WO2003038078A2 - Controsome-associated kinases - Google Patents

Abstract

The application relates to centrosome-associated kinases, and in particular to centrosome-associated kinases having decreased or no kinase activity. Nucleic acid molecules encoding such kinases are provided together with methods of identifying inhibitors or activators of cell-cycle progression comprising: (a) providing a cell comprising a centrosome-associated kinase; (b) adding a compound to the cell; and (c) determining whether the compound stimulates or inhibits cell cycle progression. This allows cell-cycle regulation to be studied and new compounds capable of regulating the cell-cycle progression to be identified.

Description

Enzymes and Enzyme Inhibition

The invention relates to centrosome associated kinases, and in particular, to the inhibition thereof.

Cell division in mammalian cells proceeds through a series of events which together make up the cell cycle. These events are normally regulated such that cells divide only when appropriate to the needs of the organism. This regulation is achieved by a combination of mechanisms reliant either on signals extrinsic to the cell, or on intrinsic molecular pathways. The two essential components of the cell cycle in mammalian cells are replication of the genetic material and division of the cell to produce two daughter cells. These fundamental events are usually separated by growth periods during which the majority of protein synthesis occurs and are regulated in somatic cells such that successive divisions are separated by periods of chromosomal replication (Norbury and Nurse (1992), Annual Review of Biochemistry, 61, 441-470).

During cell division, referred to as mitosis in somatic cells, the replicated chromosomes are partitioned equally to the two daughter cells. The process of cell division involves condensation of the genetic material to form mitotic chromosomes, dissolution of the nuclear membrane, assembly of a physical scaffold, the mitotic spindle, to which the chromosomes attach and movement of the chromosomes to opposite poles of the cell.

Subsequently, the two separated populations of chromosomes become encompassed by

reformed nuclear membranes and the two daughter cells separate. The mitotic spindle is composed of filaments known as microtubules. Microtubules are composed of tubulins and associated proteins and are nucleated by a structure which is

detectable in the majority of cell types in higher animals and is known as the centrosome.

Post-mitotic cells contain a single centrosome and, in diploid organisms such as mammals, two copies of each chromosome. These cells are referred to as being in the Gl phase of the cell cycle and containing a 2n complement of chromosomes. During each cell cycle, the centrosome is replicated, the chromosomes are replicated leading to a 4n chromosomal complement, and the two daughter centrosomes move to opposite poles of the cell and nucleate formation of a bipolar spindle at mitosis. One centrosome becomes partitioned to each daughter cell at division, as does one set of chromosomes, resulting in return to the 2n state. Thus, the centrosome can be regarded as an essential part of the mechanism by which cell division is accomplished in mammalian cells.

The centrosome is intrinsically involved in the cell cycle. Centrosome components include centrosome associated kinases, an example of which is Mak V, first reported by Korobko (Korobko et al ., (1997) Doklady Akademii Navk, 354, 554-556), and later renamed Hunk (Gardner et al., (2000) Genomics, 63, 46-59), although the function of the protein was not recognised at that time.

An antiserum raised against a synthetic peptide representing residues 431 to 452 of Mak-V/Hunk (SwissProt accession number 088866) has now been shown by the inventors to recognise specifically Mak-V/Hunk protein by enzyme-linked immunosorbent assay. This antiserum was used in indirect immunofluorescence analysis of cultured mouse

tumour cells including the embryonal carcinoma line PCC3 (Jakob, et al., (1973) Ann. Microbial (Paris), 124, 269-282) and the melanoma line B16F10 (Fidler, (1975) Cancer

Res., 35, 218-224). Staining with anti-Mak N unexpectedly resulted in fluorescence of a

single or double spot located close to the cell nucleus (Figure 1 A). Confirmation of the

identity of the staining location as the centrosome was achieved by double labelling of the

same cells with an antiserum directed against γ tubulin, which is highly concentrated at the

centrosome (Zheng, et al, (1991) Cell, 65, 817-823).

Unsuccessful attempts to mutate Mak V have been discussed (Craig, (1999) PhD.. Thesis, University of Warwick). The author of this thesis did not produce any mutations which showed any conclusive activity for the kinase.

The inventors have now found that mutated Mak N exhibits reduced localisation to the centrosomes, leading to an interruption of the cell cycle. Specifically, they have found that kinase activity, that is the ability to phosphorylate a substrate such as one or more arnino acids on the centrosome, is essential for cell cycle regulation.

This has the potential to allow conditions in which cell cycle regulation is malfunctioning, such as cancers to be studied. It also presents a potentially new site of cell-cycle regulation to be studied.

Centrosome-associated kinases preferably have the following characteristics:

1. They are localised to the centrosome either completely or predominantly during part or all of the cell cycle. This can be demonstrated either by immunohistochemistry using antibodies specific for the kinase, or by subcellular fractionation of cells and demonstrating co-localisation of the kinase with other centrosome components, or by expressing GFP

fusion proteins with the kinase.

2. They have detectable homology to proteins demonstrated to exhibit biochemical kinase activity or themselves have demonstrated kinase activity.

According to the invention there is provided an isolated centrosome associated kinase, or a fragment thereof comprising a centrosome binding site, wherein the kinase has decreased or no kinase activity. The centrosome associated kinase is preferably not Mak V (seq.DD No. 2). The decrease in activity may be due to a naturally occurring - or artificially introduced - mutation in the centrosome-associated kinase.

The presence of a centrosome binding site may be identified, for example, by labelling the polypeptide or fragment with, e.g. a fluorescent label, inducing the labelled polypeptide or fragment into a cell, and seeing if the polypeptide is localised at the centrosome in the cell.

The kinase may be 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99% homologous to an amino acid molecule encoding the sequence shown in seq.ID No. 1.

Kinase activity is preferably measured against naturally-occurring non-mutated, e.g. wild type, centrosome associated kinase. Wild ype Mak V is shown in Seq. ID 2. Preferably there is 20%, 40%, 60% or 80%, most preferably 100% reduction in kinase activity compared with the naturally occurring kinase. Preferably, kinase activity is compared with wild type Mak N activity.

In a preferred embodiment the centrosome associated kinase comprises a mutation in the

ATP binding domain. Preferably the mutation is a deletion or a point mutation. A deletion is where one or more amino acids have been deleted. A point mutation is where one or more naturally occurring amino acids have been changed and replaced by different amino acids. ATP binding sites may be identified by sequence homology with known ATP binding domains.

Also provided is a nucleic acid molecule encoding the centrosome associated kinase or fragment of the invention. Nucleic acids complementary to such nucleic acid molecules are also provided. The nucleic acid may be single or double stranded, DNA or RNA, naturally or non-naturally occurring.

Preferably the nucleic acid molecule is 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% or 100% homologous to the Mak V sequence shown in seq. ID 3. The nucleic acid molecule may be capable of hybridising to the sequence shown in seq. ID 3 under high stringency conditions typically 50 mM sodium phosphate, 1% sodium dodecyl sulphate, pH 6.8, 65°C.

Vectors comprising nucleic acid molecules, according to the invention, are also provided. Vectors are molecules which serve to transfer nucleic acids of interest into a cell. Suitable vectors include, but are not limited to, bacterial or eukaryotic vectors such as

plasmids or cosmids, phage vectors such as lambda phage, viral vectors such as adenoviral

vectors or baculoviral vectors, and other vectors known in the art.

The vector preferably comprises suitable regulatory sequences to allow the nucleic acid molecule of the invention to be expressed in a suitable host cell to produce protein encoded by the nucleic acid molecule. Typically, the vector comprises a suitable promoter and terminator sequences, or other sequences such as poly A sequences, operably linked to the nucleic acid molecule. Such regulatory sequences are well known in the art.

The vector may also comprise a gene to allow the vector to be selected within a cell, such as an antibiotic resistance gene or a nutritional gene. Such genes are well known in the art.

The vector may comprise a reporter gene to allow the expression of the nucleic acid molecule into protein to be monitored. Preferably this is fused onto the nucleic acid molecule of the invention.

The promoter, which is operably linked to the centrosome associated kinase, may be a regulatable promoter such as lactose inducible expression, available from Stratagene. Alternatively, a system for regulating gene expression such as ecdysone-inducible expression (available from InVitrogen), tetracycline regulated expression (from Clontech)

may be used. Both allow the production of kinase to be induced or suppressed to control kinase production in a host cell. The reporter gene is preferably Green Fluorescent Protein (GFP), which is known in the

art. This fluoresces and enables the position of the kinase to be identified.

A further reporter system which may be used is lacZ gene from E.coli. This encodes the

β-galactosidase enzyme. This catalyses the hydrolysis of β-galactoside sugars such as

lactose. The enzymatic activity in cell extracts can be assayed with various specialised

substrates, for example X-gal, which allow enzyme activity quantitation using a

spectrophotometer, fluorometer or a luminometer.

Alternatively, the reporter gene may be secreted alkaline phosphatase. This is a secreted enzyme which may be assayed from a supernatent by methods known in the art.

Also provided is a host cell comprising the vector. The cell may be bacterial, yeast or eukaryotic.

The invention further provides a method of identifying an inhibitor or activator of cell cycle progression comprising

a) providing a cell comprising a centrosome associated kinase,

b) adding a compound to the cell,

c) determining whether the compound stimulates or inhibits cell cycle progression.

The centrosome associated kinase may be as defined above, or may be Mak V, and may be

heterologous. Preferably the centrosome associated kinase is non-naturally occurring within the cell and/or is regulatable by means of an inducable or suppressible promoter.

The centrosome kinase may be provided within a vector as defined above.

Also provided is an inhibitor, or activator of centrosome associated kinase identifiable or identified by the method of the invention. Preferably the inhibitor or activator affects

centrosome binding.

The inhibitor or activator may be used in the manufacture of a medicament to treat uncontrolled cell cycle progression, such as cancer.

The invention also provides the use of such an inhibitor or activator to control cell cycle progression in vitro, for example in cultured cells and animal tissues.

The invention will now be described in detail with reference to the following figures:

Figure 1 shows Immunofluorescent labelling of B16F10 cells with anti-Mak (A) or anti-gamma tubulin (B), demonstrating Mak V localisation at the centrosome.

Figure 2 shows Green Fluorescent protein fluorescence in B16F10 melanoma cells

transfected with Mak V:GFP (a) or GFP alone and shows the centrosome. Figure 3 shows fluorescent activated cell sorter analysis of cells expressing Mak V fusion

proteins: A) wildtype Mak V: GFP. B) K91R Mak V: GFP.

The centrosomal location of Mak V was confirmed by constructing recombinant DNA

expression vectors, in which the coding sequence of Mak V was fused to that of green fluorescent protein (GFP). When transfected into cultured cells, the fusion protein became localised to the centrosome, in contrast to the localisation of GFP alone, which was evenly distributed throughout the cytoplasm. B16-F10 murine melanoma cells were cultured in RPMI 1640 medium supplemented with 5% fetal calf serum. After trypsinization in trypsin/EDTA (0.25% trypsin/lmM EDTA) for 5 min., cells were seeded onto glass coverslips in 12- well plates. When cells reached 50-60% confluence, they were transiently transfected using FuGENE 6 transfection Reagent (Roche, UK) with pEGFP:Mak V DNA. 24 to 30 hours after transfection, cells were fixed in methanol at -20°C for 5 min. (alternatively in 4% paraformaldehyde in PBS for 10 min. and subsequent permeabilization with methanol or 0.5% Triton X-100 for 5 min.) and incubated in PBS for 5 min. to allow cell rehydration. The coverslips were incubated with anti-Mak V appropriately diluted in 10% goat serum in PBS for 2 hours at room temperature. For washing, the cover slips were rinsed 5 times in PBS, soaked twice in PBS for 5 mins. each time, and rinsed a further 5 times in PBS. The cover slips were incubated with appropriate secondary antibodies (for

anti-γ-tubulin, goat anti-mouse TRITC; for anti-Mak V, goat anti-rabbit FITC) for 1 hour

at room temperature, followed by extensive washes as described above. The cover slips were mounted on sides with Vectashield (Vector) and viewed under UV light of the

appropriate wavelength. DNA staining was performed using lμg/ml DAPI. Figure 1 shows the results of immunofluorescent staining of B16F10 cells with anti-Mak V

(A) or anti-gamma tubulin. Mak V is shown to be localised at the centrosomes.

This was confirmed using the GFP Mak V construct (Figure 2) also showing that Mak V is localised at the centrosome.

The significance of the centrosome associated kinases was tested by producing variants of the protein using coding sequences harbouring point mutations designed to inhibit the kinase activity of the protein. In particular, a lysine residue at position 91 in the Mak V polypeptide was mutated to arginine. This mutation was chosen because it has been shown, in a number of different kinases to render the kinase inactive. The mutation is referred to as K91R Mak V, and is predicted to act as a trans-dominant inhibitor of endogenous Mak V activity.

The sequence of the mutated protein is shown as seq.ID No 1.

An expression vector constructed to direct the synthesis of a fusion protein between K91R and GFP was transfected into cultured tumour cells. Wild type Mak V fused to the C terminus of GFP was also expressed in cells. The construct pEGFP:Mak V directs expression of wild type Mak V fused to GFP, whilst pEGFP:KDMak V directs expression of K91R Mak V fused to GFP. B16F10 melanoma cells were synchronised in late G2 phase by treatment with 500nM nocodazole (Sigma). For release of the nocodazole block, cells were washed three times in PBS and incubated in fresh medium. Two hours later the cells were transiently transfected with pEGFP:Mak V or pEGFP:KDMak V. The cells were incubated for 24 hours, and then fixed with 4% paraformaldehyde at room temperature for 10 minutes and mounted on slides. EGFP expressing cells were counted using a fluorescent

microscope and divided into three groups on the basis of the appearance of the

centrosomes: single centrosome (Gl phase), duplicated centrosomes (S phase) and

separated centrosomes (G2 phase). A significantly higher proportion of the cells expressing the mutant protein contained a single centrosome. The analysis indicated that the expression of EGFP:Mak V in cells resulted in the accumulation of cells in the Gl phase of the centrosome cycle. The results are displayed in table 1.

Table 1.

In order to examine the effect of K91R Mak V on cell cycle progression, transfected cells were analysed by fluoresence activated cell sorting. B16F10 melanoma cells were again transfected with either wild type Mak V:GFP fusion protein, or with the K91R:GFP fusion protein. The cells were incubated for 24 hours, and then stained with the DNA-binding fluorescent dye propidium iodide. The DNA content of GFP expressing cells was examined by Fluorescent Activated Cell Sorter (FACS) analysis. The results are shown in Figure 3. The analysis showed a significantly higher proportion of the K91R:GFP expressing cells to contain 2n DNA, indicating them to be in the Gl phase of the cell cycle.

The combination of the results of the two experiments indicates that inhibition of centrosome associated kinase activity interferes with normal centrosomal function,

resulting in the impedance of cell cycle progression. In designing assays for Mak V, the first requirement is a substrate, either a protein or a

synthetic peptide, which can be shown to be phosphorylated by Mak V. Once such a

substrate has been identified, addition of potential inhibitors or activators to the reaction

will identify those which are effective in preventing the kinase reaction. The usual approach to performing the assay is to incubate the kinase enzyme and the substrate together with ATP. The ATP acts as a donor for the phosphate group which is transferred by the kinase onto the substrate. To analyse the outcome, it is necessary to be able to measure the proportion of the substrate which has become phosphorylated during the incubation. This is done by a variety of methods. In many cases the phosphate which is transferred from ATP, known as the terminal or gamma phosphate of ATP, is radiolabelled, i.e. Is either ³²P or ³³P. In this case, the substrate needs to be separated from the unused ATP and the amount of radiolabel incorporated into the substrate measured. Approaches to the separation include the use of gel electrophoresis or filter binding to separate the protein or peptide substrate from the ATP. The amount of radioactivity and thus phosphate incorporated by the action of the kinase is measured by scintillation counting or using an instrument such as a Phosphorlmager to quantify radioactivity.

An alternative approach is to immobilise the substrate onto a surface before conducting the assay and then detecting phosphorylated substrate using an antibody which recognises specifically phosphotyrosine or phosphoserine (i.e. The phosphorylated amino acid resulting from kinase action). This approach has the advantage that it is easily utilised in a proximity detection assay, which means that automation is possible. Such assay systems are marketed by PerkinElmer. Essentially, the substrate is bound to the bottom of the wells

of a multiwell plate, which also contain a scintillant. After incubation with kinase and ATP (which in this case is not required to be radiolabelled), with or without potential inhibitors, the substrate, which may now be phosphorylated, is incubated with a radiolabelled antibody which binds to the phosphorylated amino acid. Thus, the radiolabel on the antibody is in proximity of the scintillant and the signal can be detected by a

scintillation counter.

SEQUENCE LISTING <110> University of Warwick <120> Enzymes and Enzyme Inhibition <130> De/p704314GB

<140> <141>

<160> 3

<170> Patentln Ver. 2.1

<210> 1

<211> 714

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Mutated Mak V protein, based on Mak V from Mus Sp.

<400> 1

Met Pro Ala Ala Ala Gly Asp Gly Leu Leu Gly Glu Pro Ala Ala Pro 1 5 10 15

Gly Gly Asp Gly Gly Ala Glu Asp Thr Thr Arg Pro Ala Ala Ala Cys 20 25 30

Glu Gly Ser Phe Leu Pro Ala Trp Val Ser Gly Val Ser Arg Glu Arg 35 40 45

Leu Arg Asp Phe Gin His His Lys Arg Val Gly Asn Tyr Leu lie Gly 50 55 60

Sez Arg Lys Leu Gly Glu Gly Ser Phe Ala Lys Val Arg Glu Gly Leu 65 70 75 80

His Val Leu Thr Gly Glu Lys Val Ala lie Arg Val lie Asp Lys Lys 85 90 95

Arg Ala Lys Lys Asp Thr Tyr Val Thr Lys Asn Leu Arg Arg Glu Gly 100 105 110

Gin He Gin Gin Met He Arg His Pro Asn He Thr Gin Leu Leu Asp 115 120 125

He Leu Glu Thr Glu Asn Ser Tyr Tyr Leu Val Met Glu Leu Cys Pro 130 135 140

Gly Gly Asn Leu Met His Lys He Tyr Glu Lys Lys Arg Leu Asp Glu 145 150 155 160

Ala Glu Ala Arg Arg Tyr He Arg Gin Leu He Ser Ala Val Glu His 165 170 175

Leu His Arg Ala Gly Val^" Val His Arg Asp Leu Lys lie Glu Asn Leu 180 185 190

Leu Leu Asp Glu Asp Asn Asn He Lys Leu He Asp Phe Gly Leu Ser 195 200 205 sn Cys Ala Gly He Leu Gly Tyr Ser Asp Pro Phe Ser Thr Gin Cys 210 215 220

Gly Ser Pro Ala Tyr Ala Ala Pro Glu Leu Leu Ala Arg Lys Lys Tyr 225 230 235 240

Gly Pro Lys He Asp Val Trp Ser He Gly Val Asn Met Tyr Ala Met 245 250 255

Leu Thr Gly Thr Leu Pro Phe Thr Val Glu Pro Phe Ser Leu Arg Ala 260 265 270

Leu Tyr Gin Lys Met Val Asp Lys Ala Met Asn Pro Leu Pro Thr Gin 275 280 285

Leu Ser Thr Gly Ala Val Asn Phe Leu Arg Ser Leu Leu Glu Pro Asp 290 295 300

Pro Val Lys Arg Pro Asn He Gin Gin Ala Leu Ala Asn Arg Trp Leu 305 310 315 320

Asn Glu Asn Tyr Thr Gly Lys Val Pro Cys Asn Val Thr Tyr Pro Asn 325 330 335

Arg He Ser Leu Glu Asp Leu Ser Pro Ser Val Val Leu His Met Thr 340 345 350

Glu Lys Leu Gly Tyr Lys Asn Ser Asp Val He Asn Thr Val Leu Ser 355 360 365

Asn Arg Ala Cys His He Leu Ala He Tyr Phe Leu Leu Asn Lys Lys 370 375 380

Leu Glu Arg Tyr Leu Ser Gly Lys Ser Asp He Gin Asp Ser He Cys 385 390 395 400

Tyr Lys Thr Gin Leu Tyr Gin He Glu Lys Cys Arg Ala Thr Lys Glu 405 410 415

Pro Tyr Glu Ala Ser Leu Asp Thr Trp Thr Arg Asp Phe Glu Phe His 420 425 430

Ala Val Gin Asp Lys Lys Pro Lys Glu Gin Glu Lys Arg Gly Asp Phe 435 440 445

Leu His Arg Pro Phe Ser Lys Lys Leu Asp Lys Asn Leu Pro Ser His 450 455 460

Lys Gin Pro Ser Pro Ser Leu He Thr Gin Leu Gin Ser Thr Lys Ala 465 470 475 480

Leu Leu Lys Asp Arg Lys Ala Ser Lys Ser Gly Phe Pro Asp Lys Asp 485 490 495

Ser Phe Val Cys Arg Asn Leu Phe Arg Lys Thr Ser Asp Ser Asn Cys 500 505 510

Val Ala Ser Ser Ser Met Glu Phe He Pro Val Pro Pro Pro Arg Thr 515 520 525

Pro Arg He Val Lys Lys Leu Glu Pro His Gin Pro Gly Pro Gly Ser 530 535 540

Ala Ser He Leu Pro Lys Glu Glu Pro Leu Leu Leu Asp Met Val Arg 545 550 555 560

Ser Phe Glu Ser Val Asp Arg Glu Asp His He Glu Leu Leu Ser Pro 565 570 575

Ser His His Tyr Arg He Leu Ser Ser Pro Val Ser Leu Ala Arg Arg 580 585 590

Asn Ser Ser Glu Arg Thr Leu Ser Gin Gly Leu Leu Ser Gly Ser Thr 595 600 605

Ser Pro Leu Gin Thr Pro Leu His Ser Thr Leu Val Ser Phe Ala His 610 615 620

Glu Glu Lys Asn Ser Pro Pro Lys Glu Glu Gly Val Cys Ser Pro Pro 625 630 635 640

Pro Val Pro Ser Asn Gly Leu Leu Gin Pro Leu Gly Ser Pro Asn Cys 645 650 655

Val Lys Ser Arg Gly Arg Phe Pro Met Met Gly He Gly Gin Met Leu 660 665 670

Arg Lys Arg His Gin Ser Leu Gin Pro Ser Ser Glu Arg Ser Leu Asp 675 680 685

Ala Ser Met Ser Pro Leu Gin Pro He Ala Pro Ser Ser Leu Ser Phe 690 695 700

Asp Met Ala Asp Gly Val Lys Gly Gin Cys 705 710

<210> 2 <211> 2142 <212> DNA <213> Mus sp.

<220>

<223> Wild Type MAK V

<4O0> 2 atgccggcag cggcggggga cgggctcttg ggcgagccgg cggcaccggg gggcgatgga 60 ggcgcggagg acacgaccag gccggcggcg gcctgcgagg gaagtttcct gcccgcctgg 120 gtgagcggcg tgtcccgcga gcggctccgg gacttccagc accacaagcg cgtgggcaac 180 tacctcatcg gcagcaggaa gctgggagag ggctccttcg ccaaggtgcg cgaggggctg 240 cacgtgctga cgggagaaaa ggtagctatc aaggtcatcg ataagaaaag agccaagaaa 300 gacacctacg tcaccaaaaa cctgcgtcga gaggggcaga tccagcagat gatccgacac 360 cccaacatca cacagctcct ggacatcttg gagacagaga acagctacta cctggtcatg 420 gagctgtgtc ctggtggcaa cctcatgcac aagatctacg aaaagaaacg gttggatgaa 480 gccgaggccc gcagatacat ccggcaactc atctctgcgg tggaacacct gcaccgtgcg 540 ggggtggttc acagagactt gaagatagag aatttgctac tagatgaaga caataatatc 600 aagctgattg actttggctt gagcaactgt gcagggatcc taggttactc ggatccattc 660 agcacacagt gtggcagccc tgcctatgct gcgccagaac tgcttgccag gaagaaatat 720 ggccccaaaa ttgatgtctg gtcaataggc gtgaacatgt atgccatgct gacggggacc 780 ctacctttca ctgtggagcc tttcagcctg agggctctgt atcagaagat ggtggacaaa 840 gcaatgaatc ccctgccgac ccagctctcc acaggggccg tcaactttct gcgctccctc 900 ctggaaccag accctgtgaa gaggccgaat atccagcaag cgctggcgaa tcgctggttg 960 aatgagaatt acactggaaa ggtgccctgc aatgtcacct atcccaacag gatttctttg 1020 gaagacctga gtcccagcgt ggtgctgcac atgactgaaa agctgggcta taagaacagt 1080 gacgtcat-ca acacggtgct ctccaaccgc gcctgccaca tcctggccat ctacttcctg 1140 ttgaacaaga aacttgagcg ctatttgtca gggaaatcag atatccaaga tagcatctgc 1200 tacaagaccc agctctacca gatagagaag tgcagagcca ccaaggagcc ctatgaggcc 1260 tccctggata cctggacgag ggactttgaa ttccatgctg tgcaggataa aaagcccaaa 1320 gaacaagaaa aaagaggtga ttttctccac cgtccgtttt ccaagaagtt ggacaagaac 1380 ctgccttctc acaaacagcc atcgccctcg ctgatcacac agctccagag taccaaagcc 1440 ctgctcaaag acaggaaggc ctccaagtca ggcttccccg acaaagattc cttcgtctgc 1500 cgcaatcttt tccgaaaaac ctctgattcc aattgtgtgg cttcttcttc catggaattc 1560 atccctgtcc cacctcccag gacaccaagg attgtaaaga aactagagcc acaccaacca 1620 gggccgggaa gtgccagcat cctccccaag gaagagcccc tgctgctgga tatggtacgc 1680 tcctttgagt ctgtggatcg agaggaccac atagaactgc tgtccccttc tcaccattat 1740 aggatcctga gctcgcctgt gagcctggct cgtaggaatt ctagtgagag gacactctcc 1800 caggggctgc tgtccggaag tacctcacct ctccaaactc cactgcattc cacgctggtc 1860 tcttttgccc acgaagaaaa gaacagcccc ccgaaagagg agggtgtgtg ttcaccgcct 1920 cccgttccca gtaatggcct cctgcagcct ctggggagcc ccaactgtgt gaagagcagg 1980 ggacggttcc ccatgatggg catcggacag atgctgagga agcggcacca gagcctgcag 2040 ccttcctcag agaggtccct ggacgccagc atgtcccctc tgcagcccat agccccctcc 2100 agcctctcct ttgacatggc cgacggtgtc aagggccagt gt 2142

<210> 3

<211> 2142

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Mutated Mak V

<400> 3 atgccggcag cggcggggga cgggctcttg ggcgagccgg cggcaccggg gggcgatgga 60 ggcgcggagg acacgaccag gccggcggcg gcctgcgagg gaagtttcct gcccgcctgg 120 gtgagcggcg tgtcccgcga gcggctccgg gacttccagc accacaagcg cgtgggcaac 180 tacctcatcg gcagcaggaa gctgggagag ggctccttcg ccaaggtgcg cgaggggctg 240 cacgtgctga cgggagaaaa ggtagctatc cgggtcatcg ataagaaaag agccaagaaa 300 gacacctacg tcaccaaaaa cctgcgtcga gaggggcaga tccagcagat gatccgacac 360 cccaacatca cacagctcct ggacatcttg gagacagaga acagctacta cctggtcatg 420 gagctgtgtc ctggtggcaa cctcatgcac aagatctacg aaaagaaacg gttggatgaa 480 gccgaggccc gcagatacat ccggcaactc atctctgcgg tggaacacct gcaccgtgcg 540 ggggtggttc acagagactt gaagatagag aatttgctac tagatgaaga caataatatc 600 aagctgattg actttggctt gagcaactgt gcagggatcc taggttactc ggatccattc 660 agcacacagt gtggcagccc tgcctatgct gcgccagaac tgcttgccag gaagaaatat 720 ggccccaaaa ttgatgtctg gtcaataggc gtgaacatgt atgccatgct gacggggacc 780 ctacctttca ctgtggagcc tttcagcctg agggctctgt atcagaagat ggtggacaaa 840 gcaatgaatc ccctgccgac ccagctctcc acaggggccg tcaactttct gcgctccctc 900 ctggaaccag accctgtgaa gaggccgaat atccagcaag cgctggcgaa tcgctggttg 960 aatgagaatt acactggaaa ggtgccctgc aatgtcacct atcccaacag gatttctttg 1020 gaagacctga gtcccagcgt ggtgctgcac atgactgaaa agctgggcta taagaacagt 1080 gacgtcatca acacggtgct ctccaaccgc gcctgccaca tcctggccat ctacttcctg 1140 ttgaacaaga aacttgagcg ctatttgtca gggaaatcag atatccaaga tagcatctgc 1200 tacaagaccc agctctacca gatagagaag tgcagagcca ccaaggagcc ctatgaggcc 1260 tccctggata cctggacgag ggactttgaa ttccatgctg tgcaggataa aaagcccaaa 1320 gaacaagaaa aaagaggtga ttttctccac cgtccgtttt ccaagaagtt ggacaagaac 1380 ctgccttctc acaaacagcc atcgccctcg ctgatcacac agctccagag taccaaagcc 1440 ctgctcaaag acaggaaggc ctccaagtca ggcttccccg acaaagattc cttcgtctgc 1500 cgcaatcttt tccgaaaaac ctctgattcc aattgtgtgg cttcttcttc catggaattc 1560 atccctgtcc cacctcccag gacaccaagg attgtaaaga aactagagcc acaccaacca 1620 gggccgggaa gtgccagcat cctccccaag gaagagcccc tgctgctgga tatggtacgc 1680 tcctttgagt ctgtggatcg agaggaccac atagaactgc tgtccccttc tcaccattat 1740 aggatcctga gctcgcctgt gagcctggct cgtaggaatt ctagtgagag gacactctcc 1800 caggggctgc tgtccggaag tacctcacct ctccaaactc cactgcattc cacgctggtc 1860 tcttttgccc acgaagaaaa gaacagcccc ccgaaagagg agggtgtgtg ttcaccgcct 1920 cccgttccca gtaatggcct cctgcagcct ctggggagcc ccaactgtgt gaagagcagg 1980 ggacggttcc ccatgatggg catcggacag atgctgagga agcggcacca gagcctgcag 2040 ccttcctcag agaggtccct ggacgccagc atgtcccctc tgcagcccat agccccctcc 2100 agcctctcct ttgacatggc cgacggtgtc aagggccagt gt 2142

Claims

Claims

1. An isolated polypeptide encoding a centrosome associated kinase, or a fragment thereof comprising a centrosome binding site, wherein the polypeptide has decreased or no kinase activity.

2. An isolated polypeptide according to claim 1, having at least 80% homology with the amino acid shown in seq.ID No. 1.

3. An isolated polypeptide according to claim 1 or claim 2 comprising a mutation in an ATP binding domain of the kinase.

4. A nucleic acid molecule selected from a group consisting of:

(a) nucleic acid molecules encoding a polypeptide according to any one of claims 1 to 3;

(b) nucleic acid molecules having at least 80% homology with the nucleic acid sequence shown in Seq.ID No. 3;

(c) nucleic acid molecules, the complementary strand of which hybridises to a nucleic acid of (a) or (b); and

(d) nucleic acid molecules, the sequence of which differs from the sequence of a nucleic acid molecule of (c) due to the degeneracy of the genetic code; wherein a polypeptide encoded by the nucleic acid of sequence (a), (b), (c) or (d) comprises a centrosome binding site and has decreased or no kinase activity.

5. A vector comprising a nucleic acid molecule according to claim 4.

6. A host cell comprising a nucleic acid molecule according to claim 4 or a vector according to claim 5.

7. A method of identifying an inhibitor or activator of cell cycle progression comprising:

(a) providing a cell comprising a centrosome associated kinase; (b) adding a compound to the cell; and

(c) determining whether the compound stimulates or inhibits cell cycle progression.

8. A method according to claim 7, wherein the centrosome-associated kinase is non-naturally occurring in the cell.

9. A method according to claim 7 or claim 8, wherein the centrosome-associated kinase is a polypeptide according to claims 1 to 3 or Mak V.

10. A method according to any one of claims 7 to 9, wherein the centrosome-associated kinase is regulatable by means of an inducable or suppressible promotor.

11. An inhibitor or activator of centrosome-associated kinase identified by a method according to any one of claims 7 to 10.

12. An inhibitor or activator according to claim 11 in the manufacture of a medicament o treat uncontrolled cell cycle progression.