WO1999009181A1 - Human stress-activated protein kinase, skk4 - Google Patents

Abstract

A substantially pure stress-activated protein kinase comprising the amino acid sequence (A) or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative. The stress-activated protein kinase is useful in screening assays for drugs.

Description

HUMAN STRESS-ACTIVATED PROTEIN KINASE, SKK4

The present invention relates to polypeptides, polynucleotides and uses thereof, in particular to members of the stress-activated protein kinase kinase (SKK) family.

Seven mitogen-activated protein (MAP) kinase family members are activated by cellular stresses (chemical, heat and osmotic shock, ultraviolet radiation, inhibitors of protein synthesis), bacterial lipopolysaccharide (LPS), and the cytokines interleukin-1 (ILl) and tumour necrosis factor (TNF), and have therefore been termed stress- activated protein kinases or SAPKs (reviewed in Cohen, 1997; also Goedert et al (1997) EMBO J 16, 3563-3571; Kumar et al (1997) Biochem. Biophys. Res. Comm. 235, 533-538). The three isoforms of SAPK1 [also called c-Jun N-terminal kinases (JNKs)] phosphorylate Ser- 63 and Ser-73 in the activation domain of c-Jun (Pulverer et al, 1991), thereby increasing its transcriptional activity. The same sites in c-Jun also become phosphorylated when cells are exposed to the stresses and cytokines that activate SAPK1 (Pulverer et al, 1991; Hibi et al, 1993; Derijard et al, 1994; Kyriakis et al, 1994), suggesting that c-Jun is a physiological substrate for SAPK1.

SAPK2a [also termed ρ38 (Han et al, 1994), p40 (Freshney et al, 1994), RK (Rouse et al, 1994), CSBP (Lee et al, 1994) and Mxi2 (Zervos et al, 1995)] is inhibited very specifically by the pyridinyl imidazoles SB 203580 and SB 202190 (Lee et al, 1994; Cuenda et al, 1995; reviewed in Cohen, 1997) which have been exploited to identify several physiological substrates. These include four protein kinases, namely MAP kinase- activated protein kinase-2 (MAPKAP-K2, Rouse et al, 1994) and the closely related MAPKAP-K3 (McLaughlin et al, 1996; Ludwig et al (1996); Clifton et al, 1996), as well as MAP kinase interacting protein kinases- 1 and -2 (Mnkl and Mnk2) (Waskiewicz et al, 1997; Fukunaga and Hunter, 1997).

Physiological substrates of MAPKAP-K2/K3 include heat shock protein (HSP) 27 (Cuenda et al, 1995; Huot et al, 1995) and the transcription factor CREB (Tan et al, 1996), whereas transcription factor eIF4E is a physiological substrate of Mnkl/2 (Waskiewicz et al, 1997). SAPK2a also mediates the stress-induced phosphorylation and activation of the CEBPβ- related transcription factor CHOP (Wang and Ron, 1996) and the ternary complex factor Elk-1 (Price et al, 1996).

Based on the effects of SB 203580, the activation of SAPK2a is rate- limiting in the LPS-induced production of ILl and TNF in monocytes (Lee et al, 1994), in the TNF-stimulated transcription of IL6 and GM-CSF in fibroblasts (Beyaert et al, 1996), in the ILl-induced stimulation of glucose uptake in epithelial cells (Gould et al, 1995), in collagen-induced platelet aggregation (Saklatvala et al, 1996) and in the stress-induced transcription of c-Jun and c-Fos in fibroblasts (Hazzalin et al, 1996; Price et al, 1996).

The SAPK2a catalysed phosphorylation of Elk-1 (Price et al, 1996) and the MAPKAP-K2 catalysed phosphorylation of CREB (Tan et al, 1996) are both likely to contribute to the stress-induced transcription of c-fos (Ginty et al, 1994).

Recently, two additional SAP kinases were identified, called SAPK2b [or p38β (Jiang et al, 1996)] and SAPK3 (Mertens et al, 1996) [also called ERK6 (Lechner et al, 1996) and p38γ (Li et al, 1996)]. The amino acid sequence of S APK2b is 73 % identical to S APK2a and it is inhibited by SB 202190 and SB 203580 at similar concentrations to SAPK2a. In contrast, the amino acid sequence of SAPK3 is only 60% identical to SAPK2a and SAPK3 is not inhibited by SB 203580 (Cuenda et al, 1997). SAPK2b and SAPK3 have been introduced into mammalian cells by transient transfection and shown to be activated in response to pro-inflammatory cytokines and stressful stimuli in a manner similar to SAPKl and SAPK2a. The physiological roles of SAPK2b and SAPK3 are unknown. The mRNAs encoding these enzymes are present in all mammalian tissues examined (Jiang et al, 1996; Mertens et al, 1996; Goedert et al., 1997), with the mRNA encoding SAPK3 being highest in skeletal muscle. Expression of wild-type SAPK3 and an inactive mutant in the muscle cell line C2C12 enhanced and inhibited differentiation into myotubes, respectively (Lechner et al, 1996). In vitro, SAPK2b and SAPK3 phosphorylated several proteins that are also substrates for SAPK2a. SAPK2b was reported to phosphorylate die transcription factor ATF2 more efficiently than SAPK2a (Jiang et al., 1996) but, since the stress- and cytokine- induced phosphorylation of ATF2 in fibroblasts is unaffected by SB 203580 (Hazzalin et al, 1996; Beyaert et al., 1996), neither SAPK2a nor SAPK2b appears to be rate-limiting for ATF2 phosphorylation in vivo, in contrast to earlier studies using transfection- based approaches (Gupta et al, 1995). The substrate specificity of SAPK3 in vitro was similar to mat of SAPK2a, except mat it was much less effective in activating MAPKAP-K2/K3 and (like SAPKl, but unlike SAPK2a) phosphorylated ATF2 at Ser90, as well as at Thr69 and Thr71 (Cuenda et al, 1997). However, whether SAPKl and/or SAPK3 are rate- limiting for ATF2 phosphorylation in vivo is unknown. SAPK4 is the seventh MAP kinase family member. This enzyme, which also contains a TGY sequence in the activation domain, shows about 60% identity to SAPK2a, SAPK2b and SAPK3 and its mRNA is widely expressed in human tissues. SAPK4 is activated by me same stimuli that activate other SAP kinases, has an in vitro substrate specificity similar to that of SAPK3 and, like SAPK3, is not inhibited by SB 203480 or SB 202190.

Five chromatographically distinct SAP kinase kinases (SAPKKs or SKKs) have been identified in mammalian cells (Meier et al, 1996; Cuenda et al, 1996). In vitro, SKK1 [also termed MKK4 (Derijard et al, 1995), SEK1 (Sanchez et al, 1994) and XMEK2 (Yashar et al, 1993)] activates all four SAPKs (Sanchez et al, 1994; Derijard et al, 1995; Doza et al, 1995; Jiang et al, 1996; Cuenda et al, 1997), although SAPK2b and SAPK3 are phosphorylated less efficiently. SKK2 [also termed MKK3 (Derijard et al, 1995)] and SKK3 (Cuenda et al, 1996) [also called MKK6 (Han et al, 1996; Moriguchi et al, 1996; Raingeaud et al, 1996) and MEK6 (Stein et al, 1996)] activate SAPK2a but not SAPKl, while SKK3 was the only detectable activator of SAPK3 induced by pro-inflammatory cytokines and stressful stimuli in human epithelial KB cells or human embryonic kidney 293 cells (Cuenda et al, 1997). SKK3 was also the most efficient activator of SAPK2b in co-transfection experiments (Jiang et al, 1996), and was the only detectable activator of SAPK4 induced by pro- inflammatory cytokines and stressful stimuli in KB cells [Goedert et al (1997) EMBO J 16, 3563-3571]. SKK4 and SKK5 activate SAPKl but not SAPK2a (Meier et al, 1996) or SAPK3 (Cuenda et al, 1997). The cloning of a mouse MAP kinase kinase family member termed MKK7 has recendy been reported in Tournier et al (1997) PNAS 94, 7337-7342.

The importance of different SKKs in activating SAPKl was not previously resolved. SKK1/MKK4 is the only activator of SAPK1/JNK that can be detected biochemically in extracts prepared from PC 12 cells that have been exposed to adverse stimuli (Meier et al (1996)), and in 293 cells the activation of transfected SAPKl /JNK by anisomycin can be prevented by overexpressing a catalytically inactive form of SKK1/MKK4 (Sanchez et al (1994) Nature 372, 794-798). In addition, heat shock or anisomycin failed to activate SAPKl /JNK in murine embryonic stem cells lacking

SKK1/MKK4 (Nishina et al (1997) Nature 385, 350-353, Yang et al

(1997) PNAS 94, 3004-3009). SAPKl /JNK is activated strongly by cotransfection with MEK kinase (MEKK) (Yan et al (1994) Nature 372, 798-800). MEK Kinase is an upstream activator of SKK1 mat is activated by osmotic shock in rat 3Y1 fibroblasts and PC 12 cells (Yan et al (1994)

Nature 372, 798-800; Matsuda et al (1995) J. Biol. Chem. 270, 12781-

12786)).

Altiiough SKK1/MKK4 is clearly essential for the activation of SAPK1/JNK in some cells, the dominant activator of SAPK1/JNK detected biochemically in human epithelial KB cells (Meier et al (1996) or in Rat 3Y1 fibroblasts (Moriguchi et al (1995) is a chromatographically distinct enzyme(s). In Meier et al, (1996), SKK4 was partially resolved from SKK3 by elution from Mono S using a sodium chloride gradient, but purification of SKK4 was not achieved. Moriguchi et al, (1995) identified multiple SAP kinase kinases using a method employing Q-sepharose and heparin-sepharose chromatography. This enabled the presence of a kinase that may correspond to SKK4 as identified in Meier et al, (1996) to be detected, but purification of the kinase was not reported.

Moreover, the activation of SAPKl/JNK by UV radiation and osmotic shock in murine embryonic stem cells lacking SKK1/MKK4 was either uriimpaired (Nishina et al (1997)) or only inhibited partially (Yang et al (1997)). These observations indicate that mammalian cells contain at least one further activator of SAPKl/JNK distinct from SKK1/MKK4.

We here report the cloning of a novel SAP kinase kinase family member that we call SKK4. The mRNA is widely expressed in human tissues. The amino acid sequence of this enzyme is most similar (62.4% identity) to a fruit fly (Drosophila) MKK homologue (HEP) which geneticists have shown to lie upstream of JNK in a pathway essential for embryonic development of the fruit fly. It is also related to the human SKK1, SKK2 and SKK3 enzymes, wim 47.7%, 41.4% and 39.3% amino acid sequence identity respectively. It has 36% amino acid sequence identity with MKK1, and 35% with MKK2.

We report that SKK4 activates SAPKl/JNK in vitro, but not SAPK2a/p38, SAPK3/ERK6 or SAPK4. In human epithelial KB cells SKK4 and SKK1/MKK4 (another activator of SAPKl/JNK) are both activated by stressful stimuli, but only SKK4 is activated strongly by promflammatory cytokines. Recombinant SKK4 can be activated by incubation with MgATP and MEKK. The identification of SKK4 explains why the major SAPKl/JNK activator detected in many mammalian cell extracts is chromatographically separable from SKK1/MKK4. SKK4 may be a more attractive target for an anti-inflammatory drug than SAPKl/JNK because inhibitors of SKK4 may prevent the activation of SAPKl/JNK by proinflammatory cytokines, without affecting the activation of SAPKl/JNK by other agents.

A first aspect of the invention provides a substantially pure polypeptide comprising the amino acid sequence

M E S I E I D Q K L Q E I M K Q T G Y L

T I G G Q R Y Q A E I N D L E N L G E M

G S G T c G Q V W K M R F R K T G H V I

A V K Q M R R S G N K E E N K R I L M D

L D V V L K S H D C P Y I V Q C F G T F

I T N T D V F I A M E L M G T C A E K L

K K R M Q G P I P E R I L G K M T V A I

V K A L Y Y L K E K H G V I H R D V K P s N I L L D E R G Q I K L C D F G I S G

R L V D S K A K T R s A G C A A Y M A P

E R I D P P D P T K P D Y D I R A D V w

S L G I s L V E L A T G Q F P Y K N C K

T D F E V L T K V L Q E E P P L L P G H

M G F S G D F Q S F V K D c L T K D H R

K R P K Y N K L E H S F I K R Y E T L

E V D V A S W F K D V M A K T E S P R T

S G V L S Q P H L P F F R, or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative. This polypeptide is considered to be a stress-activated protein kinase kinase.

The polypeptide with the amino acid sequence as shown above is herein referred to as SKK4 (stress-activated protein kinase kinase 4). 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.

Thus, the invention also includes compositions comprising the said polypeptide and a contaminant wherein the contanrinant comprises less than 70% of the composition by weight, preferably less than 50% of the composition, more 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 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.

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.

By "fragment of said polypeptide" we include any fragment which retains activity or which is useful in some other way, for example, for use in raising antibodies or in a binding assay. 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. Similarly, the said polypeptide may be fused to an oligo-histidine tag 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.

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. Variants of SKK4 do not include polypeptides which have d e amino acid sequence of human SKK1 , SKK2, SKK3 or D. melanogaster HEP.

By "conservative substitutions" is intended combinations such as Gly, Ala; Val, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.

It is particularly preferred if the polypeptide variant has an amino acid sequence which has at least 98.8% identity with the amino acid sequence given above, more preferably at least 99.1 % , still more preferably at least 99.4% , yet more preferably at least 99.7% , and most preferably at least 99.9% 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 Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.

A particular embodiment of the invention provides a substantially pure human SKK4 polypeptide which consists of the amino acid sequence

M E S I E I D Q K L Q E I M K Q T G Y L T I G G Q R Y Q A E I N D L E N G E M G S G T C G Q V W K M R F R K T G H V I A V K Q M R R S G N K E E N K R I M D L D V V K S H D C P Y I V Q C F G T F

I T N T D V F I A M E L M G T C A E K L K K R M Q G P I P E R I G K M T V A I V K A Y Y L K E K H G V I H R D V K P S N I L D E R G Q I K L C D F G I S G R L V D S K A K T R S A G C A A Y M A P

E R I D P P D P T K P D Y D I R A D V S G I S L V E A T G Q F P Y K N C K T D F E V L T K V L Q E E P P L L P G H M G F S G D F Q S F V K D C L T K D H R K R P K Y N K L L E H S F I K R Y E T L

E V D V A S W F K D V M A K T E S P R T S G V L S Q P H P F F R, or naturally occurring allelic variants thereof. The amino acid sequence is also shown as me translation of a polynucleotide sequence in Figure 1.

It is particularly preferred, although not essential, tiiat 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 SKK4 with respect to the phosphorylation of SAPKl. It is more preferred if the variant or fragment or derivative or fusion of die 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 SKK4 with respect to the phosphorylation of SAPKl. 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.

A second aspect of the invention provides a recombinant polynucleotide encoding a polypeptide as defined in the first aspect of tihe invention 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 in the first aspect of the invention, except that the following Expressed Sequence Tags (ESTs) are also excluded:

AA256025, AA019720, AA252650, H85962, AA194205, W58120, (all human)

AA451434, AA194047 (mouse)

In one preferred embodiment me polynucleotide comprises me nucleotide sequence ATGGAGAGCATTGAGATTGACCAGAAGCTGCAGGAGATCATGAAGCAGACGGGCTACC

TGACCATCGGGGGCCAGCGCTACCAGGCAGAAATCAACGACCTGGAGAACTTGGGCGA GATGGGCAGCGGCACCTGCGGCCAGGTGTGGAAGATGCGCTTCCGGAAGACCGGCCAC GTCATTGCCGTTAAGCAAATGCGGCGCTCCGGGAACAAGGAGGAGAACAAGCGCATCC TCATGGACCTGGATGTGGTGCTGAAGAGCCACGACTGCCCCTACATCGTGCAGTGCTT TGGGACGTTCATCACCAACACGGACGTCTTCATCGCCATGGAGCTCATGGGCACCTGC

GCTGAGAAGCTCAAGAAGCGGATGCAGGGCCCCATCCCCGAGCGCATTCTGGGCAAGA TGACAGTGGCGATTGTGAAGGCGCTGTACTACCTGAAGGAGAAGCACGGTGTCATCCA CCGCGACGTCAAGCCCTCCAACATCCTGCTGGACGAGCGGGGCCAGATCAAGCTCTGC GACTTCGGCATCAGCGGCCGCCTGGTGGACTCCAAAGCCAAGACGCGGAGCGCCGGCT GTGCCGCCTACATGGCACCCGAGCGCATTGACCCCCCAGACCCCACCAAGCCGGACTA

TGACATCCGGGCCGACGTATGGAGCCTGGGCATCTCGTTGGTGGAGCTGGCAACAGGA CAGTTTCCCTACAAGAACTGCAAGACGGACTTTGAGGTCCTCACCAAAGTCCTACAGG AAGAGCCCCCGCTTCTGCCCGGACACATGGGCTTCTCGGGGGACTTCCAGTCCTTCGT CAAAGACTGCCTTACTAAAGATCACAGGAAGAGACCAAAGTATAATAAGCTACTTGAA CACAGCTTCATCAAGCGCTACGAGACGCTGGAGGTGGACGTGGCGTCCTGGTTCAAGG ATGTCATGGCGAAGACTGAGTCACCGCGGACTAGCGGCGTCCTGAGCCAGCCCCACCT

GCCCTTCTTCAGG,

or a variant, fragment, fusion or derivative mereof. The nucleotide sequence is shown in Figure 1 together wim the translation of the relevant open reading frame.

The invention also includes a polynucleotide comprising a fragment of the recombinant polynucleotide of me second aspect of me invention. Preferably, the polynucleotide comprises a fragment which is at least 10 nucleotides in lengm, 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.

The polynucleotide or recombinant polynucleotide may be DNA or RNA, preferably DNA. The polynucleotide may or may not contain nitrons in 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 me protein encoded by the said polynucleotide or (ii) an antisense sequence corresponding to me 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 i munogenicity of the protein or which may improve or otherwise modulate its 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 polynucleotides can be obtained by the application of known techniques to the teachings contained herein, such modified polynucleotides are witiiin me scope of the claimed invention.

Moreover, it will be recognised by tiiose skilled in the art that the polynucleotide sequence (or fragments tiiereof) of me invention can be used to obtain o er polynucleotide sequences tiiat hybridise with it under conditions of high stringency. Such polynucleotides includes any genomic DNA. Accordingly, me polynucleotide of me invention includes polynucleotide mat shows at least 89.5% , preferably 92%, and more preferably at least 95% and most preferably at least 99% homology with the polynucleotide identified in d e method of me 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.

Per cent homology can be determined by, for example, me GAP program of the University of Wisconsin Genetic Computer Group.

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 me art that the higher the temperature or the lower the SSC concentration the more stringent the hybridisation conditions. By "high stringency" we mean 2XSSC and 65°C. 1XSSC is 0.15M NaCl/0.015M sodium citrate. Polynucleotides which hybridise at high stringency are included within the scope of the claimed invention.

"Variations" of d e polynucleotide also include polynucleotide in which relatively short stretches (for example 20 to 50 nucleotides) have a high degree of homology (at least 95% and preferably at least 99 or 99.9%) witii equivalent stretches of die polynucleotide of me invention even tiiough me overall homology between me two polynucleotides may be much less. This is because important active or binding sites may be shared even when me general architecture of the protein is different.

A furtiier aspect of the invention provides a replicable vector comprising a recombinant polynucleotide encoding a said polypeptide or a variant, fragment, derivative or fusion of said polypeptide or a fusion of said variant, fragment or derivative.

A variety of methods have been developed to operably link polynucleotides, especially DNA, to vectors for example via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to me DNA segment to be inserted to me vector DNA. The vector and DNA segment are men joined by hydrogen bonding between me complementary homopolymeric tails to form recombinant DNA molecules. Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3' -ends with their polymerizing activities.

The combination of these activities tiierefore generates blunt-ended DNA segments. The blunt-ended segments are men incubated wim a large molar excess of linker molecules in me presence of an enzyme mat is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, me products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved wim e appropriate restriction enzyme and ligated to an expression vector mat has been cleaved wim an enzyme that produces termini compatible witii those of the DNA segment.

Synmetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.

A desirable way to modify me DNA encoding a polypeptide of die invention is to use me polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491. This method may be used for introducing me DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in me art. In mis method me DNA to be enzymatically amplified is flanked by two specific primers which themselves become incorporated into me amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in me art.

The DNA (or in the case of retroviral vectors, RNA) is then expressed in a suitable host to produce a polypeptide comprising me compound of me invention. Thus, me DNA encoding me polypeptide consti ting me compound of me invention may be used in accordance wim known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is men used to transform an appropriate host cell for the expression and production of the polypeptide of me invention. Such techniques include tiiose disclosed in US Patent Nos. 4,440,859 issued 3 April 1984 to Rutter et al, 4,530,901 issued 23 July 1985 to Weissman, 4,582,800 issued 15 April 1986 to Crowl, 4,677,063 issued 30 June 1987 to Mark et al, 4,678,751 issued 7 July 1987 to Goeddel, 4,704,362 issued 3 November 1987 to Itakura et al, 4,710,463 issued 1 December 1987 to Murray, 4,757,006 issued 12 July 1988 to Toole, Jr. et al, 4,766,075 issued 23 August 1988 to Goeddel et al and 4,810,648 issued 7 March 1989 to Stalker, all of which are incorporated herein by reference.

The DNA (or in me case of retroviral vectors, RNA) encoding me polypeptide constituting me compound of me invention may be joined to a wide variety of omer DNA sequences for introduction into an appropriate host. The companion DNA will depend upon me nature of me host, me manner of the introduction of me DNA into die host, and whetiier episomal maintenance or integration is desired.

Generally, me DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to me appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, altiiough such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of die hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co- transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to tiiose skilled in die art in view of die teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.

Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells. The vectors include a prokaryotic replicon, such as the ColEl ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed tiierewith.

A promoter is an expression control element formed by a DNA sequence tiiat permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.

Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and p7/r99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.

A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, d e highest level of expression being found in T antigen-producing cells, such as COS-1 cells.

An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses tiie glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of d e cloned gene. Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).

The present invention also relates to a host cell transformed with a polynucleotide 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, 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 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.

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 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, 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.

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 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 mixmre suspended in 2.5X PEB using 6250V per cm at 25μFD.

Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.

Successfully transformed cells, ie cells mat contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from die 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 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.

In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known irnmunological methods when d e recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed witii an expression vector produce 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 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 variant, fragment or derivative me 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 known in the art.

The invention also includes a polypeptide, or a variant, fragment, derivative or fusion tiiereof, or fusion of a said variant or fragment or derivative obtainable by the above method of the invention.

A still further aspect of the invention provides an antibody reactive towards a polypeptide of the invention. Examples of such antibodies are given in Example 1.

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.

Suitable monoclonal antibodies which are reactive towards die 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).

In a preferred embodiment die antibody is raised using any suitable peptide sequence obtainable from the given amino acid sequence of SKK4. It is preferred if polyclonal antipeptide antibodies are made.

It is particularly preferred if the antibody does not react substantially with another stress-activated protein kinase kinase such as SKK1, SKK2 or SKK3. Accordingly, it may be preferred if peptides based on die SKK4 sequence are used which vary significantly from any peptides found in any other stress-activated protein kinases such as SKK1, SKK2 or SKK3.

Peptides in which one or more of the amino acid residues are chemically modified, before or after the peptide is synthesised, may be used providing that the function of the peptide, namely the production of specific antibodies in vivo, remains substantially unchanged. Such modifications include forming salts with acids or bases, especially physiologically acceptable organic or inorganic acids and bases, forming an ester or amide of a terminal carboxyl group, and attaching amino acid protecting groups such as N-t-butoxycarbonyl. Such modifications may protect the peptide from in vivo metabolism. The peptides may be present as single copies or as multiples, for example tandem repeats. Such tandem or multiple repeats may be sufficiently antigenic themselves to obviate the use of a carrier. It may be advantageous for the peptide to be formed as a loop, witii the N-terrninal and C-terminal ends joined togetiier, or to add one or more Cys residues to an end to increase antigenicity and/or to allow disulphide bonds to be formed. If the peptide is covalentiy linked to a carrier, preferably a polypeptide, tiien the arrangement is preferably such that the peptide of the invention forms a loop.

According to current immunological theories, a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system. It is thought that the best carriers embody (or, together with the antigen, create) a T-cell epitope. The peptides may be associated, for example by cross-linking, with a separate carrier, such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin. More recently developed carriers which induce T-cell help in the immune response include the hepatitis-B core antigen (also called the nucleocapsid protein), presumed T-cell epitopes such as Thr-Ala-Ser-Gly-Val-Ala-Glu-Thr-Thr-Asn-Cys, beta- galactosidase and the 163-171 peptide of interleukin-1. The latter compound may variously be regarded as a carrier or as an adjuvant or as both. Alternatively, several copies of the same or different peptides of the invention may be cross-linked to one another; in this situation there is no separate carrier as such, but a carrier function may be provided by such cross-linking. Suitable cross-linking agents include those listed as such in the Sigma and Pierce catalogues, for example glutaraldehyde, carbodiimide and succinimidyl 4-(N-maleimidomethyl)cyclohexane-l- carboxylate, the latter agent exploiting the -SH group on the C-terminal cysteine residue (if present).

If the peptide is prepared by expression of a suitable nucleotide sequence in a suitable host, then it may be advantageous to express die peptide as a fusion product with a peptide sequence which acts as a carrier. Kabigen's "Ecosec" system is an example of such an arrangement.

The peptide of the invention may be linked to other antigens to provide a dual effect.

Peptides may be synthesised by the Fmoc-polyamide mode of solid-phase peptide syntiiesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of die 4,4'-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent). The peptide-to-resin cleavable linked agent used is die acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N,N-dicyclohexyl-carbodiimide/l- hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon completion of syntiuesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used are ethanedithiol, phenol, anisole and water, the exact choice depending on me constituent amino acids of the peptide being synthesised. Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with dietiiyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilisation of die aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.

A further aspect of the invention provides a method of identifying a compound that modulates the activity of a polypeptide as defined in the first aspect of the invention, the method comprising contacting a compound witii the polypeptide or a suitable variant, fragment, derivative or fusion tiiereof or a fusion of a variant, fragment or derivative thereof and determining whether the protein kinase activity of the said 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.

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 interactions between the said polypeptide and its substrate are substantially the same as between human SKK4 and its substrate in vivo. An example of a substrate of said polypeptide is SAPKl . In one embodiment, the compound decreases the activity of said polypeptide. For example, the compound may bind substantially reversibly or substantially irreversibly to die 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 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.

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 ti e active site so as to aid die binding of die said polypeptide to its substrate. In a still furtiier example, me 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.

Conveniently, the method makes use of the fact that SKK4 phosphorylates SAPKl as described in Example 1 or Example 2, but any suitable substrate may be used.

Conveniently, the method makes use of an assay which may be substantially the same as that described in Example 1. In Example 1, phosphorylation of ATF2 by SAPKl is measured. Alternatively but still preferably, phosphorylation of GST-c-Jun( 1-194) by SAPKl may be measured. It is preferred mat the SKK4 is recombinant SKK4.

A still further aspect of the invention provides a method of identifying a compound which binds to SAPKl (or other substrate of the polypeptide as defined in the first aspect of the invention) and enhances or prevents its activation by the polypeptide as defined in the first aspect of the invention, the method comprising deteπnining whether a compound enhances or prevents the interaction of SAPKl (or other substrate of the polypeptide as defined in the first aspect of the invention) or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative with the polypeptide as defined in die first aspect of the invention or deternrining whether the compound substantially blocks activation of SAPKl (or otiier substrate of the polypeptide as defined in the first aspect of the invention) or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative by the polypeptide as defined in the first aspect of the invention.

A still further aspect of the invention provides a method of identifying a compound which modulates the activation of the polypeptide as defined in the first aspect of the invention by an "upstream activator" , for example MEKK. By "upstream 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 SKK4. The method comprises deterrnining whether a compound enhances or disrupts the interaction between (a) a polypeptide as defined in the first aspect of the invention or a suitable fragment, variant, derivative or fusion tiiereof or a suitable fusion of a fragment, variant or derivative and (b) an "upstream activator" , for example MEKK, or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or deteiτnining whether the compound substantially blocks activation of the said polypeptide or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion by an "upstream activator" or a suitable variant, derivative, fragment or fusion thereof.

MEKK is a known activator of SKK1 that is shown here to also be an activator of the polypeptide of die invention, known as SKK4. Prior to the present invention, it was not known how SKK4 could be activated. By "activation of SKK4" it is meant ti at the abtiity of SKK4 to phosphorylate SAPKl is increased following the treatment of SKK4, for example by MgATP and MEKK.

The sequence of MEKK is given in Lange-Carter et al (1993) Science 260, 315-319, and its expression as a histidine-tagged fusion protein and purification using nickel-nitrilotriacetate-agarose is described in Meier et al (1996) and Example 1.

Thus a further aspect of the invention is the use of MEKK for the activation of the polypeptide of die invention. A still further aspect of the invention provides a method of identifying a polypeptide tiiat interacts with the protein kinase (polypeptide) of die invention, die method comprising 1) contacting a) the said protein kinase as defined in the first aspect of the invention or a suitable variant fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof with b) a composition that may contain a polypeptide that interacts with the said protein kinase, 2) detecting the presence of a complex containing the said protein kinase and a polypeptide, and optionally 3) identifying any polypeptide bound to d e said protein kinase.

In one embodiment, the composition may comprise material from cells. In particular, the cells may be selected from the following types: 1) cells which do not have SKK4 activity even when stimulated, 2) cells which have SKK4 activity after exposure to a stimulus, but which have not been so exposed and 3) cells of type 2 after exposure to the stimulus. Polypeptides tiiat are found in a subset only of types 1-3 are of particular interest and may be characterised further. Such a peptide may be an activator of SKK4. Alternatively, it may be an inactivator of SKK4.

It will be appreciated tiiat the method may be performed within a cell, for example using the yeast two hybrid system as is well known in the art. In this example, cDNAs copied from mRNA from the three cell types described above would be used.

A still further aspect of the invention provides a method of identifying a compound which blocks die activation of the polypeptide as defined in the first aspect of the invention by an interacting polypeptide, for example MEKK, die metiiod comprising deteπriining whether a compound enhances or disrupts the interaction between (a) a protein kinase as defined in the first aspect of the invention or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative and (b) said interacting polypeptide or a suitable variant, derivative, fragment or fusion thereof or a suitable fusion of a variant, derivative or fragment, or deterrnining whether the compound substantially blocks activation of the polypeptide according to the first aspect of the invention or a suitable variant, fragment, derivative or fusion thereof, or a fusion of a said fragment, derivative or fusion by said interacting polypeptide or a suitable variant, derivative, fragment or fusion thereof.

Conveniently, the said polypeptide according to the first aspect of the invention or fragment, derivative, variant or fusion thereof used in the method is one which is produced by recombinant DNA technology. Similarly, it is preferred if die SAPKl or fragment, derivative, variant or fusion tiiereof used in the method of identifying compounds tiiat modulate activity of the said protein kinase is one which is produced by recombinant DNA technology. Similarly, it is preferred if MEKK or other "upstream activator" or fragment, derivative, variant or fusion thereof used in the method is one which is produced by recombinant DNA technology.

It will be appreciated tiiat it may be necessary to activate the polypeptide of me invention prior to its use in assays. In a preferred embodiment the polypeptide of the invention (SKK4) is activated in vitro by treating the polypeptide witii MEKK and MgATP, as described in Example 1. It is particularly preferred if die SKK4 is the recombinant polypeptide produced according to die methods of die invention. It will be appreciated tiiat by "suitable" we mean that the said components in the method are those that have interactions or activities which are substantially the same as those of SKK4 or SAPKl or other substrates, or the upstream activator such as MEKK as the case may be but which may be more convenient to use in an assay. For example, fusions of SKK4 or SAPKl are particularly useful since said fusion may contain a moiety which may allow the fusion to be purified readily.

It will be appreciated that the methods described may be performed in cells. "Reporter gene" constructs may be prepared by methods known to those skilled in the art, using the teaching herein. For example, a reporter gene construct may be made with a c-jun-dependent promoter sequence. This construct may be introduced togedier with an SKK4 construct into a cell line, in the parent cell line of which SAPKl is activated in response to known stimuli, and in which die endogenous SKK4 gene has been inactivated. Alternatively die reporter gene construct could be introduced into the cell line in which SAPKl is activated in response to known stimuli. The expression of the reporter gene will be dependent on the activity of SKK4 and tiius the effect of compounds can be measured. In a further example, the reporter gene may be fatal to the cells, or alternatively may allow cells to survive under otherwise fatal conditions. Cell survival can tiien be measured, for example using colorimetric assays for mitochondrial activity, such as reduction of WST-1 (Boehringer). WST-1 is a formosan dye that undergoes a change in absorbance on receiving electrons via succinate dehydrogenase. In a further embodiment the yeast two-hybrid system is used. The enhancement or disruption of the interaction between the said polypeptide of die invention and SAPKl or an interacting polypeptide as defined above, or suitable derivatives, fragments, fusions or variants can be measured in vitro using methods well known in the art of biochemistry and include any methods which can be used to assess protein-protein interactions.

The said interaction can also be measured within a cell, for example using the yeast two hybrid system as is well known in the art.

It will be appreciated that the invention provides screening assays for drugs which may be useful in modulating the activity of SKK4 or its interactions with upstream activators. The compounds identified in the methods may themselves be useful as a drug or tiiey may represent lead compounds for the design and synthesis of more efficacious compounds.

It will be appreciated that screening assays which are capable of high throughput operation will be particularly preferred. Examples may include the cell based assays described and protein-protein binding assays. A further example is an SPA-based (Scintillation Proximity Assay) system as described in Example 2.

A further aspect of the invention provides a compound identifiable by die screening methods of d e invention. A still further aspect provides such a compound for use in medicine.

Before the present invention it was not appreciated how diverse stimuli may lead to activation of SKK4. It was therefore not known that the SKK4 signalling pathway could potentially be modulated in such a way that responses to particular stimuli could be selectively affected. The motivation to attempt to identify compounds that modulate die signalling pathway was therefore lacking, as it would have appeared likely tiiat such compounds could interfere with beneficial roles of the SKK4 signalling pathway, in addition to potentially detrimental ones.

We show here that SKK4 becomes activated in response to proinflammatory cytokines (interleukin-1, tumour necrosis factor) in human epithelial cells, whereas the other known activator of SAPKl (SKK1/MKK4) is not. Since overproduction and/or inappropriate production of interleukin-1 and tumour necrosis factor is known to be a major cause of inflammatory diseases, such as rheumatoid arthritis, drugs tiiat inhibit SKK4 are likely to suppress a number of the intracellular effects of these cytokines and may therefore be useful for treating inflammation.

A further aspect of the invention is the use of any of the screening methods of the invention in the identification of a molecule that may be useful in treating inflammatory disease.

It is believed that a compound identifiable by any of die screening methods of die invention may be useful in treating inflammatory disease. Inflammatory diseases include rheumatoid artiiritis, psoriasis, septic shock, asthma and inflammatory bowel disease.

Thus, a further aspect of the invention provides a metiiod of treating a patient with an inflammatory disease the method comprising administering to the patient an effective amount of a compound identifiable by the screening methods of die invention.

A still further invention provides a use of a compound identifiable by die screening metiiods of the invention in the manufacture of a medicament for treating an inflammatory disease in a patient.

Thus, a further aspect of the invention provides a method of treating a patient with an inflammatory disease the method comprising administering to tiie patient an effective amount of a compound identifiable by die screening methods of the invention.

It is further believed tiiat such compounds are useful in treating disease in which apoptosis is involved. For example, such compounds may suppress apoptosis, which may aid cell survival during or following cell damaging processes. Examples of such diseases include, but are not limited to, ischaemic disease, for example stroke and myocardial infarction, neural injury and myocardial infarction.

Some such compounds may aid apoptosis. Conditions in which aiding apoptosis may be of benefit include resolution of inflammation.

Thus, a further aspect of the invention provides a method of treating a patient with an ischaemic disease die method comprising adrmnistering to d e patient an effective amount of a compound identifiable by die screening methods of die invention. A still further invention provides a use of a compound identifiable by the screening methods of the invention in the manufacture of a medicament for treating an ischaemic disease in a patient.

Thus, a further aspect of the invention provides a method of treating a patient with an ischaemic disease the method comprising administering to the patient an effective amount of a compound identifiable by die screening methods of the invention.

The aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method including oral and parenteral (e.g. subcutaneous or intramuscular) injection. The treatment may consist of a single dose or a plurality of doses over a period of time.

Whilst it is possible for a compound of d e invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of die invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.

Thus, the invention also provides pharmaceutical compositions comprising the compound identifiable by die screening metiiods of the invention and a pharmaceutically acceptable carrier.

Further aspects of the invention provide a use of a polypeptide (protein kinase) as defined in the first aspect of the invention in a screening assay for compounds which inhibit the activity of the said protein kinase or which block interactions of said protein kinase.

A further aspect of the invention provides a kit of parts tiiat are useful in carrying out the screening methods.

The present invention will now be described in more detail witii reference to the following Figures and Examples in which:

Figure 1 : the nucleotide and predicted amino acid sequence of human SAP kinase kinase-4 (SKK4). Nucleotides are numbered in the 5' to 3' direction and amino acids are shown in single-letter code above d e nucleotide sequence. The in-frame termination codon is marked by an asterisk.

Figure 2: the sequence comparison of human SKK4, human SKK1 , human SKK2, human SKK3 and HEP from D. melanogaster . Amino acids were aligned and gaps were introduced to maximise the homology. Amino acid identities between at least tiiree of the five sequences are indicated by boxed regions.

Figure 3: activation of SKK4 and SKK1/MKK4 in KB cells. (A) KB cells were incubated in the absence (control) or presence of the indicated agonist as described in Section 2.7 of Example 1 and SKK4 (open bars) and SKK1/MKK4 (filled bars) irnmunoprecipitated from die ly sates and assayed for their ability to activate SAPK1/JNK1. The results are shown as means ± SEM for six dishes of ells witii each agonist (two separate experiments). The basal activity of SKK4 and SKK1/MKK4 in unstimulated cells was 5.0 ± 0.4 mU/mg and 2.0 ± 0.2 mU/mg, respectively. Abbreviations: - IL-1, interleukin-1; TNFα, tumour necrosis factor α; EGF, epidermal growth factor; PMA, phorbol myristate acetate. (B) SKK4 and SKK1/MKK4 were i munoprecipitated from extracts prepared from UV-irradiated KB cells. Where indicated, the antibodies were incubated witii the peptides used to raise die anti-SKK4 or anti-SKKl/MKK4 antibodies. The results are presented as means ± SEM for at least three experiments.

Figure 4: GST-SKK4 is activated by MEKK and activates SAPKl/JNK, but not SAPK2a/p38. GST-SKK4 (0.25 μM) and GST-SKK1/MKK4 (0.1 μM) were incubated witii MgATP in the presence or absence of MEKK and then assayed for their ability to activate SAPKl/JNK (A) or SAPK2a/p38 (B) (Section 2.8 of Example 1). The activation of SAPKl/JNK and SAPK2a/p38 were monitored by die phosphorylation of GST-ATF2[19-96].

Figure 5: SKK4 immunoprecipitated from extracts prepared from UV- irradiated KB cells activates SAPKl/JNK, but not SAPK2/p38. SKK4 and SKK1/MKK4 immunoprecipitates were incubated with MgATP and SAPK2a/p38 or SAPKl/JNK. At the end of the reaction SAPK2a/p38 and SAPKl/JNK activity was tiien measured using ATF2 as substrate. The results are expressed as means ± SEM for three experiments.

Example 1: Purification, characterisation, cloning and expression of SKK4, and generation of antibodies binding to SKK4 Summary. A cDNA was cloned and expressed that encodes human stress-activated protein kinase kinase-4 (SKK4), a novel MAP kinase kinase family member whose mRNA is widely expressed in human tissues. SKK4 activated SAPKl/JNK in vitro, but not SAPK2a/p38, SAPK2b/p38β, SAPK3/ERK6 or SAPK4. It appears to be the mammalian homologue of HEP, an activator of SAPKl/JNK in Drosophila. In human epithelial KB cells SKK4 and SKK1/MKK4 (another activator of SAPKl/JNK) were both activated by stressful stimuli, but only SKK4 was activated strongly by proinflammatory cytokines. The identification of SKK4 explains why the major SAPKl/JNK activator detected in many mammalian cell extracts is chromatographically separable from SKK1/MKK4.

Key words. SAPK, JNK, p38, MAP kinase, cytokine, stress

1. Introduction. Seven mitogen-activated protein kinase (MAPK) family members have been identified that are activated strongly by adverse stimuli (eg chemical, heat and osmotic shock, ultraviolet radiation and die protein synthesis inhibitor anisomycin) or by signals produced/released during infection (eg lipopolysaccharide and d e proinflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor (TNF)), but only weakly (in most cell contexts) by polypeptide growth factors or phorbol esters (reviewed in [1]). For these reasons, they are termed stress-activated protein kinases (SAPKs).

The three isoforms of SAPKl/JNK bind tightly to the activation domain of c-Jun and are the only SAPKs that phosphorylate (at significant rates) the residues in this transcription factor that become phosphorylated in vivo in response to adverse stimuli and proinflammatory cytokines [204]. The two isoforms of SAPK2/p38 (SAPK2a/p38 and SAPK2b/p38β) are inhibited specifically by die pyridinyl imidazoles SB 203580 and SB 202190 at submicromolar concentrations [4-6] and their physiological substrates include the transcription factors Elkl [7], SAP1 [7], CHOP [8] and MEF2C [9], as well as MAPK-activated protein kinase-2 (MAPKAP- K2) [10], MAPKAP]K3 [11,12], MAPK-interacting protein kinase-1 (Mnkl) and Mnk2 [13,14]. Intracellular targets for MAPKAP- K2/MAPKAP-K3 include heat shock protein 27 [5,15], tyrosine hydroxylase [16] and the transcription factor CREB [17], while one of the substrates for Mnkl/Mnk2 is eukaryotic initiation factor eIF4E [13]. SAPK3/ERK6 [18,20] and SAPK4 [4,6] have only been identified recently and their physiological substrates are unknown. SAPK3 and SAPK4 are 60% identical to each other or to SAPK2a and SAPK2b, but they are not inhibited by SB 203580 and SB 202190 [4,6,18].

The SAPKs are activated by MAPK kinase (MKK) family members, termed here SAPK kinases (SKKs). The major activator of SAPK2a/p38, SAPK2b/p38β, SAPK3 and SAPK4 that has been detected biochemically is SKK3/MKK6 [4,18,21]23], although SAPK2/p38 can also be activated by SKK2/MKK3 in vitro [24]. Neither SKK2/MKK3 nor SKK3/MKK6 are capable of activating SAPKl/JNK, which is activated by a distinct enzyme termed SKK1/MKK4 [24,25]. SKK1/MKK4 is the only activator of SAPKl/JNK that can be detected biochemically in extracts prepared from PC 12 cells that have been exposed to adverse stimuli [21], and in 293 cells the activation of transfected SAPKl/JNK by anisomycin can be prevented by overexpressing a catalytically inactive form of SKK1/MKK4 [25] . In addition, heat shock or anisomycin failed to activate SAPKl/JNK in murine embryonic stem cells lacking SKK1/MKK4 [26,27]. SKK1/MKK4 can activate SAPK2/p38 in vitro [24,28] but, in contrast to SAPKl/JNK which is activated strongly by cotransfection with MEK kinase (MEKK) [29] (an upstream activator of SKK1/MKK4), SAPK2/p38 is not activated [24] or activated much more weakly [30]. Moreover the activation of SAPK2/p38 by osmotic shock and anisomycin is unimpaired in murine embryonic stem cells lacking SKK1/MKK4 [26,27]. For these reasons, SKK1/MKK4 does not seem to be rate- limiting for the activation of SAPK2/p38 in vivo.

Although SKK1/MKK4 is clearly essential for the activation of SAPKl/JNK in some cells, the dominant activator of SAPKl/JNK detected biochemically in human epithelial KB cells [21] or in Rat 3Y1 fibroblasts [31] is a chromatographically distinct enzyme(s). Moreover, the activation of SAPKl/JNK by UV radiation and osmotic shock in murine embryonic stem cells lacking SKK1/MKK4 was either unimpaired [26] or only inhibited partially [27] . These observations indicate that mammalian cells contain at least one further activator of SAPKl/JNK distinct from SKK1/MKK4. In this Example we have cloned a novel MKK family member, termed here SKK4, that is activated by stressful stimuli and proinflammatory cytokines and which activates SAPKl/JNK, but not otiier SAPKs.

2. Materials and Methods

2.1 Materials. An E. coli plasmid encoding a fusion protein comprising glutathione S-transferase (GST) linked to residues 19-96 of activating transcription factor-2 (GST-ATF2[19-96]) was provided by Dr N Jones (ICRF, London) and a plasmid encoding GST-SKK1/MKK4 by Dr J Woodgett (Ontario Cancer Institute, Toronto). A plasmid encoding the maltose-binding protein (MalE) linked to the Xenopus homologue of SAPK2a/p38 (MalE-Mpk2) was a gift from Dr A R Nebreda (EMBL, Heidelberg, Germany), while a plasmid encoding MEKK preceded by six histidine residues (6-His-MEKK) was provided by Dr G Johnson (National Jewish Centre for Immunology and Respiratory Medicine, Denver, USA). All plasmids were transformed into E. coli strain BL21 (DE3), and expressed and purified as described [4,18,21]. 6-His SAPKl/JNKlγ [32] was expressed and purified as reported previously [33]. PKI, the specific peptide inhibitor of cAMP-dependent protein kinase (TTYADFIASHGRTGRRNAIHD) was synthesised by Mr F B Caudwell in the MRC Protein Phosphorylation Unit in Dundee and other peptides by Dr Graham Bloomberg, University of Bristol, UK.

2.2 cDNA cloning and sequencing. Two oligonucleotides corresponding to part of die EST encoded by GenBank ID H85962, (forward, 5 '-GACGGATCCGACCCCACCAAGCCGGACTTT; reverse, 5 '-GGCCAAGCTTGTCTTTGACGAAGGACTGGAA) were used to amplify a 209 base pair fragment from a λgtlO human skeletal muscle cDNA library (Clontech). The nucleotide sequence of this product was identical to that in the database. The PCR fragment was ³²P- labelled by random priming and used to probe die same cDNA library at high stringency. One strongly hybridising plaque was identified after screening 500,000 plaques. This clone was isolated, the phage DNA purified, and the insert cloned into pBluescript (Stratagene) and sequenced using an Applied Biosystems model 373A DNA sequencer. 2.3 Expression of GST-SKK4 in E. coli. The open reading frame of SKK4 was amplified by PCR and subcloned as a BamHI/EcoRI fragment into the expression vector pGEX4T-3 (Pharmacia) followed by transformation into E. coli strain BL21 (DE3). Expression and purification of GST-SKK4 were carried out as described in [34]. The purified protein was dialysed against 50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 0.03% (m/v) Brij35, 0.1 % (v/v) 2-mercaptoethanol and 50% (v/v) glycerol, and stored unfrozen at -20 °C at 2 mg/ml.

2.4 Preparation of anti-SKK4 and anti-SKKl/MKK4 antibodies.

Peptides were synthesised corresponding to the C-terminal 12 residues of human SKK4 (GVLSQPHLPFFR) and the last 14 residues of SKK1/MKK4 (DQMPATPSSPMYVD) [24], coupled to botii bovine serum albumin and keyhole limpet haemocyanin using glutaraldehyde and injected into sheep at the Scottish Antibody Production Unit (Carluke, Ayrshire, UK). The anti-SKK4 and anti-SKKl/MKK4 antibodies were purified on peptide antigen-CH-Sepharose columns [22] .

2.5 Cell culture and cell lysis. KB cells were cultured [18] and exposed for 15 min to osmotic stress (0.5 M sorbitol) or proinflammatory cytokines (20 ng/ml IL-lα or 100 ng/ml TNFα), or for 30 min to anisomycin (10 μg/ml), or for 10 min to 100 ng/ml epidermal growth factor (EGF) or 300 ng/ml phorbol myristate acetate (PMA). UV-C irradiation was carried out at 60 J/m2 and the cells then incubated for a further 30 min at 37 °C. Cells were lysed as described [21].

2.6 Immunoprecipitation of SKK4 and SKK1/MKK4. KB cell lysates (250 :g) were incubated for 60 min at 4 °C with 10 μg of affinity purified antibody coupled to 5 μl protein G-Sepharose. The protein G- Sepharose immunoprecipitates were washed twice witii 1 ml lysis buffer containing 0.5 M NaCl, twice with lysis buffer without NaCl and assayed as described below. In control experiments, antibodies bound to protein G-Sepharose were incubated for 30 min at 4 °C with the peptide immunogen (1 mg/ml) prior to the addition of cell lysate.

2.7 Assay of SKK1/MKK4 and SKK4 immunoprecipitates.

Immunoprecipitates ( -6.5 μl) were incubated on a shaking platform for 30 min at 30 °C with 1 :1 of 20 μM 6-His-SAPKl/JNK in 50 mM Tris/HCl (pH 7.4), 0.1 mM EGTA, 0.03% (m/v) Brij-35, 0.1 % (v/v) 2- mercaptoethanol and 5% (v/v) glycerol, and the reactions initiated witii 2.5 μl of 40 mM magnesium acetate-0.4 mM unlabelled ATP. After 30 min, the active SAPKl/JNK generated was assayed by adding 40 μl of a solution containing 31.25 mM Tris/HCl (pH 7.4), 0.125 mM EGTA, 1.25 mM sodium orthovanadate, 3.1 μM PKI, 0.1 % (by vol) 2- mercaptoethanol and 0.25 mg/ml GST-ATF2[ 19-96], 12.5 mM magnesium acetate and 0.125 mM [(γ³²P]ATP. After 30 min at 30 °C reactions were stopped by adding 5 :1 of 6% (m/v) SDS, 400 mM Tris/HCl (pH 6.8), 50% (v/v) glycerol, 5% (v/v) 2-mercaptoethanol and

0.2% (m/v) bromophenol blue. A 40 μl aliquot was withdrawn, electrophoresed on a 10% SDS/polyacrylamide gel and autoradiographed.

The ³²P-labelled band corresponding to GST-ATF[ 19-96] was excised and counted. One unit of SKK1/MKK4 or SKK4 activity was that amount which increased the activity of SAPKl/JNK by 1 U/min. One Unit of SAPKl/JNK was that amount which incorporated 1 nmol of phosphate into GST-ATF2[19-96] in one min. SKK1/MKK4 and SKK4 were also assayed for their ability to activate Xenopus SAPK2a. The assay was identical except tiiat SAPK2a (2 μM) replaced SAPKl/JNK.

2.8 Activation and assay of expressed GST-SKK4 and GST-

SKK1/MKK4. Each GST fusion protein (4 μM) was activated by incubation for 60 min at 30 °C with MEKK (1 μM) in 50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 0.1 % (v/v) 2-mercaptoethanol, 10 mM magnesium acetate and 0.1 M unlabelled ATP. Activated GST-SKK4 and GST-SKK1/MKK4 were then measured by their ability to activate SAPKl/JNK or SAPK2a/p38, which were assayed by the phosphorylation of ATF2. The assays (50 μl) were carried out at 30 °C and comprised 0.1 μM GST-SKK4 or GST-SKK1/MKK4, 0.2 μM 6-His SAPKl/JNK, 0.2 mg/ml GST-ATF2[ 19-96] , 50 mM Tris/HCl pH 7.5, 0.1 mM EGTA, 0.1 % (v/v) 2-mercaptoethanol, 10 mM magnesium acetate and 0.1 mM [γ³²P]ATP. The reactions were terminated by spotting 40 μl on to 2 x 2 cm squares of phosphocellulose p81 paper followed by immersion into 75 mM phosphoric acid. After washing the papers several times in phosphoric acid followed by immersion in acetone, the papers were dried and counted.

3. Results

3.1 Molecular cloning of SKK4. To identify novel members of the SKK family, we used the DNA sequence encoding SKK1/MKK4 to interrogate a number of EST databases. This search identified a 250 bp sequence (GenBank ID H85962) tiiat was distinct from any previously identified SKK homologue, but was much more closely related to members of this family than to any other protein kinase. A suitable PCR probe (see Methods) was therefore used to screen a human skeletal muscle cDNA library. This resulted in the isolation of a 1.2 kb clone that encoded a novel MKK family member, hereafter termed SKK4. The nucleotide and deduced amino acid sequence of human SKK4 is shown in Fig 1. The open reading frame encodes a protein of 333 residues, with a predicted molecular mass of 37.9 kDa. It possesses all the conserved amino acid domains (I-XI) characteristic of protein kinases and shows 62% identity with the Drosophila MKK homologue HEP (see Discussion), 48% identity with human SKK1/MKK4, 39% identity with human SKK2/MKK3, and 41 % identity with human SKK3/MKK6 (Fig 2). The sequence identity witii MKK1 and MKK2 is 36% and 35%, respectively. Ser 185 and Thr 189 in kinase subdomain VIII are in an equivalent position to the Ser/Thr residues in other MKK family members, whose phosphorylation is required to generate enzymatic activity.

Hybridisation of ³²P-labelled SKK4 cDNA to multiple tissue Northern blots (Clontech) showed an RNA transcript of ~ 4 kb that was present in heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas (data not shown). The presence of ESTs in databases revealed tiiat SKK4 is also expressed in retina and germinal centre B-cells. Thus SKK4 and mRNA is widely expressed in mammalian cells.

3.2 SKK4 is activated by proinflammatory cytokines and stressful stimuli. An antibody was raised against the unique C-terminal sequence of SKK4 (Section 2.4) and used to immunoprecipitate SKK4 from KB cell extracts after stimulation with a variety of agonists (Fig 3). These studies revealed that SKK4 was activated rapidly in KB cells in response to the same stimuli that trigger the activation SAPKl/JNK [21], namely proinflammatory cytokines and stressful stimuli (UV radiation, osmotic shock and anisomycin). SKK4 was activated weakly by EGF, but not by PMA. SKK4 was also activated by stressful stimuli in COS cells (data not shown). SKK4 was activated to a greater extent by interleukin-1 (IL-1) and tumour necrosis factor (TNFα) than by UV irradiation, osmotic shock or anisomycin.

SKK1/MKK4, another activator of SAPKl/JNK (see Introduction) is also present in KB cells and, like SKK4, was activated by UV irradiation, osmotic shock and anisomycin. However, in contrast to SKK4, SKK1/MKK4 was hardly activated by interleukin-1, TNFα or EGF (Fig 3A).

The specificities of the antibodies used in these experiments were established by the finding that immunoprecipitation of SKK4 was prevented by incubating die anti-SKK4 antibody with the SKK4 peptide immunogen used to generate it, but not with the SKK1/MKK4 peptide immunogen. Conversely, immunoprecipitation of SKK1/MKK4 was prevented by incubating the anti-SKKl/MKK4 antibody with the SKK1/MKK4 peptide immunogen, but not the SKK4 peptide immunogen (Fig 3B).

3.3 Activation and substrate specificity of SKK4. GST-SKK4 was expressed in E. coli (Section 2.3) and 10 mg of purified enzyme could be isolated from 500 ml of bacterial culture. The preparation showed a single protein-staining band with the predicted molecular mass of 64 kDa (data not shown). GST-SKK4 was inactive, but could be activated by incubation with MgATP and MEKK (Fig 4), an enzyme that also activates SKK1/MKK4 [29]. The activated GST-SKK4 was able to activate SAPKl/JNK in vitro, but not SAPK2a/ρ38 (Fig 4), SAPK2b/p38β, SAPK3/ERK6 or SAPK4 (data not shown). In contrast, activated GST- SKK1/MKK4 activated SAPK2a/p38 as well as SAPKl/JNK (Fig 4).

SKK4 and SKK1/MKK4 immunoprecipitated from the lysates of UV- stimulated KB cells had the same specificity as the bacterially expressed enzymes. Thus SKK4 activated SAPKl/JNK but not SAPK2a/p38, while SKK1/MKK4 activated both enzymes (Fig 5).

4. Discussion

In this Example we have cloned a novel MKK homologue that is activated strongly by proinflammatory cytokines and stressful stimuli, and has therefore been termed SKK4. SKK4 expressed in E. coli and activated in vitro, or immunoprecipitated from KB cell extracts activated SAPKl/JNK, but was unable to activate other SAPKs, suggesting that SAPKl/JNK may be a physiological substrate of SKK4. This view is reinforced by the striking similarity between SKK4 and the Drosophila MKK homologue, termed HEP (Fig 2). HEP was identified by genetic dissection of a signalling pathway that is required for dorsal closure during early embryonic development and shown to be situated "upstream" of

Drosophila JNK [35-37]. Thus HEP is almost certainly the enzyme responsible for activating JNK in Drosophila. The generation of mice lacking SKK4 will be needed to find out if SKK4 also plays an essential role in mammalian embryonic development. SKK4 is activated in vitro by MEKK (Fig 4) one of many protein kinases [38] that have been shown to activate SKK1/MKK4 in vitro and to trigger the activation of SAPKl/JNK in cotransfection experiments. These observations, and the find that overexpression of inactivate mutants of SKK1/MKK4 prevent the activation of SAPKl/JNK by MEKK, other upstream activities and stressful stimuli [25,29,38], suggested tiiat SKK1/MKK4 mediates the activation of SAPKl/JNK in vivo. However, since the inactive mutant of SKK1/MKK4 probably exerts its dominant negative effect by binding to SAPKl/JNK, it remained possible tiiat MEKK and other upstream activators trigger the activation of SAPKl/JNK via another MKK homologue. This view was confirmed by biochemical experiments which show that the major activator(s) of SAPKl/JNK in several cell extracts is chromatographically separable from SKK1/MKK4 [21 ,31], and by the finding that UV radiation and osmotic shock can still trigger the activation of SAPKl/JNK in stem cells from mice that do not express SKK1/MKK4 [26,27] . The present work indicates that one of the additional SAPKl/JNK activators is likely to be SKK4. Whether mammalian cells express additional MKK homologues that activate SAPKl/JNK remains to be established.

The present work (Fig 3) shows that SKK4 is activated strongly by proinflammatory cytokines as well as stressful stimuli in KB cells, whereas SKK1/MKK4 is activated by stress stimuli only. These observations suggest that SKK4 may be a more attractive target for an anti-inflammatory drug than SAPKl/JNK because inhibitors of SKK4 may prevent the activation of SAPKl/JNK by proinflammatory cytokines, without affecting the activation of SAPKl/JNK by other agents. References

1. Cohen, P. (1997) Trends Cell Biol. (in press).

2. Kyriakis, J.M. and Avruch, J. (1996) Bioessays 18, 567-577. 3. Karin, M. (1995) J. Biol. Chem. 270, 16483-16486.

4. Goedert, M. et al (1997) EMBO J. 16, 3563-3571.

5. Cuenda, A. et al (1995) FEBS Lett. 364, 229-233.

6. Kumar, S. et al (1997) Biochem. Biophys. Res. Comm. 235, 533- 538. 7. Price, M.A. et al (1996) EMBO J. 15, 6652-6563.

8. Wang, X.Z. and Ron, D. (1996) Science 111, 1347-1349.

9. Han, J. et al (1997) Nature 386, 296-299.

10. Rouse, J. et al (1994) Cell 78, 1027-1037.

11. McLaughlin, MM. et al (1996) J. Biol. Chem. 271 , 8488-8492. 12. Clifton, A.D. et al (1996) FEBS Lett. 392, 209-214.

13. Waskiewicz, A.J. et al (1997) EMBO J. 16, 1909-1920.

14. Fukunaga, R. and Hunter, T. (1996) EMBO J. 16, 1921-1933.

15. Guay, J. et al (1997) J. Cell Sci. 110, 357-368.

16. Thomas, G. et al (1997) Eur. J. Biochem. (in press). 17. Tan, Y. et al (1996) EMBO J. 15, 101-114.

18. Cuenda, A. et al (1997) EMBO J. 16, 295-305.

19. Lechner, C. et al (1996) Proc. Natl. Acad. Sci. USA 93, 4355- 4359.

20. Mertens, S. et al (1996) FEBS Lett. 383, 273-276. 21. Meier, R. et al (1996) Eur. J. Biochem. 236, 796-805.

22. Cuenda, A. et al (1996) EMBO J. 15, 4156-4164.

23. Moriguchi, T. et al (1996) /. Biol. Chem. 271, 26981-26988.

24. Derijard, B. et al (1995) Science 267, 682-684. 25. Sanchez, I. et al (1994) Nature 372, 794-798.

26. Nishina, H. et al (1997) Nature 385, 350-353.

27. Yang, D. et al (1997) Proc. Natl. Acad. Sci. USA 94, 3004-3009.

28. Doza, Y.N. et al (1995) FEBS Lett. 364, 223-228. 29. Yan, M. et al (1994) Nature 372, 798-800.

30. Xu, S. et al (1996) Proc. Natl. Acad. Sci. USA 93, 5291-5295.

31. Moriguchi, T. et al (1995) J. Biol. Chem. 270, 12969-12972.

32. Derijard, B. et al (1994) Cell 76, 1025-1037.

33. Goedert, M. et al (1997) FEBS Lett. 409, 57-62. 34. Alessi, D.R. et al (1995) Methods Enzymol. 255, 279-290.

35. Gilse, B. et al (1995) Cell 83, 451-461.

36. Riesgo-Escovar, J.R. et al (1996) Gene Dev. 10, 2759-2768.

37. Sluss, H.K. et al (1996) Gene Dev. 10, 2745-2758.

38. Fanger, G.R. et al (1997) Curr. Opin. Genetic. Dev. 7, 67-74. 39. Raingeaud, J. et al (1996) Mol. Cell. Biol. 16, 1247-1255.

Example 2: Alternative protein kinase assays

SKK4 is assayed routinely by phosphorylation of SAPKl , as described in Meier et al, (1996). 10 μl of a solution containing SKK4 and inactive SAPKl (equivalent to lOOmU before inactivation with PTP IB) in 20 mM sodium glycerol 2-phosphate pH 7.4, 1.0 mM EGTA, 0.03% (by mass) Brij-35, 5 % (by vol.) glycerol, 1 mM benzamadine, 0.1 % (by vol.) 2- mercaptoethaonol, 1 mM sodium orthovanadate, 1 μM microcystin-LR was incubated for 3 min at 30 °C, and the reaction initiated with 2 μl 60 mM MgCl₂, 0.6 mM unlabeled ATP. After 30 min at 30 °C, a 5 μl aliquot was withdrawn and assayed for SAPKl activity as described below. Control incubations were performed in which SKK4 or inactive SAPKl were omitted. One unit (U) of SKK4 was that amount which increased SAPKl activity by 1 U/min.

SAPKl is assayed by phosphorylation of GST-c-Jun( 1-194), as described in Meier et al, (1996). A solution (40 μl) containing SAPKl and 200 μg/ml GST-c-Jun(l-194) in 25 mM Tris/HCl, 20 mM sodium glycerol 2- phosphate pH 7.4, 0.1 mM EGTA, 1 mM sodium orthovanadate, 2.5 μM PKI, was incubated for 3 min at 30 °C and d e assay initiated witii 10 μl 50 mM MgCl₂, 0.5 mM [γ-³²P]ATP (5xl0⁵ cpm nmol).

After a further 20 min, a 40 μl aliquot was withdrawn, spotted on to a square (1.5 x 1.5 cm) of Whatman P81 phosphocellulose paper and immersed immediately in 75 mM phosphoric acid. After washing the papers several times in 75 mM phosphoric acid followed by immersion in acetone, P radioactivity incorporated into c-Jun(l-194) was measured. One unit (U) of SAPKl activity was that amount which catalysed the phosphorylation of 1 nmol GST-c-Jun(l-194) in 1 min.

Alternatively, a Scintillation Proximity Assay (SPA) system (Amersham International) is used to assess the incorporation of P radioactivity into cJun(l-194). In this system, the sample is mixed with beads comprising scintillant and antibodies that bind GST-c-Jun(l-194). Conveniently this is done in a 96-well format. The plate is then counted using a suitable scintillation counter, using known parameters for P SPA assays. Only ³²P that is in proximity to the scintillant, i.e. only that bound to GST-c- Jun(l-194) that is then bound by the antibody, is detected. Example 3: Assay for compounds which modulate SKK4 activity

An assay is set up with SAPKl, as described in Example 1 or Example 2.

Compounds are tested in the assay and those that give rise to inhibition or activation of SKK1 are selected for further study. To confirm that the any effects observed are not due to effects on SAPKl, compounds are tested for effects on activated and inactive SAPKl .

Example 4: Assay for polypeptides that interact with SKK4

A yeast two hybrid assay system is set up to identify polynucleotides encoding polypeptides that are capable of associating with SKK4 in a stable enough manner to allow transcriptional activation to occur. The polynucleotides are (in separate experiments) cDNAs copied from mRNA from cells that are capable of expressing SKK4, before or after stimulation capable of activating SKK4, and from cells which do not express SKK4. Interactions which are found in a subset only of these cell types are of particular interest.

The polypeptide encoded by the polynucleotide is deteirnined by sequencing the insert by the Sanger metiiod as described in Example 1 to obtain a predicted amino acid sequence.

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Claims

1. A substantially pure polypeptide comprising the amino acid sequence

M E S I E I D Q K L Q E I M K Q T G Y L

T I G G Q R Y Q A E I N D L E N L G E M

G s G T C G Q V W K M R F R K T G H V I

A v K Q M R R s G N K E E N K R I L M D

L D V v L K S H D C P Y I V Q C F G T F

I T N T D V F I A M E L M G T C A E K L

K K R M Q G P I P E R I L G K M T V A I

V K A L Y Y K E K H G V I H R D V K P s N I L L D E R G Q I K L C D F G I s G

R L V D S K A K T R S A G C A A Y M A P

E R I D P P D P T K P D Y D I R A D V w

S L G I s L V E L A T G Q F P Y K N C K

T D F E V T K V L Q E E P P L L P G H

M G F S G D F Q s F V K D c L T K D H R

K R P K Y N K L L E H S F I K R Y E T L

E V D V A S W F K D V M A K T E s P R T

S G V S Q P H P F F R

or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant or fragment or derivative.

2. A variant of the polypeptide according to Claim 1 wherein the amino acid sequence of said variant has at least 99.1 % identity with the given amino acid sequence.

3. A variant of the polypeptide according to Claim 2 wherein the amino acid sequence of said variant has at least 99.4% identity witii the given amino acid sequence.

4. A variant of the polypeptide according to Claim 3 wherein the amino acid sequence of said variant has at least 99.7% identity with the given amino acid sequence.

5. A recombinant polynucleotide encoding a polypeptide as defined in any one of Claims 1 to 4 or encoding a variant or fragment or derivative or fusion of said polypeptide or a fusion of a said variant or fragment or derivative.

6. A recombinant polynucleotide comprising the nucleotide sequence

ATGGAGAGCATTGAGATTGACCAGAAGCTGCAGGAGATCATGAAGCAGACGGGCTACC TGACCATCGGGGGCCAGCGCTACCAGGCAGAAATCAACGACCTGGAGAACTTGGGCGA GATGGGCAGCGGCACCTGCGGCCAGGTGTGGAAGATGCGCTTCCGGAAGACCGGCCAC GTCATTGCCGTTAAGCAAATGCGGCGCTCCGGGAACAAGGAGGAGAACAAGCGCATCC

TCATGGACCTGGATGTGGTGCTGAAGAGCCACGACTGCCCCTACATCGTGCAGTGCTT TGGGACGTTCATCACCAACACGGACGTCTTCATCGCCATGGAGCTCATGGGCACCTGC GCTGAGAAGCTCAAGAAGCGGATGCAGGGCCCCATCCCCGAGCGCATTCTGGGCAAGA TGACAGTGGCGATTGTGAAGGCGCTGTACTACCTGAAGGAGAAGCACGGTGTCATCCA CCGCGACGTCAAGCCCTCCAACATCCTGCTGGACGAGCGGGGCCAGATCAAGCTCTGC

GACTTCGGCATCAGCGGCCGCCTGGTGGACTCCAAAGCCAAGACGCGGAGCGCCGGCT GTGCCGCCTACATGGCACCCGAGCGCATTGACCCCCCAGACCCCACCAAGCCGGACTA TGACATCCGGGCCGACGTATGGAGCCTGGGCATCTCGTTGGTGGAGCTGGCAACAGGA CAGTTTCCCTACAAGAACTGCAAGACGGACTTTGAGGTCCTCACCAAAGTCCTACAGG AAGAGCCCCCGCTTCTGCCCGGACACATGGGCTTCTCGGGGGACTTCCAGTCCTTCGT

CAAAGACTGCCTTACTAAAGATCACAGGAAGAGACCAAAGTATAATAAGCTACTTGAA CACAGCTTCATCAAGCGCTACGAGACGCTGGAGGTGGACGTGGCGTCCTGGTTCAAGG ATGTCATGGCGAAGACTGAGTCACCGCGGACTAGCGGCGTCCTGAGCCAGCCCCACCT GCCCTTCTTCAGG, or a variant, fragment, fusion or derivative thereof.

7. A polynucleotide according to Claim 5 or 6 which contains no introns.

8. A replicable vector comprising a polynucleotide as defined in any one of Claims 5 to 7.

9. A host cell comprising a recombinant polynucleotide or a replicable vector as defined in any one of Claims 5 to 8.

10. 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 9 which expresses said 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.

11. 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 10.

12. An antibody reactive towards a polypeptide as defined in any one of Claims 1 to 4 and 11.

13. Antibody reactive towards a polypeptide or a variant or fragment or derivative thereof according to Claim 12 wherein the antibody does not react substantially with another stress-activated protein kinase kinase.

14. A method of identifying a compound that modulates the activity of a polypeptide as defined in Claim 1, the method comprising contacting a compound witii the said polypeptide or a suitable variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative tiiereof and determining whether the activity of the said polypeptide is changed compared to the activity of the said protein kinase or said variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof in the absence of said compound.

15. A method according to Claim 14 in which the activity is decreased.

16. A method according to Claim 14 in which the activity is increased.

17. A method of identifying a compound which binds to SAPKl (or other substrate of the polypeptide as defined in Claim 1) and enhances or prevents its activation by die polypeptide as defined in Claim 1, the method comprising determining whether a compound enhances or prevents the interaction of SAPKl (or other substrate of the polypeptide as defined in Claim 1) or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative with the polypeptide as defined in Claim 1 or determining whether the compound substantially blocks activation of SAPKl (or other substrate of the polypeptide as defined in Claim 1) or a suitable fragment, variant, derivative or fusion tiiereof or a suitable fusion of a fragment, variant or derivative by the polypeptide as defined in claim 1.

18. A method of identifying a compound which blocks die activation of a polypeptide as defined in Claim 1 by an interacting polypeptide, such as MEKK, the method comprising determimng whether a compound enhances or disrupts the interaction between (a) a polypeptide as defined in the first aspect of the invention or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative and (b) die interacting polypeptide, such as MEKK, 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 or a suitable variant, fragment, derivative or fusion tiiereof, or a fusion of a said fragment, derivative or fusion by the interacting polypeptide, such as MEKK, or a suitable variant, derivative, fragment or fusion thereof.

19. The use of MEKK for the activation of the polypeptide as defined in Claim 1.

20. A method of identifying a polypeptide that interacts with a polypeptide as defined in Claim 1, the method comprising 1) contacting a) the polypeptide as defined in Claim 1 with b) a composition that may contain such an interacting polypeptide, 2) detecting the presence of a complex containing the polypeptide as defmed in Claim 1 and an interacting polypeptide, and optionally 3) identifying any interacting polypeptide bound to the said polypeptide as defined in Claim 1.

21. A polypeptide identifiable by the method of Claim 20.

22. A method of identifying a compound which blocks the activation of a polypeptide as defined in Claim 1 by a polypeptide as defined in Claim 21 the method comprising determining whether a compound enhances or disrupts the interaction between (a) a polypeptide as defined in Claim 1 and (b) said polypeptide as defined in Claim 21 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 as defmed in Claim 1 by said polypeptide as defined in Claim 21 or a suitable variant, derivative, fragment or fusion thereof.

23. A compound identifiable by the method of any one of Claims 14-18, 20, 22.

24. A compound according to Claim 23 for use in medicine.

25. A method of treating a patient with an inflammatory or ischaemic disease the method comprising administering to the patient an effective amount of a compound according to Claim 23.

26. Use of a polypeptide as defined in Claim 1 in a screening assay for compounds which modulate the activity of the said polypeptide or which modulate the activation of said polypeptide.

27. A kit of parts comprising a polypeptide as defined in Claim 1 and a means for carrying out the method as defined in any one of Claims 14-18, 20, 22.

28. Any novel stress-activated protein kinase as herein disclosed.