1-639) [Droso... +2 247 4.0e-36 2 60 giJ2822146 (AC002299) Protein kinase C beta (5' partia... +2 237 4.le-36 2 giJ2065190 (Y12237) protein kinase C [Hydra vulgaris] +2 242 4.3e-36 2 WO 00/36135 PCT/GB99/04228 122 giJ163530 (M13973) protein kinase C [Bos taurus] +2 242 4.5e-36 2 giJ1673 (X04796) PKC-gamma (AA 1-672) [Oryctolagus ... +2 242 4.5e-36 2 giJ2297395 (A41791) unnamed protein product [unidentif... +2 242 4.5e-36 2 giJ35483 (X52479) protein kinase C alpha (AA 1-672) ... +2 242 4.5e-36 2 5 gil200363 (M25811) protein kinase C-alpha [Mus musculus] +2 242 4.5e-36 2 gi|49939 (X52685) protein kinase C (Mus musculus] +2 242 4.5e-36 2 gi|56914 (X07286) protein kinase C alpha (AA 1-672) ... +2 242 4.5e-36 2 giJ55132 (X52684) protein kinase C [Mus musculus] +2 242 4.5e-36 2 giJ3114956 (Y13099) Serine/Threonine protein kinase (S... +2 248 4.5e-36 2 10 gif1487920 (Z75953) F57F5.5 [Caenorhabditis elegant] +2 237 5.3e-36 2 giJ1000127 (U33053) PRK1 [Homo sapiens] +2 250 6.1e-36 2 gi|914098 (S75546) protein kinase PRK1 [human, fetal ... +2 250 6.1e-36 2 giJ825505 (D26181) P1M (Homo sapiens] +2 250 6.1e-36 2 giJ1671 (X04795) P1C-beta (AA 1-671) (Oryctolagus c... +2 237 8.5e-36 2 15 gil35489 (X06318) PKC beta 1 (AA 1-671) [Homo sapiens] +2 237 8.5e-36 2 giJ206175 (M19007) protein kinase C beta-i [Rattus no... +2 237 8.5e-36 2 gi156959 (X04439) protein kinase C (AA 1-671) [Rattu... +2 237 8.5e-36 2.. 20 Genbank search results using 396076E03.s2.seq (search 3 and search 4) Blast Results 25 Smallest Sum Reading High Probability (X028) roei knae aph Frame2).. Scr+2N 30 Sequences producing High-scorig Segment Pairs: +2 giJ663254 (Z48149) probable protein kinase [Saccharom... -3 417 8.2e-102 2 giJ1419952 (Z74842) ORF YOL100w [Saccharomyces cerevis... -3 417 8.2e-102 2 gi1927745 (U33050) Ydr49Ocp; CA : 0.11 [Saccharomyces... -3 409 2.e-99 2 giJ1431588 (X99280) protein kinase [Schizosaccharomyce... -3 409 2.e-92 2 35 gi12832892 (AL021730) hypothetical protein kinase phos. .. -3 405 9.9e-81 2 giJ2407613 (AF017995) 3-phosphoinositide dependent pro... -3 280 1.1e-79 3 giJ2505936 (Y15056) PkB kinase 1Homo sapiens] -3 280 1.1e-79 3 giJ2665356 (Y15748) PkB kinase [Rattus norvegicus . -3 278 2.3e-79 3 giJ927730 (U33050) Ydr466wp; CAI: 0.11 [Saccharomyces.. . -3 277 5.3e-74 4 40 giJ2980851 (Y07908) serine/threonine protein kinase [D... -3 307 4.7e-63 2 gi22435556 (AF026207) Similar to protein kinase [Caeno... -3 259 5.6e-49 2 giJloo0o69 (115210) rac-alpha serine/threonine kinase ... -3 197 3.9e-48 3 giJ3043598 (ABO1l1O9) KIAA0537 protein [Homo sapiens] -3 233 4.4e-46 5 giJ1401040 (D49836) RAC-PK gamma [Rattus norvegicus] -3 198 2.2e-45 4 45 giJ35479 (X07767) protein kinase catalytic subunit t... -3 188 6.2e-45 3 giJl9l175 (M63311) cAMP-dependent protein kinase aiph... -3 188 6.2e-45 3 giJ200367 (M12303) cAMP-dependent protein kinase cata... -3 190 1.2e-44 3 giJ3875l3 (M19960) cAMP-dependent protein kinase alph... -3 190 1.2e-44 3 giJ1890142 (D83380) catalytic subunit of cAMP-dependen... -3 185 1.3e-44 3 50 gi12253148 (Z82096) ZK909.2a [Caenorhabditis elegans] -3 185 1.9e-44 3 giJ2299249 (A44422) unnamed protein product [Eimeria in... -3 148 2.3e-44 5 giJ56912 (X57986) cAMP-dependent protein kinase subu... -3 188 2.4e-44 3 gi2288120 (X53261) cAMP-dependent protein kinase cata... -3 184 2.5e-44 3 giI22s3149 (Z82096) ZK909.2b [Caenorhabditis elegans] -3 185 3.4e-44 3 55 giJ633 (X67154) protein kinase [Bos taurus] -3 188 4.8e-44 3 giJ276o82l (L06196) serine/threonine protein kinase [E... -3 200 5.3e-44 3 giJ1673493 (Z81140) WlOG6.2 [Caenorhabditis elegans] -3 269 1.1e-43 2 giligl (X05998) C-beta subunit (338 AA) [Sus scrofa] -3 187 1.4e-43 3 giJ2632252 (Y12464) serine/threonine kinase (Sorghum b... -3 185 1.6e-43 4 60 giJ773642 (U23730) vinclozolin resistance protein [Us... -3 191 2.6e-43 4 WO 00/36135 PCT/G B99/04228 123 gi1200642 (M94335) protein kinase (Mus musculus] -3 132 2.8e-43 5 gi|2213667 (U83459) Riml~p [Saccharomyces cerevisiae] -3 248 2.8e-43 4 giJ836721 (D50617) YFLO33C [Saccharomyces cerevisiae] -3 248 2.8e-43 4 gi|603542 (X83510) RAC protein kinase DRAC-PK66 [Dros... -3 187 3.7e-43 4 5 gil398924 (Z26242) Daktl serine-threonine protein kin... -3 187 3.7e-43 4 giJ166600 (M93023) SNFl-related protein kinase [Arabi... -3 193 4.0e-43 4 giJ1742969 (X94757) ser/thr protein kinase [Arabidopsi... -3 193 4.0e-43 4 gi|1279425 (Z71757) calmodulin-domain protein kinase (... -3 145 4.4e-43 5 gi|178326 (M95936) protein serine/threonine kinase CH... -3 191 6.5e-43 4 10 giJ903942 (U29608) LATS [Drosophila melanogaster] -3 210 7.3e-43 4 gij755008 (L39837) tumor suppressor [Drosophila melan... -3 210 7.3e-43 4 gif1335643 (X83510) RAC protein kinase DRAC-PK85 [Dros... -3 187 9.3e-43 4 gi|337491 (M77198) rac protein kinase-beta (Homo sapi... -3 191 1.2e-42 4 gi|287807 (X65687) serine/threonine protein kinase [M... -3 127 1.4e-42 5 15 giJ485403 (D30040) RAC protein kinase alpha [Rattus n... -3 127 1.4e-42 5 gij496385 (D26602) protein kinase [Nicotiana tabacum] -3 189 1.4e-42 4 giJl743009 (Y10036) SNF1-related protein kinase [Cucum... -3 192 1.8e-42 4 giJ1935916 (U83797) StubSNF1 protein [Solanum tuberosu ] -3 196 7.0e-42 4 giJ191177 (M63312) cAMP-dependent protein kinase beta... -3 184 1.8e-41 3 20 gi1200387 (J02626) cAMP-dependent protein kinase beta... -3 184 1.8e-41 3 gif220704 (D10770) cAMP-dependent protein kinase cata... -3 184 1.8e-41 3 giJ163532 (J02647) protein kinase beta-catalytic subu... -3 183 1.8e-41 3 giJ189983 (M34181) cAMP-dependent protein kinase cata... -3 183 1.8e-41 3 iJ942578 (U22445) serine/threonine kinase [Mus muscu... -3 188 3.0e-41 4 25 gij 210068 (M80675) gag:akt fusion protein [AKT8 retro... -3 127 5.5e-41 5 giJl72l75 (J02665) protein kinase [Saccharomyces cere... -3 186 5.5e-41 3 giJ173009 (M17072) cAMP-dependent protein kinase subu... -3 186 5.5e-41 3 giJl008352 (Z49439) ORF YJL164c [Saccharomyces cerevis... -3 186 5.5e-41 3 gif162787 (M60482) cAMP-dependent protein kinase I-b... -3 183 5.5e-41 3 30 giJ4075l6 (Z2G878) unknown [Saccharomyces cerevisiae] -3 187 5.6e-41 3 giJ48629l (Z28166) ORF YKL66c [Saccharomyces cerevis... -3 187 5.6e-41 3 giJ156233 (M37120) cAMP-dependent protein kinase cata... -3 178 7.4e-41 3 gil173009 (M17073) cAMP-dependent protein kinase subu... -3 188 8.3e-41 3..

Claims

1. A method of identifying a compound which modulates the activity to different extents of (a) a host yeast cell protein kinase or kinases and (b) an 5 protein kinase derivable from a source other than the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases, wherein a compound is exposed to 1) a first host yeast cell wherein the yeast cell is capable of expressing the said host yeast cell protein kinase or kinases and is not capable of expressing the 10 said equivalent protein kinase and 2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said host yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from a source other 15 than the host yeast cell and the effect of the compound on the phenotype of the said yeast cells is measured, wherein either (1) the host yeast cell is a pathogenic yeast and the source other than the host yeast cell is any source other than the host yeast cell 20 or (2) the host yeast cell is any yeast and the source other than the host yeast cell is not a mammal.

2. A method of identifying a compound which modulates the activity to 25 different extents of (a) a protein kinase derivable from a first source and (b) a protein kinase derivable from a second source, both said protein kinases being equivalent to the same host yeast cell protein kinase or kinases, wherein a compound is exposed to WO 00/36135 PCT/GB99/04228 125 1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from the first source and 5 2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from the second source and the effect of the compound on the phenotype of the said yeast cells is measured. 10

3. A method according to claim 1 or 2 wherein the host yeast cell other than the host yeast cell that is a pathogenic yeast is from any one of the genera Saccharomyces, including Saccharomyces cerevisiae, Candida, including Candida albicans, Pichia, Kluyveromyces, Torulopsis, Hansenula, 15 Schizosaccharomyces, Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, Aspergillus, including Aspergillus fumigatus, Cryptococcus, including Cryptococcus neoformans, and Histoplasma, including Histoplasma capsulatum. 20

4. A method according to any one of claims 1 to 3 wherein a yeast host cell which is not capable of expressing the said host yeast cell protein kinase or kinases is substantially not capable of growing unless the said yeast host cell is capable of expressing the said protein kinase derivable from a source other than 25 the said host yeast cell that is equivalent to the said host yeast cell protein kinase or kinases.

5. A method according to any one of claims 1 to 4 wherein at least one protein kinase from a source other than the host yeast cell is a human protein kinase. WO 00/36135 PCT/GB99/04228 126

6. A method according to any one of claims 1 to 5 wherein the said host yeast cell protein kinase or kinases is Pkh1 and/or Pkh2, wherein Pkh1 is the polypeptide encoded by open reading frame YDR490c of S. cerevisiae or 5 equivalent open reading frame in yeast other than S. cerevisiae and Pkh2 is the polypeptide encoded by open reading frame YOL100w of S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae.

7. A method according to any one of claims 1 to 6 wherein the said protein 10 kinase equivalent to the said host yeast cell protein kinase or kinases is PDK1.

8. A method according to any one of claims 1 to 5 wherein the said host yeast cell protein kinase or kinases is Ypk1 and/or Yrk2. 15 9. A method according to any one of claims 1 to 5 and 8 wherein the said protein kinase equivalent to the said host yeast cell protein kinase or kinases is serum and glucocorticoid induced protein kinase (SGK) or protein kinase B (PKB). 20 10. The method of any one of claims 2 to 9 wherein the first source is a human and the second source is a pathogenic yeast from any one of the genera Candida spp, Blastomyces spp, for example B. dermatitidis, Coccidioides spp, for example C. immitis, Histoplasma spp, for example H. capsulatum, Sporothrix spp, for example S. schenckii, Aspergillus spp, for example A. 25 fumigatus, A. flavus, A. niger, Phialophora compacta (Fonsecaea compacta), P. pedrosoi (F. pedrosi), P. verrucosa, Cladosporium carrionii, Rhinocladiella aquaspersa, Cryptococcus spp, for example C. neoformans, Cephalosporium spp, Fusarium spp, Histoplasma spp, for example H. capsulatum, Pneumocystis carinii, Rhizopus spp, Rhizomucor spp, Madurella spp, for WO 00/36135 PCT/GB99/04228 127 example M. mycetomatis, M. grisea, Pseudallescheria boydii, Paracoccidioides spp, for example P. brasiliensis, Prototheca spp, for example P. wickerhamii, Epidermophyton spp, Microsporum spp, Trichophyton spp, Malassezia spp, for example M. furfur (Pityrosporum orbiculare) 5

11. A method of identifying a compound that modulates (inhibits) the activity of PDK1 derivable from a first source, wherein a compound is exposed to 1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing a yeast polypeptide that is a functional equivalent of human PDK1 (Pkh1 and 10 Pkh2) and (b) is capable of expressing PDK1 derivable from the said first source and optionally 2) a second host yeast cell wherein the yeast cell is capable of expressing a yeast polypeptide that is a functional equivalent of human PDK1 (Pkhl and/or 15 Pkh2) and the effect of the compound on the viability of the said yeast cell or cells is measured, and a compound that affects the viability of the first said yeast cell, or optionally that affects the viability of the first said yeast cell and the said second yeast cell differently, is identified. 20

12. A method of identifying a compound that modulates (inhibits) the activity of a functional equivalent of Ypkl and/or Ykr2 derivable from a first source, wherein a compound is exposed to 1) a first host yeast cell wherein the yeast cell is (a) not capable of expressing a 25 yeast polypeptide that is a functional equivalent of Ypk1 and/or Yrk2 and (b) is capable of expressing a functional equivalent of Ypk1 and/or Ykr2 (for example SGK) derivable from the said first source and optionally WO 00/36135 PCT/GB99/04228 128 2) a second host yeast cell wherein the yeast cell is capable of expressing a yeast polypeptide (for example, an endogenous polypeptide) that is a functional equivalent of Ypkland/or Yrk2 and the effect of the compound on the viability of the said yeast cell or cells is 5 measured, and a compound that affects the viability of the first said yeast cell, or optionally that affects the viability of the first said yeast cell and the said second yeast cell differently, is identified.

13. A yeast cell that is not capable of expressing Pkh1 and Pkh2 or any 10 functional equivalent thereof.

14. A yeast cell that is not capable of expressing endogenous Pkhl and/or Pkh2. 15 15. A yeast cell according to claim 14 that is capable of expressing a functional equivalent of Pkhl and/or Pkh2 that is not endogenous Pkhl or Pkh2.

16. The yeast cell of claim 15 wherein the said functional equivalent is 20 human PDK1 or a variant, fusion or derivative thereof.

17. A yeast cell according to any one of claims 13 to 15 wherein the open reading frame encoding Pkh1 or Pkh2 is disrupted by insertion of a selectable marker. 25

18. A yeast cell wherein one or more genes encoding a functional equivalent of human PDK1 is mutated such that the yeast cell is not capable of expressing the said functional equivalent of human PDK1. WO 00/36135 PCT/GB99/04228 129

19. A yeast cell according to claim 18 wherein each such gene encoding a functional equivalent of human PDK1 is mutated such that the yeast cell is not capable of expressing a functional equivalent of human PDK1. 5 20. A method of identifying a compound that modulates (inhibits) the activity of PDK1 wherein a yeast cell according to any one of claims 13 to 19 is used.

21. Use of a yeast cell according to any one of claims 13 to 19 in a method 10 of identifying a compound that modulates (inhibits) the activity of PDK1.

22. The method of claim 20 or use of claim 21 wherein the PDK1 is mammalian PDK1. 15 23. The method of claim 20 or use of claim 21 wherein the PDK1 is a yeast PDK1, for example Candida PDK1.

24. A protein kinase derivable from yeast capable of phosphorylating a polypeptide comprising the consensus sequence Arg-Xaa-Arg-Xaa-Xaa 20 (Ser/Thr)-Hyd.

25. A protein kinase derivable from yeast capable of being phosphorylated by Pkhl or Pkh2 or PDKl. 25 26. A protein kinase according to claim 24 or 25 wherein the said protein kinase is Ypk1 from S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae, for example Candida spp or Ykr2 from S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae, for example Candida spp. WO 00/36135 PCT/GB99/04228 130

27. A variant, derivative, fragment or fusion or a fusion of a variant, derivative or fragment of a protein kinase as defined in claim 26 that is capable of being phosphorylated by Pkh1 or Pkh2 or mammalian, preferably human, 5 PDK1 and/or capable of phosphorylating a polypeptide comprising the consensus sequence Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr-Hyd.

28. A yeast, for example S. cerevisiae or Candida, cell wherein one or more endogenous genes encoding a functional equivalent of human SGK is mutated 10 such that the yeast cell is not capable of expressing the said functional equivalent of human SGK.

29. A yeast cell according to claim 28 wherein the said gene is Ypk1 or Yrk2. 15

30. A yeast cell according to claim 27 or 29 wherein each such endogenous gene encoding a functional equivalent of human SGK or Ypk1 or Yrk2 is mutated such that the yeast cell is not capable of expressing an endogenous functional equivalent of, for example, human SGK or Ypk1 or Yrk2. 20

32. A method of identifying a compound which blocks the activation of a polypeptide that is a functional equivalent of Ypk1 and/or Ykr2 and is not SGK, PKBca or p70S6 kinase by an interacting polypeptide, for example Pkhl, 25 Pkh2 or PDK1, the method comprising determining whether a compound enhances or disrupts the interaction between (a) a polypeptide that is a functional equivalent of Ypk1 and/or Ykr2 that is not SGK, PKBa or p70S6 kinase or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative and (b) the interacting polypeptide, WO 00/36135 PCT/GB99/04228 131 or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or determining whether the compound substantially blocks activation of the said polypeptide that is a functional equivalent of Ypkl and/or Ykr2 or a suitable variant, fragment, derivative or 5 fusion thereof, or a fusion of a said fragment, derivative or fusion by the interacting polypeptide, or a suitable variant, derivative, fragment or fusion thereof.

33. The use of Pkhl or Pkh2 or a suitable variant, fragment, derivative or 10 fusion thereof, or a fusion of a said fragment, derivative or fusion thereof that is not PDK1 to phosphorylate and/or activate a polypeptide that is Ypk1 and/or Ykr2 or SGK or PKBca or a functional equivalent thereof or suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion. 15

34. The use of PDK1 or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion thereof to phosphorylate and/or activate a polypeptide that is Ypk1 and/or Ykr2 or SGK or a functional equivalent thereof or suitable variant, fragment, derivative or 20 fusion thereof, or a fusion of a said fragment, derivative or fusion that is not PKBa or p70S6 kinase.

35. A kit of parts comprising means useful for carrying out the method as defined in any one of Claims 1 to 10. 25

36. A kit of parts according to claim 35 comprising a first host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from a first source WO 00/36135 PCT/GB99/04228 132 and 2) a second host yeast cell wherein the yeast cell is (a) not capable of expressing the said yeast cell protein kinase or kinases and (b) is capable of expressing the said equivalent protein kinase derivable from an source other 5 than the first source.

37. Any novel protein kinase as herein disclosed.

38. A compound identifiable by the method of any one of claims 1 to 12, 20, 10 22, 23 or 32.

39. A compound according to claim 38 that is capable of inhibiting mammalian PDK1 or SGK. 15 40. A compound according to claim 38 that is capable of inhibiting a fungal functional equivalent of PDK1 (which may be Pkhl or Pkh2) or SGK (which may be Ypkl or Yrk2).

41. A compound according to any one of claims 38 to 40 for use in medicine. 20

42. Use of a compound according to claim 40 in the manufacture of a medicament for the treatment of a fungal, for example a Candida infection, for example thrush. 25 43. Use of a compound according to claim 39 in the manufacture of a medicament for the treatment of cancer.

44. A substantially pure polypeptide encoded by open reading frame YDR490c of S. cerevisiae or equivalent open reading frame in yeast other than WO 00/36135 PCT/GB99/04228 133 S. cerevisiae or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative or a substantially pure polypeptide encoded by open reading frame YOL100w of 5 S. cerevisiae or equivalent open reading frame in yeast other than S. cerevisiae. or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative wherein the polypeptide does not comprise the amino acid sequence of human PDK1 or Drosophila PDK1 (DSTPK61). 10

45. A recombinant polynucleotide suitable for expressing a polypeptide as defined in claim 44.

46. A host cell comprising a recombinant polynucleotide as defined in claim 15 45.

47. A method of making a polypeptide, or a variant, fragment, derivative or fusion thereof or fusion of a said variant or fragment or derivative the method comprising culturing a host cell as defined in Claim 46 which expresses said 20 polypeptide, or a variant, fragment, derivative or fusion thereof or fusion of a said variant or fragment or derivative and isolating said polypeptide or a variant, fragment, derivative or fusion thereof or fusion of a said variant, or fragment or derivative. 25 48. A polypeptide, or a variant, fragment, derivative or fusion thereof or fusion of a said variant or fragment or derivative obtainable by the method of Claim 47.

49. An antibody reactive towards a polypeptide as defined in claim 44 or 48.