CA2280206A1 - Cyclin-dependent protein kinase - Google Patents

Abstract

An isolated nucleic acid molecule is disclosed which encodes a novel human cyclin-dependent kinase (CDK) which comprises a novel cyclin binding domain signature sequence and lacks several heretofore conserved amino acid residues involved in regulation of the cdk/cyclin complex. Associated proteins and biologically active mutant forms are also disclosed.

Description

w0 98/35015 PCT/US98/02337 TITLE OF THE :fNVENTION
CYCLIN-DEPErfDENT PROTEIN KINASE
CROSS-REFERENCE TO RELATED APPLICATIONS
Pro~risional Application U.S. Serial Number 60/037,855 filed February 7, 1997.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes a novel human cyclin-dependent kinas~e (CDK:) comprising a novel cyclin binding domain signature sequence and lacking several heretofore conserved amino acid residues involved in regulation of the cdk/cyclin complex. The present invention also relates to associated human CDK proteins and human CDK mutant proteins.
BACKGROUND OF THE INVENTION
Cell growth and division in eukaryotic organisms is mediated through the cell cycle. The cell cycle consists of two major events separated by two central gap phases. DNA synthesis and replication occwc during the S phase while mitosis occurs during the M
phase. A first gap phase, called G1, which occurs between the M phase and the S phase:, allowf~ for accumulation of enzymes and other compounds necessary to drive DNA synthesis and genome replication.
A second gap phase, called G2, occurs between the S phase and the M
phase, allowing for controls to check for proper DNA replication prior to committing to cell division.

protein kinases (CDKs). Activation of a CDK requires binding to a cyclin regulatory subunit, and in the case of CDKl - CDK6, phosophorylation of threonine 160/161 (Thr160/161). These CDKs contain a cyclin binding site near the amino terminal portion of the protein. The activated CDK/cyclin complex phosphorylates proteins involved in various stages of the cell cycle.
The family of cyclin proteins may generally be classified as either G1 cyclins or mitotic cyclins, depending on peak expression levels.
A CDK may bind a subset of cyclins. For example, CDK4 is known to bind cyclin D1 or cyclin D3 whereas CDK2 is known to bind cyclin A, cyclin B1, cyclin B2, cyclin B3 and cyclin E. The vertebrate cyclins show homology within a region of approximately 100 amino acids, referred to as the cyclin box. This region is responsible for CDK binding and activity (Kobayashi, et al., 1992, Molec. Biol. Cell. 3: 1279-1294; Lees, et al., 1993, Molec. Cell. Biol., 1993, 13: 1194-1201). It is this region of the cyclin protein which interacts with the cyclin binding domain of a respective CDK protein.
Complete activation of a known CDK/cyclin complex requires phosphorlyation by a CDK-Activating Kinase (CAK). The vertebrate CAK has been identified as a CDK/cyclin complex, more specifically CDK7/cyclinH (Fisher and Morgan, 1994, Cell 78: 713-724).
The CAK enzyme comprises a threonine 170 residue (in human CDK7) which has been shown to be required for optimal activity (Poon, et al., 1994, J. Cell Sci. 107: 2789-2799; Fisher and Morgan, 1994, Cell 78: 713-724).
Inhibition of CDK/cyclin complexes are thought to occur via phosphorylation at threonine 14 (Thrl4) and/or tyrosine 15 (Tyrl5) of the CDK subunit. The Weel kinase has been suggested as either a Thrl4 kinase or as a Thrl4 and TyrlS kinase. Additionally, CDC25 is thought to be a dual kinase targeting both Thrl4 and/or Tyrl5 (Morgan, 1995, Nature 374: 131-134).
It would be advantageous to identify a gene encoding an additional CDK protein. A nucleic acid molecule expressing a CDK
protein would be extremely useful in screening for compounds acting as a modulator of the cell cycle. Such a compound or compounds will be useful in controlling cell growth associated with cancer or immune cell proliferation. Additionally, the recombinant form of protein expressed from such a novel gene would be useful for an in vitro assay to determine specificity toward substrate proteins, inhibitors and cyclin activators.
Additionally, an isolated and purified (~DK10 cDNA which encodes CDK-10 or an active mutant thereof zvill also be useful for the recombinant production of large quantities of respec;l~ive protein. The ability to praducE~ large quantities of the protein ~~ould be useful for the production of a therapeutic agent comprising the (~DK10 protein or a mutant Such as i;he exemplified mutant disclosed herein. A therapeutic agent comprised of C.'.DliIO protein wotdd be useful in the treatment of cell cycle and/or ~,'.;rJK.7.(? .related diseases or conditions wh.i.ch are r esponsive. The present invention addresses and meets this need.
SUMMARY OF 'CHE INVENTION
The present invention relates to an isolated nucleic acid molecule (polynu.cleotide) which encodes a novel human cyclin-dependent kinasn. This CDK comprises a novel cyclin binding domain signature sequence (Pro-Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID NO:1), lacks Thrl4 and/or Tyrl.S, and also lacks the T-loop domain containing the conserved Th.r160/161 residue.
The presenlt invention relates to biologically active fragments or mutants o:f a novel isolated nucleic acid molecule which encodes mRNA f:xpressing a novel human cyclin-dependent kinase.
Any such biologically active fragment and/or mutant will encode a protein or protein fragment comprising a novel cyclin binding domain signature sequence (Pro-Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID NO:1), which lacks Thr:l4 and/or TyrlS as well as a T-loop domain containing the conserved Trir160/lfil residue. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use.
The isolated nucleic acid molecule of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
A preferred aspect of the present invention is disclosed in SEQ ID N0:11 and Figure 1, a human DNA fragment which encodes the novel human cyclin-dependent kinase, CDK10.
The present invention also relates to a substantially purified novel cyclin-dependent kinase which comprises a novel cyclin binding domain signature sequence (Pro-Asn-Gln-Ala-Leu-Arg-Glu; SE(~ ID
NO:1), lacks Thrl4 and Tyrl5 which make up the conserved ATP
binding motif of several known CKDs, and also lacks the T-loop domain containing the conserved Thr160/161 residue.
The present invention also relates to biologically active fragments and/or mutants of a novel cyclin-dependent kinase which comprises a novel cyclin binding domain signature sequence, lacks Thrl4 and/or Tyrl5 which make up the conserved ATP binding motif of known CKDs, and also lacks the T-loop domain containing the conserved Thr160/161 residue, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use.
A preferred aspect of the present invention is disclosed in SE(1 ID N0:3 and Figure 2, the amino acid sequence of CDK10.
The open reading frame of the CDK10 coding region runs from nucleotide 210 to nucleotide 1182 of SEQ ID N0:2.
Another preferred aspect of the present invention is disclosed in SEQ ID N0:11, wherein nucleotide 588 of the wild-type form (SEQ ID NO: 2) is mutated from "G" to "A".
Another preferred aspect of the present invention is the mutant protein, (CDK10-D127N), wherein nucleotide 588 of SEQ ID
N0:11 is mutated from "G" to "A", as compared to the wild-type form (SEQ ID N0:2), which results in a change of Asp127 to Asn127 as compared to the wild-type amino acid sequence (SEQ ID N0:3), disclosed as SEfa ID N0:12.
The present invention also relates to methods of expressing the cyclin-dependent kinases disclosed herein, assays employing these cyclin-dependent kinases, cells expressing these cyclin-dependent kinases, and compounds identified through the use of these cyclin-dependent kinases, including modulators of the cyclin-dependents kinase either through direct contact with the cyclin-dependent kinase, an associated cyclin, or the CKD/cyclin complex. Such modulators identified in this process are useful as therapeutic agents for controlling cell growth or inunune cell proliferation commonly associated with cancer.
DETAILED DESCRIPTION OF THE DRAWINGS
Figure 1 shows the nucleotide sequence (SEQ ID N0:2) which comprises the full length cDNA encoding human CDK10.
Figure 2 shows the amino acid sequence (SEQ ID N0:3) of human CDKh).
Figure 3 shows the strategy utilized to generate a full-length DNA frag;m.ent encoding human CDK10.
Figure 4 shows northern blot analysis of human tissue mRNA hybridized to a 32P-labeled probe from the 3' region of the DNA fragment encoding human CDK10.
Figyire 5 shows northern blot analysis of human tissue mRNA hybridized to a 32P-labeled probe from the 3' region of the DNA fragment encoding human CDK10.
DETAILED DE~~CRIPTION OF THE INVENTION
The present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes a novel cyclin-dependent kinase which co:mprise~e a novel human cyclin binding domain (Pro-Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID NO:1), lacks Thrl4 and/or Tyrl5 which make up the can;served ATP binding motif of known CDKs, and also lacks the T-loop domain containing the conserved Thr160/161 residue.
The; present invention also relates to biologically active fragments and/or mutants of a novel isolated nucleic acid molecule which encode mRNA expressing a novel human cyclin-dependent kinase. Such a protein comprises a novel cyclin binding domain signature sequence (Pro-Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID NO:1), lacks Thrl4 and/or Tyr lS, and also lack a T-loop domain containing the conserved Thr160/I61 residue. The protein of the present invention includes but is not limited to nucleotide substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA
S which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use.
A preferred aspect of the present invention is disclosed in Figure 1 and SEf~ ID N0:2, a human cDNA encoding a novel cyclin-dependent kinase, CDK10, disclosed herein as:
lO GAAAAGGCGC AGTGGGGCCC GGAGCTGTCA CCCCTGACTC GACGCAGCTT CCGTTCTCCT
GGTGACGTCG CCTACAGGAA CCGCCCCAGT GGTCAGCTGC CGCGCTGTTG CTAGGCAACA
GCGTGCGAGC TCAGATCAGC GTGGGGTGGA GGAGAAGTGG AGTTTGGAAG TTCAGGGGCA
CAGGGGCACA GGCCCACGAC TGCAGCGGGA TGGACCAGTA CTGCATCCTG GGCCGCATCG
GGGAGGGCGC CCACGGCATC GTCTTCAAGG CCAAGCACGT GGAGACTGGC GAGATAGTTG
IS CCCTCAAGAA GGTGGCCCTA AGGCGGTTGG AAGACGGCTT CCCTAACCAG GCCCTGCGGG
AGATTAAGGC TCTGCAGGAG ATGGAGGACA ATCAGTATGT GGTACAACTG AAGGCTGTGT
TCCCACACGG TGGAGGCTTT GTGCTGGCCT TTGAGTTCAT GCTGTCGGAT CTGGCCGAGG
TGGTGCGCCA TGCCCAGAGG CCACTAGCCC AGGCACAGGT CAAGAGCTAC CTGCAGATGC
TGCTCAAGGG TGTCGCCTTC TGCCATGCCA ACAACATTGT ACATCGGGAC CTGAAACCTG
ZO CCAACCTGCT CATCAGCGCC TCAGGCCAGC TCAAGATAGC GGACTTTGGC CTGGCTCGAG
TCTTTTCCCC AGACGGCAGC CGCCTCTACA CACACCAGGT GGCCACCAGG TCTGTGGGCT
GCATCATGGG GGAGCTGTTG AATGGGTCCC CCCTTTTCCC GGGCAAGAAC GATATTGAAC
AGCTTTGCTA TGTGCTTCGC ATCTTGGGCA CCCCAAACCC TCAAGTCTGG CCGGAGCTCA
CTGAGCTGCC GGACTACAAC AAGATCTCCT TTAAGGAGCA GGTGCCCATG CCCCTGGAGG
ZS AGGTGCTGCC TGACGTCTCT CCCCAGGCAT TGGATCTGCT GGGTCAATTC CTTCTCTACC
CTCCTCACCA GCGCATCGCA GCTTCCAAGG CTCTCCTCCA TCAGTACTTC TTCACAGCTC
CCCTGCCTGC CCATCCATCT GAGCTGCCGA TTCCTCAGCG TCTAGGGGGA CCTGCCCCCA
AGGCCCATCC AGGGCCCCCC CACATCCATG ACTTCCACGT GGACCGGCCT CTTGAGGAGT
CGCTGTTGAA CCCAGAGCTG ATTCGGCCCT TCATCCTGGA GGGGTGAGAA GTTGGCCCTG

CTTCACCCTC CAAGGCCTCC CCATGGCCAC AGTGGGCCCA CACCACACCC TGCCCCTTAG
CCCTTGCGAG GGTTGGTCTC GAGGCAGAGG TCATGTTCCC AGCCAAGAGT ATGAGAACAT
CCAGTCGAGC AGAGGAGATT CATGGCCTGT GCTCGGTGAG CCTTACCTTC TGTGTGCTAC
TGACGTACCC ATCAGGACAG TGAGCTCTGC TGCCAGTCAA GGCCTGCATA TGCAGAATGA

GAGTAAGGTG TCCTTATGTT GGAAACTCAA GTGGAAGGAA GATTTGGTTT GGTTTTATTC
TCAGAGCCAT TAAACACTAG TTCAGTATGT GAGATATAGA TTCTAAAAAC CTCAGGTGGC

TCTGCCTTAT GTCTG,TTCCT C'.CTTCATTTC TCTCAAGGGA AATGGCTAAG GTGGCATTGT
CTCATGGCTC TCGTTTTTGG CiGTCATGGGG AGGGTAGCAC CAGGCATAGC CACTTTTGCC
CTGAGGGACT CCTGTGTGCT TCACATCACT GAGCACTCAT TTAGAAGTGA GGGAGACAGA
AGTCTAGGCC CAGGGATGGC TCCAGTTGGG GATCCAGCAG GAGACCCTCT GCACATGAGG
S CTGGTTTACC AACATCTACT C'CCTCAGGAT GAGCGTGAGC CAGAAGCAGC TGTGTATTTA
AGGAAACAAG CGTTCCTGGA ~I,TTAATTTAT AAATTTAATA AATCCCAATA TAATCCCAAA
AA,AAAAAAAA AAAAAATTCC TGCGGCCGCA AGGA (SEQ ID N0:2).
The presenl; invention also relates to a substantially purified novel cyclin-dependent kinase which comprises a novel cyclin binding domain F>ignature sequence (Pro-Asn-Gln-Ala-Leu Arg-Glu; SEQ ID N0:1), lacks Thrl4 and/or Tyrl5 as well as the T-loop domain containing the conserved Thr160/I61 residue. Any such nucleic acid. may be isolated and characterized from a mammalian cell, including but not limited to human, human and 1S rodent. A human form :is an especially preferred form, such as the isolated cDNA exemplified herein as set forth in SEQ ID N0:2 and a dominant negative mutant form as set forth in SEQ ID N0:12.
The present; invention also relates to biologically active fragments and/or mutants of a novel cyclin-dependent kinase which comprises the navel cyclin binding domain (Pro-Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID N0:1), lacks Thrl4 and/or Tyrl5 which make up the conserved ATP banding motif of known CDKs, and also lacks the T-loop domain containing the conserved Thr160/161 residue, including but nol; necessarily limited to amino acid substitutions, 2S deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use.
Any such nucleic: acid may be isolated and characterized from a mammalian cell, including but not limited to human, human and rodent, with a human form being an especially preferred form.
A preferred aspect of the present invention is disclosed in SEQ ID N0:3 and Figure 2, the amino acid sequence of CDK10. The open reading frame of the CDK10 coding region runs from nucleotide 210 to nucleotide 1182 of SEfa l:D N0:2. The amino acid sequence of the novel 3S cyclin-dependent kinase, CDK10, is disclosed herein as:
MDQYCILGRI GEGAHGIVFK AKHVETGEIV ALKKVALRRL EDGFPNQALR
EIKALQEMED NQS.'VVQLK~~V FPHGGGFVLA FEFMLSDLAE VVRHAQRPLA

WO 9835015. PCTIUS 9 8 / 0 2 3 3 7 PCTNS98/02337 ~p'~~~ 0 3 SEP 1998 QAQVKSYLQM LLKGVAFCHA NNIVHRDLKP ANLLISASGQ LKIADFGLAR
VFSPDGSRLY THQV'ATRSVCi CIMGELLNGS PLFPGKNDIE QLCYVLRILG
TPNPQVWPEL TELPDYNKIS FKEQVPMPLE EVLPDVSPQA LDLLGQFLLY
PPHQRIAASK ALLHQYFFTA PLPAHPSELP IPQRLGGPAP KAHPGPPHIH
S DFHVDRPLEE SLLN'PELIRF' FILEG (SEQ ID N0:3).
Another preferred aspect of the present invention is disclosed in SEQ IUD NO:11, wherein nucleotide 588 of the wild-type form (SEfa ID NO: 2) is mutated from "G" to "A".
Another preferred aspect of the present invention is the ,..,~ 10 mutant protein, (CDK10-I)127N), wherein nucleotide 588 of SEQ ID
NO:11 is mutated from "G" to "A", as compared to the wild-type form (SEQ ID N0:2), which results in a change of Asp127 to Asn127 as compared to the wild-type amino acid sequence (SEQ ID N0:3), disclosed as SEQ ID N0:12.
1S The present invention also relates to methods of expressing the cyclin-dependent kinases disclosed herein, assays employing these cyclin-dependent ~:inases, cells expressing these cyclin-dependent kinases, and compounds identified through the use of these cyclin-dependent kinases, including modulators of the cyclin-dependents 20 kinase either through direct contact with the cyclin-dependent kinase, an associated cyclin, or the CKD/cyclin complex. Such modulators -~..
__:-e identified in this process .are useful as therapeutic agents for controlling cell growth or immune cell proliferation associated with human cancers. Additionally, an isolated and purified CDK10 cDNA which 2S encodes CDK-10 or an active mutant thereof will also be useful for the recombinant production o:f large quantities of respective protein. The ability to produce large quantities of the protein would be useful for the production of a the~rapeutiic agent comprising the CDK10 protein or a mutant such as the exemplified mutant disclosed herein. A therapeutic 30 agent comprised of CDK10 protein would be useful in the treatment of cell cycle and/or C~DK10 related diseases or conditions which are CDK10 responsive or possibly a therapeutic agent comprised of a mutant, including but not limited to CDK10-D127N, which may be useful in the treatment of cell cycle diseases or conditions which are responsive to the 3S regulatory effects of the mutant kinase.
The isolated nucleic acid molecule of the present invention may include a deo~xyribormcleic acid molecule (DNA), such as genomic d,N~ENDED SHEET

DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, siingle stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
It i;; known that there is a substantial amount of redundancy in t:he various colons which code for specific amino acids. Therefore., this invention is also directed to those DNA
sequences which contain alternative colons which code for the eventual translation of 'the identical amino acid. For purposes of this specification, a e~equence bearing one or more replaced colons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated proteiin which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine m~~y not cause a change in functionality of the polypeptide. Therefore, this invention is also directed to those DNA
sequences which express RNA comprising alternative colons which code for the eventual translation of the identical amino acid, as shown below:
A=Ala=Alanine: colons GCA, GCC, GCG, GCU
C=Cys=Cysteine: codon~s UGC, UGU
D=Asp=Aspartic: acid: c;odons GAC, GAU
E=Glu=Glutamic acid: colons GAA, GAG
F=Phe=Phenylal.anine: colons UUC, UUU
G=Gly=Glycine: colons GGA, GGC, GGG, GGU
H=His =Histidine: colons CAC, CAU
I=Ile =Isoleucin~e: colons AUA, AUC, AUU
K=Lys=Lysine: colons AAA, AAG
L=Leu=Leucine: colons UUA, UUG, CUA, CUC, CUG, CUU
M=Met=Methiorune: colon AUG
N=Asp=Asparag;ine: co~dons AAC, AAU
P=Pro=Proline: ~codons CCA, CCC, CCG, CCU
Q=Gln=Glutamine: colons CAA, CAG
R=Arg=ArgininE~: colons AGA, AGG, CGA, CGC, CGG, CGU
S=Ser=Serine: colons AGC, AGU, UCA, UCC, UCG, UCU

T=Thr=Threonine: codons ACA, ACC, ACG, ACU
V=Val=Valine: codons GUA, GUC, GUG, GUU
W=Trp=Tryptophan: codon UGG
Y=Tyr=Tyrosine: codons UAC, UAU
Therefore, the present invention discloses codon redundancy which may result in differing DNA molecules expressing an identical protein. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide. Methods of altering the DNA sequences include but are not limited to site directed mutagenesis.
Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide. Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis.
Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
As used herein, a "biologically active equivalent" or "functional derivative" of a wild type CDK possesses a biological activity that is substantially similar to the biological activity of the wild type CDK10 protein. The term "functional derivative" is intended to include the "fragments," "mutants," "variants," "degenerate variants," "analogs" and "homologues" or to "chemical derivatives" of the wild type CDK10 protein. The term "fragment" is meant to refer to any polypeptide subset of wild type CDK10. The term "mutant" is meant to refer to a molecule that may be substantially similar to the wild type form but possesses distinguishing biological characteristics. Such altered characteristics include but are in no fd way limited to altered enzymatic activity, altered cyclin binding altered substrate binding, altered substrate affinity and altered sensitivity to chemical compounds affecting biological activity.
An exemplified mutant i.s CDK10-D127N, wherein a single base mutation at nucleotide 588 of SEMI ID N0:2 results in a single amino acid substitution at residue 127, from aspartic acid to asparagine.
This mutation ah~ers kinase activity of CDK10-D127N as compared to the wild type CD K10 protein. The term "variant" is meant to refer to a molecule substaritially similar in structure and function to either the entire wild type ~~rotein or to a fragment thereof. A molecule is "substantially similar" to a wild type CDK10-like protein if both molecules have substantially similar structures or if both molecules possess similar b~iologicail activity. Therefore, if the two molecules possess substantiially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the fwo amino acid sequences are not identical.
The term "analog" refers to a molecule substantially similar in function to either the entire wild type CDK10-like protein or to a fragment thereof.
"Substantial homology" or "substantial similarity", when referring to nucleic acids means that the segments or their complementary ,strands,, when optimally aligned and compared, are identical with appropriate nucleotide insertions or deletions, in at least 75% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize to a strand or its complement.
The term "substantial homology", when referring to polypeptides, indicates that the polypeptide or protein in question exhibits at least about 30% homology with the naturally occurring protein in question, usually at least about 65% homology.
The nucleic: acids claimed herein may be present in whole cells or in cell lysates or in a partially purified or substantially purified form. A nucleic acid i.s considered substantially purified when it is purified away from environmental contaminants. Thus, a nucleic acid sequence isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
Any of a variety of procedures may be used to clone CDK10. These methods include, but are not limited to, (1) a RACE
PCR cloning technique (Frohman, et al., 1988, Proc. Natl. Acad.
Sci.85: 8998-9002). 5' and/or 3' RACE may be performed to generate a full length cDNA sequence. This strategy involves using gene-specific oligonucleotide primers for PCR amplification of CDK10 cDNA. These gene-specific primers are designed through identification of an expressed sequence tag (EST) nucleotide sequence which has been identified by searching any number of publicly available nucleic acid and protein databases;

(2) direct functional expression of the CDK10 cDNA following the construction of an CDK10-containing cDNA library in an appropriate expression vector system; (3) screening a CDK10-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled degenerate oligonucleotide probe designed from the amino acid sequence of the CDK10 protein; (4) screening a CDK10-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the CDK10 protein. This partial cDNA is obtained by the specific PCR amplification of CDK10 DNA
fragments through the design of degenerate oligonucleotide primers from the amino acid sequence known for other CDK
kinases which are related to the CDK10 protein; (5) screening an CDK10-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the CDK10 protein. This strategy may also involve using gene-specific oligonucleotide primers for PCR amplification of CDK10 cDNA
identified as an EST as described above; or (6) designing 5' and 3' gene specific oligonucleotides using SEQ ID N0:2 as a template so that either the full length cDNA may be generated by known RACE techniques, or a portion of the coding region may be generated by these same known RACE techniques to generate and isolate a portion of the coding region to use as a probe to screen one of numerous types of cDNA and/or genomic libraries in order to isolate a full length version of the nucleotide sequence encoding CDK10.
It is. readily apparent to those skilled in the art that other types of lix~raries, as well as libraries constructed from other cells types or spE~cies types, may be useful for isolating a CDK10-encoding DNA or a CDK10 homologue. Other types of libraries include, but are not limited to, cDNA libraries derived from other cells or cell lines other than human cells or tissue such as murine cells, rodent cells or any other such vertebrate host which may contain a CDK10-encoding DNA. Additionally a CDK10 gene ma~~ be isolated by oligonucleotide- or polynucleotide-based hybridization scrE~ening of a vertebrate genomic library, including but not limited to a human genomic library, a murine genomic library and a rodent genomic library, as well as concomitant human genomic DNA libraries.
It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from cells or cell lines which have CDK:10 actiwity. The selection of cells or cell lines for use in preparing a cDNA library to isolate a CDK10 cDNA may be done by first me~3suring cell associated CDK10 activity using any known assay for CDK activity.
Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA
library construction techniques can be found for example, in Sambrook, et al., 1989, .iVlolecukzr Cloning: A Laboratory Manual;
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Complementary DNA libraries may also be obtained from numerous commercial sources, including but not limited to Clontech Laboratories, Inc. and Stratagene.
It is, also readily apparent to those skilled in the art that DNA encoding CDK10 may also be isolated from a suitable genomic DNA literary. Construction of genomic DNA libraries can be performed by standard techniques well known in the art.
Well known genomic DNA library construction techniques can be found in Sambrook, et al., supra.
In order to clone the CDK10 gene by one of the preferred methods, the amino acid sequence or DNA sequence of CDK10 or a homologous protein may be necessary. To accomplish this, the CDK10 or a homologous protein may be purified and partial amino acid sequence determined by automated sequenators. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids can be determined for the PCR amplification of a partial CDK10 DNA fragment. Once suitable amino acid sequences have been identified, the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides.
Only one member of the set will be identical to the CDK10 sequence but others in the set will be capable of hybridizing to CDK10 DNA
IS even in the presence of DNA oligonucleotides with mismatches.
The mismatched DNA oligonucleotides may still sufficiently hybridize to the CDK10 DNA to permit identification and isolation of CDK10 encoding DNA. Alternatively, the nucleotide sequence of a region of an expressed sequence may be identified by searching one or more available genomic databases. Gene-specific primers may be used to perform PCR amplification of a cDNA of interest from either a cDNA library or a population of cDNAs. As noted above, the appropriate nucleotide sequence for use in a PCR-based method may be obtained from SEIa ID N0:2, either for the purpose of isolating overlapping 5' and 3' RACE products for generation of a full-length sequence coding for CDK10, or to isolate a portion of the nucleotide sequence coding for CDK10 for use as a probe to screen one or more cDNA- or genomic-based libraries to isolate a full-length sequence encoding CDK10 or CDK10-like proteins.
In an exemplified method, the RACE PCR technique (Frohman, et al., 1988, Proc. Nactl. Acad. Sci 85: 8998-9002) is used for cloning a 5'coding region of CDK10 encoding DNA. First round PCR used adapter-ligated human placenta cDNA template (from Clontech), gene-specific primer PK22L234, (5'-TGATGCAGCCCACAGACCTG-3 ; SEQ ID NO: 4) and an adapter primer AP1 1~

(5'-CCATCCTAA'TACCxACTCACTATAGGGC-3'; SEQ ID N0:5).
PCR amplif"lcation was performed using the ElongaseTM.
Thermal cycling was completed and a portion of this first PCR
reaction was added to a aecond PCR reaction as DNA template.
This PCR reaction also differed from the first PCR reaction in that the nested gene specific primer PK22L161 (5'-GCCGTCTGGGGAAAAGA-3'; SEQ ID N0:6) and the nested adapter primer h~P2 (5'-ACTCACTAT~~1GGGC;TCGAGCGGC-3', SEQ ID N0:7) were utilized.
An a.pproxirnately 600 by DNA product was identified from a 1% agarose electrophoresis gel, excised, and purified using a Qiagen PCR-spun column (Qiaquick T""). This fragment was used directly for IDNA sequencing using PK22L161 and AP2 primers, and for cloning into pCR2.1 using the Invitrogen TA-cloning kit.
A DhTA fragment 3' to and overlapping the 600 by 5' fragment was idE~ntified by searching public nucleic acid and protein database;. This 3' fragment is an approximately 1.8 Kb cDNA insert available a;s a NotI-HindIII fragment in a typical phagemid vector. This cDNA clone is readily identified by Genbank Accession No. H17727, Image Clone ID No. 50484, Washington University Clone ID No. ym40a06, and GBD Clone ID
No. 423294. This cDNA was isolated from a library constructed from human infant brain mRNA. This construct is available from Research Genetics,. Inc., 2130 Memorial Parkway SW, Hunstville, AL 3.5801 (h.ttp://www. resgen.com).
A full lengfh CDK10 coding region was assembled in pLITMUS28 (New England Biolabs) as an expression cassette with a BamHI site appended ;just 5' to the ATG translational start codon. A BamHl~:-XbaI fragment bearing CDK10 was recloned into pcDNA3.1 e:~pression vector (Invitrogen) and a BamHI-NcoI
fragment bearing; CDKIiD was recloned into pBlueBacHis2 baculovirus expression vector (Invitrogen). A similar construct was generated which contains dominant-negative single base pair mutation of CDK:10. This mutant was generated from pLITMUS28::CDK10 using the Stratagene "Quik Change" kit and primers 22U-D 127N
(5'-CAACATTGTACATCGGAACCTGAAACCTGCC-3'; SEQ ID
NO: 8) and 22L-D127N
(5'-GGCAGGTTTCAGGTTCC-GATGTACAATGTTG-3'; SEQ ID
NO: 9). Both mutant constructions were subcloned into pcDNA3.1 (as a BamHI-XbaI fragment) and pBlueBacHis2 (as a BamHI-NcoI fragment), respectively.
The sequence for the 5' upstream sequences, coding region and 3' untranslated sequences for the human full-length cDNA encoding CDK10 is shown in SEQ ID N0:2. The deduced amino acid sequence of CDK10 from the cloned cDNA is shown in SEQ ID N0:3. Inspection of the determined cDNA sequence reveals the presence of a single open reading frame that encodes a 325 amino acid protein. The open reading frame of the CDK10 coding region runs from nucleotide 210 to nucleotide 1182 of SEQ ID N0:2.
The nucleotide sequence which encodes a preferred mutant form (Asp127 to Asn127), is disclosed as SEQ ID N0:11.
The amino acid sequence for this preferred mutant form, CDK10-D127N, is disclosed in SEQ ID N0:12.
A variety of mammalian expression vectors may be used to express recombinant CDK10 in mammalian cells.
Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency.
Expression vectors may include, but are not limited to, cloning Ib vectors, modified cloning vectors, specifically designed plasmids or viruses.
Conirnercially available mammalian expression vectors which may be suitable for recombinant CDK10 expression, include but are not limited to, pcDNA3.1 (Invitrogen), pBlueBacHis2 (Invitrogen), pLITMUS~;B, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pS(15 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 371.99), p)R,SVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC x7460), .and ~,ZD35 (ATCC 37565).
A variety of bacterial expression vectors may be used to express recombinant CI)K10 in bacterial cells. Commercially available bacterial exprcasion vectors which may be suitable for recombinant CDlKlO expression include, but are not limited to pCR2.1 (Invitrogen), pE~~lla (Novagen), lambda gtll (Invitrogen), pcDNAII
(Invitrogen), pK1~223-3 (Pharmacia).
A variety of fungal cell expression vectors may be used to express recombinant CI)K10 in fungal cells. Commercially available fungal cell expression vectors which may be suitable for recombinant CDK10 expression include but are not limited to pYES2 (Invitrogen), Pichicz expression vector (Invitrogen).
A variety of insect cell expression vectors may be used to express recombinant receptor in insect cells. Commercially available insect cell expre;>sion vectors which may be suitable for recombinant expression of CDK10 include but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen).
The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, lipofection, protoplast fusion, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce CDK10 protein. Identification of CDK10 expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-CDK10 antibodies.

Expression of CDK10 DNA may also be performed using in vitro produced synthetic mRNA or native mRNA. Synthetic mRNA or mRNA isolated from CDK10 producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.
An expression vector containing DNA encoding a CDK10-like protein may be used for expression of CDK10 in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL
171).
The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation.
The expression vector-containing cells are individually analyzed to determine whether they produce CDK10 protein. Identification of CDK10 expressing cells may be done by several means, including but not limited to immunological reactivity with anti-CDK10 antibodies, and the presence of host cell-associated CDK10 activity.
The cloned CDK10 cDNA obtained through the methods described above may be recombinantly expressed by molecular cloning into an expression vector (such as pcDNA3.1, pCR2.l, pBlueBacHis2 and pLITMUS28) containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant CDK10. Techniques for such manipulations can be found described in Sambrook, et al., supra , I~

are discussed at length in the Example section and are well known and easily available to tile artisan of ordinary skill in the art.
Expression of CDK10 DNA may also be performed using in vitro produced syntlaetic mRNA. Synthetic mRNA can be efficiently translated in various cell-fi~ee systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into :frog ooc;ytes, with microinjection into frog oocytes being preferred.
To determine i~he CDK10 cDNA sequences) that yields optimal levels of CDK10 protein, CDK10 cDNA molecules including but not limited to the following can be constructed: the full-length open reading frame of the CDKlLO cDNA and various constructs containing portions of the cDNA encoding only specific domains of the protein or rearranged domain: of the protein. All constructs can be designed to contain nane, all or portions of the 5' and/or 3' untranslated region of CDK10. CDK10 activity and levels of protein expression can be determined following the introduction, both singly and in combination, of these constructs :into appropriate host cells. Following determination of the CDK10 cDN~!~ cassette yielding optimal expression in transient assays, this CDK10 cDNA construct is transferred to a variety of expression vectors ;including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteria, and yeast cells.
Levels of CDK.10 protein in host cells is quantified by a variety of techniquE~s including, but not limited to, immunoaffinity and/or ligand aff'lniity techniques. CDK10-specific affinity beads or CDK10-specific antibodies .are used to isolate 3~S-methionine labeled or unlabelled CDK10 F~rotein. Labeled CDK10 protein is analyzed by SDS-PAGE. Unlabelled CDK10 protein is detected by Western blotting, ELISA
or RIA assays employing C;DK10 specific antibodies.
Following expression of CDK10 in a host cell, CDK10 protein may be recovered to provide CDK10 in active form. Several CDK10 purification proced»res arE~ available and suitable for use. Recombinant CDK10 may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt :fractionation, ion exchange chromatography, size ~9 exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.
In addition, recombinant CDK10 can be separated from other cellular proteins by use of an immuno-affinity column made with monoclonal or polyclonal antibodies specific for full length CDK10, or polypeptide fragments of CDK10. Additionally, polyclonal or monoclonal antibodies may be raised against a synthetic peptide (usually from about 9 to about 25 amino acids in length) from a portion of the protein as disclosed in SEIa ID N0:3. Monospecific antibodies to CDK10 are purified from mammalian antisera containing antibodies reactive against CDK10 or are prepared as monoclonal antibodies reactive with CDK10 using the technique of Kohler and Milstein (1975, Nature 256:
495-497). Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for CDK10. Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the CDK10, as described above.
CDK10 specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with an appropriate concentration of CDK10 or CDK10 synthetic peptide either with or without an immune adjuvant.
Preimmune serum is collected prior to the first immunization. Each animal receives between about 0.1 ~.g and about 1000 ~g of CDK10 associated with an acceptable immune adjuvant. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium pa~rr~um and tRNA. The initial immunization consists of the CDK10 protein or CDK10 synthetic peptide in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP) or both. Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunizaiton. Those animals receiving booster injections are generally given an equal amount of CDK10 in Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20°C.
Monoclonal antibodies (mAb) reactive with CDK10 are prepared by immutuzing inbred mice, preferably Balb/c, with CDK10.
The mice are immunized b;y the IP or SC route with about 1 ~g to about 100 fig, preferably about l0i ~,g, of CDK10 in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred. The mice receive an initial immunization on da;y 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 1 to about :100 ~g of CDK10 in a buffer solution such as phosphate buffered saline by t;he intravenous (IV) route. Lymphocytes, from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cellLs are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions whiich will allow the formation of stable hybridomas. Fusion partners may include, but are not limited to:
mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (:DMEM) by procedures known in the art.
Supernatant fluids are collected form growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such. as solid phase immunoradioassay (SPIRA) using CDK10 as the antigen. Th.e culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb.
Hybridoma cells from antibody positive wells are cloned by a technique such as the soft ag:~r technique of MacPherson, 1973, Soft Agar Techniques, in Tis:>ue Culture Methods and Applicactions, Kruse and Paterson, Eds., Academic Press.
Monoclonal antibodies are produced in vivo by injection of pristine primed Balb/c mice, approximately 0.5 ml per mouse, with about 2 x 100 to about 6 x 106 hybridoma cells about 4 days after priming.
Ascites fluid is collected at; approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
In vitro production of anti-CDK10 mAb is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufl'lcient quantities of the specific mAb. The mAb are purified by techniques known in the art.
Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, pas sive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of CDK10 in body fluids or tissue and cell extracts.
It is readily apparent to those skilled in the art that the above described methods for producing monospecific antibodies may be utilized to produce antibodies specific for CDK10 polypeptide fragments, or full-length CDK10 polypeptide.
CDK10 antibody affinity columns are made by adding the antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8). The column is washed with water followed by 0.23 M
glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing CDK10 or CDK10 fragments are slowly passed through the column. The column is then washed with phosphate buffered saline until the optical density (A2g0) falls to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6). The purified CDK10 protein is then dialyzed against phosphate buffered saline.
The novel CDK10 of the present invention is suitable for use in an assay procedure for the identification of compounds which modulate CDK10 activity. Modulating CDK10 activity, as described herein includes the inhibition or activation of the protein and also includes directly or indirectly affecting the cell cycle regulatory properties associated with CDK10 activity. Compounds which modulate i~VO 98/35015 PCT/US98/02337 CDK10 activity include agonists, antagonists, inhibitors, activators, and compounds which directly o~r indirectly affect regulation of the CDK10 activity and/or the C:DK10/c;yclin association.
The CD~K10 protein kinase of the present invention may be obtained from both native and recombinant sources for use in an assay procedure to identif~;~ CDK10 modulators. In general, an assay procedure to identif3~ CDK1~0 modulators will contain the CDKIO-protein of the present invention, native cyclin protein which will form a CDK10/cyclin complex, and a test compound or sample which contains a putative CDK10 modulator. The test compounds or samples may be tested directly on, for example, purified CDK10 protein whether native or recombinant, subcellular fractions of CDK10-producing cells whether native or recombinant, andJor whole cells expressing the CDK10 whether native or recombinant. The test compound or sample may be added to the CDK10 in the ~aresence or absence of a known CDK10 modulator. The mo~3ulatin~; activity of the test compound or sample may be determined by, for example, analyzing the ability of the test compound or sample to bind to CDK10 protein, activate the protein, inhibit CDK10 activity, inhibit or enhance the binding of other compounds to the CDK10 protein, modifying receptor regulation, or modifying an intracellular activity.
The identification of modulators of CDK10 activity are useful in treating disease ~;tates involving the cell cycle will be useful in controlling cell grovvth associated with cancer or immune cell proliferation. Other compounds may be useful for stimulating or inhibiting activity of the enzyme. These compounds could be of use in the treatment of diseases in which activation or inactivation of the CDK10 protein resmlts in either cellular proliferation, cell death, nonproliferation, induction of cellular neoplastic transformations or metastatic tumor gt~owth and hence could be used in the prevention and/or treatment of various cancers.
The present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding a CDK protE~in of the present invention or which modulates the function of a such a CDK protein. Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules. Compounds may modulate by ~3 increasing or attenuating the expression of DNA or RNA encoding the CDK protein, or the function of a CDK protein. Compounds that modulate the expression of DNA or RNA encoding the CDK protein or the biological function thereof may be detected by a variety of assays.
The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample. Kits containing modified CDK10, antibodies to CDK10, or modified CDK10 protein may be prepared by known methods for such uses.
The DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of CDK10 DNA, RNA or protein. The recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of CDK10. Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant CDK10 protein or anti-CDK10 antibodies suitable for detecting CDK10. The carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.
Pharmaceutically useful compositions comprising modulators of CDK10 may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, or modified CDK10.
Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.

The ph;armace~utical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular"
The term "chemical derivative" describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicit3~ of the base molecule. Examples of such moieties are described in a variety o~f texts, such as Remington's Pharmaceutical Sciences.
Compounds identified according to the methods disclosed herein may be used alone a.t appropriate dosages. Alternatively, co-administration or sf~quenti.~l administration of other agents may be desirable.
The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds identified according to this invention as the active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. Fo:r example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
Advani;ageousay, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore:, compounds for the present invention can be administered in int:ranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin p~~tches well known to those of ordinary skill in that art. To be adnunistered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.
The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug Isolated and purified CDK10 is also be useful for the recombinant production of large quantities of CDK10 protein. The ability to produce large quantities of the protein would be useful for the production of a therapeutic agent comprising the CDK10 protein. A
therapeutic agent comprised of CDK10 protein would be useful in the treatment of cell cycle and/or CDK10 related diseases or conditions which are CDK10 responsive.
By computer analysis of a genomic database, molecular cloning and DNA sequencing a novel member of the human CDK
gene family has been identified. This new cDNA fragment encodes a novel cyclin-dependent kinase which comprises a novel cyclin binding domain signature sequence, lacks Thrl4 and/or Tyrl5 within the conserved ATP binding motif of known CDKs, and also lacks the T-loop domain containing the conserved Thr160/161 residue.
Northern hybridization experiments with RNA from various cell and tissues indicates that CDK10 is expressed in various human tissue, including brain, testis, pituitary gland and adrenal gland derived cells or tissues.
~b The following examples are provided as illustrative of the present invention without, however, limiting the same thereto.

Isolation and Characterization of DNA Fragments Encoding CDK
A 3' portion of the CDK10 coding region was detected among the Merck-Washington University EST's as 5' EST H17727. EST "H17727"
resembled several (~DK anal MAPK genes. The pH17727 plasmid construct comprising the 3' coding region and 3' untranslated region of CDK10 is contained within a NotI-HindIII fragment of approximately 1.8 Kb, in a typical phagetnid vector. The 5' portion of this fragment overlaps the 3' end of the 600 bp. This cDNA clone is publicly available by Genbank Accession. No. H:L7727, Image Clone ID No. 50484, Washington IS University Clone II) No. yrn40a06, and GBD Clone ID No. 423294. This cDNA was isolated. from a. library constructed from human infant brain mRNA. This consi;ruct is available from Research Genetics, Inc., 2130 Memorial Parkway SW, Hunstville, AL 35801 (http://www. resgen.com).
The 5' portion of the gene was isolated by performing 5' RACE (Frohman, et al., 1988, Proc. Natl. Acad. Sci.85: 8998-9002) using MarathonT"'-ready human placenta cDNA available from Clontech (Protocol #PT1156-1, Catalog #K1802-1). Adapter-ligated double stranded cDNA generated from human placenta mRNA was used as a template for PCR amplification using a gene specific primer PK22L234 (5'-TGATGCAGCCCACAGACCTG-3'; SEQ ID NO: 4) and an adapter primer AP1 (5'-C(~ATCC'rAATACGACTCACTATAGGGC-3' SEA ID
N0:5). PCR ampli~ficatior~ was performed using the ElongaseTM long-PCR enzyme mix (stored i:n 20mM Tris-HCl (pH 8.0 at 25°C), O.lmM
EDTA, 1mM DTT, stabili~:ers and 50%(v/v) glycerol) and PCR reaction buffer obtained from Gibco-BRL. The buffer comprised 300mM Tris-S04 (pH 9.1 at 25°C), 90mM (TfH4)2504 and 1.5 mM MgS04. Two microliters of Marathon placenta cDNA template and 10 pmoles each of PK22L234 and AP1 were added to the reaction mix and brought to a total volume of 20m1 with sterile iwater. 'thermal cycling was ( 1) 94°C/30sec, 68°C/6min for 5 cycles; {2) 94°'C/30sec, 64°C/30sec, 68°C/4min for 5 cycles; and, (3) a~

94°C/30sec, 62°C/30sec and 68°C/4min for 30 cycles. One microliter from a 1/20 dilution of this first PCR reaction was added to a second PCR
reaction as DNA template. This PCR reaction also differed from the first PCR reaction in that nested primers PK22L161 (5'-GCCGTCTGGGGA.AAAGA-3'; SEQ ID N0:6) and AP2 (5'-ACTCACTATAGGGCTCGAGCGGC-3', SEQ ID N0:7) were used.
An approximately 600 by PCR product was identified from a 1% agarose electrophoresis gel, excised, and purified using a Qiagen PCR-spun column. This fragment was used directly for DNA sequencing using PK22L161 and AP2 oligonucleotide primers.
The MarathonT""-ready human placenta cDNA available from Clontech is enhanced by ligation of a double-stranded, 5' overhang adapter to the double stranded cDNA template. The 3' end of the adapter is blocked by an amine group to prevent extension during PCR
amplification. It is within the non-extended 3' region that the AP1 oligo will hybridize. Therefore, AP1 does not hybridize and extend any of the original cDNA template molecules, instead beginning extension and amplification in the second round of PCR.

Construction of a Full Length DNA Fragment Encoding CDK10 The 3' portion of a DNA fragment which encodes CDK10 is contained within a DNA plasmid vector, pH17727. This insert contains a 5' XhoI site unique to the insert and a NcoI site in the 3' unstranslated region unique to the insert. This XhoI-NcoI fragment was isolated and subcloned into XhoI-NcoI digested pLITMUS28 plasmid DNA (New England Biolabs), resulting in pLITMUS28:H17727.
The 600 by PCR fragments obtained from 5' RACE were cloned into pCR2.1 (Invitrogen) using the Invitrogen TA-cloning kit as described by the manufacturer. A PmlI restriction site is located at approximately the midpoint of the 600 by PCR product. The PmII site was used to construct a wild type form of the 600 by 5' fragment from 2 independent 5' RACE PCR clones, pPK22bo4 and pPK22do4. The PmlI-BamHI restriction fragment of pPK22bo4 (which contains a ag mutation 3' to the PmlI site) was replaced with the with the PmlI-BamHI fragment of clone pPK22do4 (which contains a mutation 5' to the PmlI site ). The resulting clone, pPK22bo4/do4, overlaps the 5' portion of pH1772? through the unique XhoI restriction site. An SpeI-BamHI-NdeI restricltion site cluster was appended just 5' to the ATG
translational start codon by PCR-amplifying the insert from clone pPK22bo4/do4 using primers PK22L661 (5'-GCCGTCTGGGGAA.AACxA-3'; SEQ ID NO: fi) and PK22U210 (5'-GGACTAGTGG~~TCCA'rATGGACCAGTACTGCATCCT-3'; SEQ ID
NO:10). The resulting PCR fragment was digested with Spe1 and XhoI
and ligated into BamHI-XhoI digested pLITMUS28:H17727, resulting in pLITMUS28:CDK10 (Figure 3).

Construction of CDF~10 Mammalian Expression Vector A Baml I-Xbal: fragment from pLITMUS28:CDK10 comprising the CKD10 coding region was subcloned into the mammalian expression vector, pcDNA3.1 (Invitrogen), which was previously digested with BamHI and XbaI. The resulting construct, pcDNA3.1:CDK10, contains a portion of the CMV promoter and a T7 primer site upstream of the CDK10 ATG translational start codon as well as the BGH polyA region downstream of the translational termination codon. Of course, other components to allow growth in E. coli and mammalian cells are present in this vector.

Construction of CDK10 Ba<:ulovirus Transfer Vector A BamIHI-Ncol fragment from pLITMUS28:CDK10 containing the CKD10 coding region was cloned into the baculovirus expression vector, pBlueBacHis2 (Invitrogen), which was previously digested with BamP~I and NcoI. The resulting construct, pBBH:CDK10, may be used to express recombinant CDK10 from insect cells by following the manufacturer's instructions (e.g., see Invitrogen Cat. No.
V375-20 for pBlueBacHis2 A, B, and C).

Construction of DNA Fragment Encoding a CDK10 Dominant-Negative Mutant The pLITMUS:CDK10 construct (see Example 2) was mutated to generate a "dominant-negative" single base pair mutation.
This mutation was generated from pLITMUS28:CDK10 using the Stratagene "Quik Change" kit and primers 22U-D127N:
(5'-CAACATTGTACATCGGAACCTGAAACCTGCC-3'; SEQ ID N0:8), and 22L-D127N: (5'-GGCAGGTTTCAGGTTCCGATGTACAATGTTG-3';
SE(a ID NO: 9), according to the manufacturer's instructions. The dominant-negative mutation changes the codon GAC (at nucleotides 588-590 of SEQ ID N0:2) to AAC (at nucleotides 588-590 of SE(a ID NO:11), thus deletion essential amino acid Asp127 to Asn127 (see SEQ ID N0:12), which inactivates kinase activity (see Example 7 and van den Heuvel &
Harlow, 1993, Science 262:2050-2054). A CDK10-D 127N construction was subcloned into pcDNA3.1 (as a BamHI-Xbal fragment), resulting in pcDNA3.1:CDK10-d127N. A CDK10-D127N construction was also subcloned into pBlueBacHis2 (as a BamHI-Nco1 fragment), resulting in pBBH:CDK10-d127N.

Tissue Distribution Of CDK10 Expression Human multiple tissue Northern Blot #7760-1, Human Brain Northern Blot II #7755-1, Human Brain Northern Blot III #7750-1, and "Human multiple tissue Northern Dot Blot were purchased from Clontech. The probe was made by PCR amplifying the NotI-HindIII
insert from pH17727 using the "Universal"

3~

-~WO 98/35015 PCT/US98/02337 {5'-CCCAGTCACGA,CGTT(~TAAA.ACG-3 ; SEQ ID N0:13) and "Reverse" (5'-AGCG~GATAACA,ATTTCACACAGG-3': SEQ ID N0:14) primers from Gibco :BRL. Twenty-five ng of the probe was labeled with 32p using a Pharmacia "Ready-to-go" random priming kit and hybridized to the four Northern blots at high stringency according to Clontech instructions.
Figure 4 and Figure 5 show Northern data indicating the presence of CDK10 i~ranscri.pts in a variety of adult human tissue (Figure 4) as well as in specific regions of the adult and fetal human brain (Figure 5). This data shows increased expression levels in the testis as well as in pituitary and adrenal glands. Expression in various regions of the brain was relatively constant, with increased expression seen in the frontal and temporal lolbes and the cerebral cortex.

Effect of CDK:D127hT on Cell Growth Human osteosarcoma cell line Saos2 (ATCC HBT-85) was grown in DMEM hi;~h glucose medium + glutamine +10% fetal calf serum (in concentrations as recommended by Gibco-BRL). Two replicates of the exl>eriment were performed sequentially. Cells were split 1:6 into 10 cm culture dishes two days prior to transfection.
Transfection was pE~rformed using the CaP04 method according to Chen and Okayama ( 1987, Mol. ocnd Cell. Biol. 7:~ 2745-2752). Ten ug of each plasmid DNA {pcDI~TA3.1, pcDNA3:CDK10, pcDNA3:CDK10-D127N) was transfected into ~6a~% confl.uent cells in each 10 cm dish. Cells were rinsed 2x with Dulb~ecco's PBS (Gibco-BRL) and 10 mL fresh medium was added. After two days, cells were trypsinized and plated in 12 well dishes in fresh medium + 500ug/mL geneticin (Gibco-BRL). At 11 and 16 days after plating, colony counts were made to determine how many transfected cells were capable of growth and colony formation (Table 1).
This data indicates that expression of the kinase inactive "dominant-negative" form of C17K10 (i.e., CDK10-D127N) impairs colony formation by analogy to the data presented in van den Heuvel and Harlow (1993, Science 262: 2050-2054).

Day 11 (Colonies) Day 16 (# Colonies) DNA construct Ren A Reu B Ren A Ren B
no DNA 2 1 0 0 cDNA3.1 23 42 16 40 cDNA3.1:CDK10 3 2 5 9 23 49 cDNA3.1:CDK-D127N 5 I 2 I

Specific Effect of the Dominant Mutant CDK:D127N on Expression of Cell Cycle Genes HeLa cervical carcinoma cells were treated for 48 hours with a control adenovirus deleted for the E1 and E3 genes or the same adenovirus which comprised the construct encoding CDK10-D127N.
Western blots were performed with a rabbit antibody raised to the C-terminal 25 amino acids of the CDK10 protein (amino acid 301 - amino acid 325 of SEQ ID NO: 3). The cell line transfected with Ad/CDK10-D127N expressed CDK10-DI27N at a 50-fold higher level than endogenous, wild type CDK10. The two infected cell populations were subjected to mRNA isolations and probes were prepared for gene expression DNA chip studies essentially as described by Lockhart, et al.
(1996, Nature Biotechnology 14:1675-1680). Among the genes which were suppressed at the mRNA level by CDK10-D127N are summarized in Table 2.
3~

i~VO 98/35015 PCT/US98/02337 GENE Ad/CDK10- Ad- Control CDC2~ib 8.3 19.1 CDK7 3.1 8.5 CKSl 2Ei.3 82.5 CKS2 17L.6 98.3 Cyclin lfi.5 41.5 B

Cyclin 4.5 11.5 poly- 186.5 664.5 Ubiquitin I Quantified arbitrary expression units measured from the fluorescence image of the oli~;onucleotide array.
These data indicate a cell cycle block by the dominant-negative mutant gene, CDK10-D 1271\f, which shows the importance of the CDK10 protein to the cell cycle. Cell cycle analysis (using a fluorescence-activated cell sorter, or FAGS) of cells treated for 48 hours with the two viruses indicate that cells are not blocked in any particular phase of the cell cycle.
The dai;a reported in the above Example sections show the importance of CDK10 in the cell cycle. Therefore, a therapeutic agent comprising the CDK:10 protein would be useful in the treatment of cell cycle and/or CDK10 related diseases or conditions which are CDK10 responsive as well as showing a potential use for a dominant-negative mutant such as CD1~10-D1:?7N, whach may be useful in the treatment of cell cycle diseases oo conditions which are responsive to the mtuant proteins ability to rc;gulate a phase or phases of the cell cycle.
3~

SEQUENCE LISTIDIG
(1) GENERAL INFORMATION:
(i) APPLICANT: Gerhold, David L.
(ii) TITLE OF INVENTION: CYCLIN-DEPENDENT PROTEIN KINASE
(iii) NUMBER OF SEQUENCES: 14 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Merck & Co., Inc.
(B) STREET: P.O. Box 2000, RY60-30 (C) CITY: Rahway (D) STATE: NJ
(E) COUNTRY: US
(F) ZIP: 07065-0907 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTV~ARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hand, J. Mark (B) REGISTRATION NUMBER: 36,545 (C) REFERENCE/DOCKET NUMBER: 19885Y
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 732/594-3905 (B) TELEFAX: 732/594-4720 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Pro Asn Gln Ala Leu Arg Glu (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2074 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ
ID N0:2:

GAAAAGGCGCAGTGGGGC~CGGAGCTGTCACCCCTGACTCGACGCAGCTTCCGTTCTCCT 60 CCCTCAAGAAGG'rGGCCCTAAGGCGGTTGGAAGACGGCTTCCCTAACCAGGCCCTGCGGG 360 AGATTAAGGCTC'rGCAGGAGATGGAGGACAATCAGTATGTGGTACAACTGAAGGCTGTGT 420 AGCTTTGCTATGTGCTTCGCATCTTGGGCACCCCAAACCCTCAAGTCTGGCCGGAGCTCA 84~) CTGAGCTGCCGGACTACAACAAGF,TCTCCTTTAAGGAGCAGGTGCCCATGCCCCTGGAGG 900 AGGTGCTGCCTGACGTCTCTCCCC'.AGGCATTGGATCTGCTGGGTCAATTCCTTCTCTACC 960 AGGCCCATCCAGGGCCCC'CCCACATCCATGACTTCCACGTGGACCGGCCTCTTGAGGAGT 1140 CGCTGTTGAACCCAGAGC'TGATTC:GGCCCTTCATCCTGGAGGGGTGAGAAGTTGGCCCTG 1200 GTCCCGTCTGCCTGCTCC'TCAGGACCACTCAGTCCACCTGTTCCTCTGCCACCTGCCTGG 1260 CCCTTGCGAGGGTTGGTC'TCGAGC>CAGAGGTCATGTTCCCAGCCAAGAGTATGAGAACAT 1380 3~ .

(2) INFORMATION
FOR SEQ
ID N0:3:

(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH:325 amino acids (B) TYPE:
amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY:
linear (ii) MOLECULE
TYPE:
protein (xi)SEQUENCE DESCRIPTION: N0:3:
SEQ
ID

MetAsp GlnTyr CysIleLeu GlyArgIle GlyGluGly AlaHisGly 1 5 ~ 10 15 IleVal PheLys AlaLysHis ValGluThr GlyGluIle ValAlaLeu LysLys ValAla LeuArgArg LeuGluAsp GlyPhePro AsnGlnAla LeuArg GluIle LysAlaLeu GlnGluMet GluAspAsn GlnTyrVal ValGln LeuLys AlaValPhe ProHisGly GlyGlyPhe ValLeuAla PheGlu PheMet LeuSerAsp LeuAlaGlu ValValArg HisAlaGln ArgPro LeuAla GlnAlaGln ValLysSer TyrLeuGln MetLeuLeu 3~

Lys Gly Val Al.a Phe Cys His Ala Asn Asn Ile Val His Arg Asp Leu Lys Pro Ala As;n Leu Leu Ile Ser Ala Ser Gly Gln Leu Lys Ile Ala Asp Phe Gly Leu Ala Arg Val Phe Ser Pro Asp Gly Ser Arg Leu Tyr Thr His Gln Val Ala '7~hr Arg Ser Va1 Gly Cys Ile Met Gly Glu Leu Leu Asn Gly Ser Pro Leu Phe Pro Gly Lys Asn Asp Ile Glu Gln Leu 1E~0 185 190 Cys Tyr Val Le~u Arg 7Cle Leu Gly Thr Pro Asn Pro Gln Val Trp Pro Glu Leu Thr Gl.u Leu I?ro Asp Tyr Asn Lys Ile 5er Phe Lys Glu Gln Val Pro Met Pro Leu Glu Glu Val Leu Pro Asp Val Ser Pro Gln Ala Leu Asp Leu Leu Gly C3ln Phe Leu Leu Tyr Pro Pro His Gln Arg Ile 24~i 250 255 Ala Ala Ser L~~s Ala heu Leu His Gln 'hyr Phe Phe Thr Ala Pro Leu 2f>0 265 270 Pro Ala His Pro Ser Glu Leu Pro Ile Pro Gln Arg Leu Gly Gly Pro Ala Pro Lys A:La His 3?ro Gly Pro Pro His Ile His Asp Phe His Val Asp Arg Pro Le:u Glu Glu Ser Leu Leu Asn Pro Glu Leu Ile Arg Pro 305 :310 315 320 Phe Ile Leu G:Lu Gly (2) INFORMATION FOR SEQ I17 N0:4:
( i ) SEQUEPdCE c~HARACT:ERISTICS
(A) LENG'PH: 20 abase pairs (B) TYPE: nuc:Leic acid ( C ) STRA1JDEDNES;S : s ing 1 a (D) TOPOhOGY: linear (ii) MOLECULE 'TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: S:

(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:

(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

3~

(2) INFORMATION FOR SEQ I17 N0:8:
( i ) SEQUENCE C:HARACTIsRISTICS
(A) LENG".!'H: 31 base pairs (B) TYPE.: nucleic acid (C) STRALJDEDNESS: single (D) TOPOhOGY: linear (ii) MOLECULE 7:'YPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi ) SEQUENCE I)ESCRIP'PION: SEQ ID NO: 3 CAACATTGTA CATCGGA)~CC TGAi~ACCTGC C 31 ( 2 ) INFORMATLON FOIL SEQ I17 NO : 9 (i) SEQUENCE CHARACTERISTICS:
(A) LENG'a'H: 31 base pairs (B) TYPE'; nucleic acid ( C ) STRAIdDEDNESS : s ing 1 a (D) TOPOhOGY: linear (ii) MOLECULE ~('YPE: other nucleir_ acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE I)ESCRIP'rION: SEQ ID N0:9:
GGCAGGTTTC AC;GTTCCGAT GTACAATGTT G 31 (2) INFORMATION FOR SEQ I1J N0:10:
( i ) SEQUENCE I~HARACT:ERISTICS
(A) LENG'~H: 36 '.base pairs (B) TYPE: nuc:Leic acid ( C ) STRA1JDEDNES;S : s ing 1 a (D) TOPOhOGY: linear (ii) MOLECULE 'TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQtJENCE :JESCRIP'TION: SEQ ID N0:10:
GGACTAGTGG ATCCATA'1'GG ACCAGTACTG CATCCT 36 3 °/

(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2074 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:11:

Nn CCAGTCGAGCAGAGGAG.ATTCATGGCCTGTGCTCGGTGAG CCTTACCTTCTGTGTGCTAC 1440 TCAGAGCCATTAAACAC'rAGTTCAGTATGTGAGATATAGA TTCTAAAAACCTCAGGTGGC 1680 CTCATGGCTCTCGTTTT'rGGGGTCATGGGGAGGGTAGCAC CAGGCATAGCCACTTTTGCC 1800 CTGAGGGACTC(:TGTGTGCTTCACATCACTGAGCACTCAT TTAGAAGTGAGGGAGACAGA 1860 CTGGTTTACCAACATCT.ACTCCCTCAGGATGAGCGTGAGC CAGAAGCAGCTGTGTATTTA 1980 AAAA.AAAAAAAAAAAAT'rCCTGCGGCCGCAAGGA 2074 (2) INFORMATION
FOR SEQ
ID N0:12:

(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH:325 amino acids (B) TYPE:
amino acid (C) STRAP1DEDNESS:
single (D) TOPOLOGY:
linear (ii) MOLECULE
'TYPE:
protein {xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Asp Gln Tyr Cys Ile Leu Gly Arg Ile Gly Glu Gly Ala His Gly 1 5 ~ 10 15 Ile Val Phe Lys Ala Lys His Val Glu Thr Gly Glu Ile Val Ala Leu Lys Lys Val Ala Leu Arg Arg Leu Glu Asp Gly Phe Pro Asn Gln Ala Leu Arg Glu Ile Lys Ala Leu Gln Glu Met Glu Asp Asn Gln Tyr Val Val Gln Leu Lys Ala Val Phe Pro His Gly Gly Gly Phe Val Leu Ala Phe Glu Phe Met Leu Ser Asp Leu Ala Glu Val Val Arg His Ala Gln Arg Pro Leu Ala Gln Ala Gln Val Lys Ser Tyr Leu Gln Met Leu Leu Lys Gly Val Ala Phe Cys His Ala Asn Asn Ile Val His Arg Asn Leu Lys Pro Ala Asn Leu Leu Ile Ser Ala Ser Gly Gln Leu Lys Ile Ala Asp Phe Gly Leu Ala Arg Val Phe Ser Pro Asp Gly Ser Arg Leu Tyr Thr His Gln Val Ala Thr Arg Ser Val Gly Cys Ile Met Gly Glu Leu Leu Asn Gly Ser Pro Leu Phe Pro Gly Lys Asn Asp Ile Glu Gln Leu Cys Tyr Val Leu Arg Ile Leu Gly Thr Pro Asn Pro Gln Val Trp Pro Glu Leu Thr Glu Leu Pro Asp Tyr Asn Lys Ile Ser Phe Lys Glu Gln Val Pro Met Pro Leu Glu Glu Val Leu Pro Asp Val Ser Pro Gln Ala Leu Asp Leu Leu Gly Gln Phe Leu Leu Tyr Pro Pro His Gln Arg Ile Ala Ala Ser Lys Ala Leu Leu His Gln Tyr Phe Phe Thr Ala Pro Leu Pro Ala His Pro Ser Glu Leu Pro Ile Pro Gln Arg Leu Gly Gly Pro Ala Pro Lys Ala His Pro Gly Pro Pro His Ile His Asp Phe His Val Asp Arg Pro Leu Glu Glu Ser Leu Leu Asn Pro Glu Leu Ile Arg Pre Phe Ile Leu Glu Gly (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LEIQGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide"
4~

(xi ) SEQUENCE DESCRIP~.L'ION: SEQ ID N0: 13 CCCAGTCACG ACGTTGT~sAA AC:G 23 (2) INFORMATION FOF; SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE; nucleic acid (C) STRArdDEDNESS: single (D) TOPOhOGY: linear (ii) MOLECULE 7:'YPE: other nucleic acid (A) DESCRIPTION: /desc = "Oligonucleotide"
(xi) SEQUENCE I)ESCRIP'.t'ION: SEQ ID N0:14:
AGCGGATAAC AATTTCAC:AC AC=G 23 u3

Claims (28)

WHAT IS CLAIMED IS:

1. A purified DNA molecule encoding a human cyclin dependent kinase wherein said protein comprises the peptide motif Pro-Asn-Gln-Ala-Leu-Arg-Glu at the amino terminal region of the protein for cyclin binding.

2. A purified DNA molecule of Claim 1 wherein said protein lacks a conserved threonine residue and conserved tyrosine residue within a conserved ATP-binding motif at the amino terminal region of said protein.

3. A purified DNA molecule of Claim 2 wherein said protein lack a conserved T-loop domain at the carboxy terminal region of said protein.

4. A purified DNA molecule of Claim 3 which comprises the nucleotide sequence as follows:
GAAAAGGCGC AGTGGGGCCC GGAGCTGTCA CCCCTGACTC GACGCAGCTT CCGTTCTCCT
GGTGACGTCG CCTACAGGAA CCGCCCCAGT GGTCAGCTGC CGCGCTGTTG CTAGGCAACA
GCGTGCGAGC TCAGATCAGC GTGGGGTGGA GGAGAAGTGG AGTTTGGAAG TTCAGGGGCA
CAGGGGCACA GGCCCACGAC TGCAGCGGGA TGGACCAGTA CTGCATCCTG GGCCGCATCG
GGGAGGGCGC CCACGGCATC GTCTTCAAGG CCAAGCACGT GGAGACTGGC GAGATAGTTG
CCCTCAAGAA GGTGGCCCTA AGGCGGTTGG AAGACGGCTT CCCTAACCAG GCCCTGCGGG
AGATTAAGGC TCTGCAGGAG ATGGAGGACA ATCAGTATGT GGTACAACTG AAGGCTGTGT
TCCCACACGG TGGAGGCTTT GTGCTGGCCT TTGAGTTCAT GCTGTCGGAT CTGGCCGAGG
TGGTGCGCCA TGCCCAGAGG CCACTAGCCC AGGCACAGGT CAAGAGCTAC CTGCAGATGC
TGCTCAAGGG TGTCGCCTTC TGCCATGCCA ACAACATTGT ACATCGGGAC CTGAAACCTG
CCAACCTGCT CATCAGCGCC TCAGGCCAGC TCAAGATAGC GGACTTTGGC CTGGCTCGAG
TCTTTTCCCC AGACGGCAGC CGCCTCTACA CACACCAGGT GGCCACCAGG TCTGTGGGCT
GCATCATGGG GGAGCTGTTG AATGGGTCCC CCCTTTTCCC GGGCAAGAAC GATATTGAAC
AGCTTTGCTA TGTGCTTCGC ATCTTGGGCA CCCCAAACCC TCAAGTCTGG CCGGAGCTCA
CTGAGCTGCC GGACTACAAC AAGATCTCCT TTAAGGAGCA GGTGCCCATG CCCCTGGAGG
AGGTGCTGCC TGACGTCTCT CCCCAGGCAT TGGATCTGCT GGGTCAATTC CTTCTCTACC
CTCCTCACCA GCGCATCGCA GCTTCCAAGG CTCTCCTCCA TCAGTACTTC TTCACAGCTC
CCCTGCCTGC CCATCCATCT GAGCTGCCGA TTCCTCAGCG TCTAGGGGGA CCTGCCCCCA

AGGCCCATCC AGGGCCCCCC CACATCCATG ACTTCCACGT GGACCGGCCT CTTGAGGAGT
CGCTGTTGAA CCCAGAGCTG ATTCGGCCCT TCATCCTGGA GGGGTGAGAA GTTGGCCCTG
GTCCCGTCTG CCTGCTCCTC AGGACCACTC AGTCCACCTG TTCCTCTGCC ACCTGCCTGG
CTTCACCCTC CAAGGCCTCC CCATGGCCAC AGTGGGCCCA CACCACACCC TGCCCCTTAG
CCCTTGCGAG GGTTGGTCTC GAGGCAGAGG TCATGTTCCC AGCCAAGAGT ATGAGAACAT
CCAGTCGAGC AGAGGAGATT CATGGCCTGT GCTCGGTGAG CCTTACCTTC TGTGTGCTAC
TGACGTACCC ATCAGGACAG TGAGCTCTGC TGCCAGTCAA GGCCTGCATA TGCAGAATGA
CGATGCCTGC CTTGGTGCTG CTTCCCCGAG TGCTGCCTCC TGGTCAAGGA GAAGTGCAGA
GAGTAAGGTG TCCTTATGTT GGAAACTCAA GTGGAAGGAA GATTTGGTTT GGTTTTATTC
TCAGAGCCAT TAAACACTAG TTCAGTATGT GAGATATAGA TTCTAAAAAC CTCAGGTGGC
TCTGCCTTAT GTCTGTTCCT CCTTCATTTC TCTCAAGGGA AATGGCTAAG GTGGCATTGT
CTCATGGCTC TCGTTTTTGG GGTCATGGGG AGGGTAGCAC CAGGCATAGC CACTTTTGCC
CTGAGGGACT CCTGTGTGCT TCACATCACT GAGCACTCAT TTAGAAGTGA GGGAGACAGA
AGTCTAGGCC CAGGGATGGC TCCAGTTGGG GATCCAGCAG GAGACCCTCT GCACATGAGG
CTGGTTTACC AACATCTACT CCCTCAGGAT GAGCGTGAGC CAGAAGCAGC TGTGTATTTA
AGGAAACAAG CGTTCCTGGA ATTAATTTAT AAATTTAATA AATCCCAATA TAATCCCAAA
AAAAAAAAAA AAAAAATTCC TGCGGCCGCA AGGA, set forth as SEQ ID NO:2

5. A DNA molecule of Claim 4 which comprises from about nucleotide 210 to about nucleotide 1185 of SEQ ID NO:2.

6. A purified DNA molecule encoding a human cyclin dependent kinase wherein said DNA molecule encodes a protein comprising the amino acids sequence as follows:
Met Asp Gln Tyr Cys Ile Leu Gly Arg Ile Gly Glu Gly Ala His Gly Ile Val Phe Lys Ala Lys His Val Glu Thr Gly Glu Ile Val Ala Leu Lys Lys Val Ala Leu Arg Arg Leu Glu Asp Gly Phe Pro Asn Gln Ala Leu Arg Glu Ile Lys Ala Leu Gln Glu Met Glu Asp Asn Gln Tyr Val Val Gln Leu Lys Ala Val Phe Pro His Gly Gly Gly Phe Val Leu Ala Phe Glu Phe Met Leu Ser Asp Leu Ala Glu Val Val Arg His Ala Gln Arg Pro Leu Ala Gln Ala Gln Val Lys Ser Tyr Leu Gln Met Leu Leu Lys Gly Val Ala Phe Cys His Ala Asn Asn Ile Val His Arg Asp Leu Lys Pro Ala Asn Leu Leu Ile Ser Ala Ser Gly Gln Leu Lys Ile Ala Asp Phe Gly Leu Ala Arg Val Phe Ser Pro Asp Gly Ser Arg Leu Tyr Thr His Gln Val Ala Thr Arg Ser Val Gly Cys Ile Met Gly Glu Leu Leu Asn Gly Ser Pro Leu Phe Pro Gly Lys Asn Asp Ile Glu Gln Leu Cys Tyr Val Leu Arg Ile Leu Gly Thr Pro Asn Pro Gln Val Trp Pro Glu Leu Thr Glu Leu Pro Asp Tyr Asn Lys Ile Ser Phe Lys Glu Gln Val Pro Met Pro Leu Glu Glu Val Leu Pro Asp Val Ser Pro Gln Ala Leu Asp Leu Leu Gly Gln Phe Leu Leu Tyr Pro Pro His Gln Arg Ile Ala Ala Ser Lys Ala Leu Leu His Gln Tyr Phe Phe Thr Ala Pro Leu Pro Ala His Pro Ser Glu Leu Pro Ile Pro Gln Arg Leu Gly Gly Pro Ala Pro Lys Ala His Pro Gly Pro Pro His Ile His Asp Phe His Val Asp Arg Pro Leu Glu Glu Ser Leu Leu Asn Pro Glu Leu Ile Arg Pro Phe Ile Leu Glu Gly, set forth as SEQ ID NO:3.

7. An expression vector for the expression of a human cyclin dependent kinase in a recombinant host cell wherein said expression vector comprises the DNA molecule of Claim 4.

8. The expression vector of Claim 7 which is selected from the group consisting of pLITMUS28:CDK10, pcDNA3.1:CDK10, and pBBH:CDK10.

9. A host cell which expresses a recombinant human cyclin dependent kinase wherein said host cell contains the expression vector of Claim 7.

10. A host cell which expresses a recombinant human cyclin dependent kinase wherein said host cell contains the expression vector of Claim 8.

11. A purified DNA molecule which comprises the nucleotide sequence as follows:
GAAAAGGCGC AGTGGGGCCC GGAGCTGTCA CCCCTGACTC GACGCAGCTT CCGTTCTCCT
GGTGACGTCG CCTACAGGAA CCGCCCCAGT GGTCAGCTGC CGCGCTGTTG CTAGGCAACA
GCGTGCGAGC TCAGATCAGC GTGGGGTGGA GGAGAAGTGG AGTTTGGAAG TTCAGGGGCA
CAGGGGCACA GGCCCACGAC TGCAGCGGGA TGGACCAGTA CTGCATCCTG GGCCGCATCG
GGGAGGGCGC CCACGGCATC GTCTTCAAGG CCAAGCACGT GGAGACTGGC GAGATAGTTG
CCCTCAAGAA GGTGGCCCTA AGGCGGTTGG AAGACGGCTT CCCTAACCAG GCCCTGCGGG
AGATTAAGGC TCTGCAGGAG ATGGAGGACA ATCAGTATGT GGTACAACTG AAGGCTGTGT
TCCCACACGG TGGAGGCTTT GTGCTGGCCT TTGAGTTCAT GCTGTCGGAT CTGGCCGAGG

TGGTGCGCCA TGCCCAGAGG CCACTAGCCC AGGCACAGGT CAAGAGCTAC CTGCAGATGC
TGCTCAAGGG TGTCGCCTTC TGCCATGCCA ACAACATTGT ACATCGGAAC CTGAAACCTG
CCAACCTGCT CATCAGCGCC TCAGGCCAGC TCAAGATAGC GGACTTTGGC CTGGCTCGAG
TCTTTTCCCC AGACGGCAGC CGCCTCTACA CACACCAGGT GGCCACCAGG TCTGTGGGCT
GCATCATGGG GGAGCTGTTG AATGGGTCCC CCCTTTTCCC GGGCAAGAAC GATATTGAAC
AGCTTTGCTA TGTGCTTCGC ATCTTGGGCA CCCCAAACCC TCAAGTCTGG CCGGAGCTCA
CTGAGCTGCC GGACTACAAC AAGATCTCCT TTAAGGAGCA GGTGCCCATG CCCCTGGAGG
AGGTGCTGCC TGACGTCTCT CCCCAGGCAT TGGATCTGCT GGGTCAATTC CTTCTCTACC
CTCCTCACCA GCGCATCGCA GCTTCCAAGG CTCTCCTCCA TCAGTACTTC TTCACAGCTC
CCCTGCCTGC CCATCCATCT GAGCTGCCGA TTCCTCAGCG TCTAGGGGGA CCTGCCCCCA
AGGCCCATCC AGGGCCCCCC CACATCCATG ACTTCCACGT GGACCGGCCT CTTGAGGAGT
CGCTGTTGAA CCCAGAGCTG ATTCGGCCCT TCATCCTGGA GGGGTGAGAA GTTGGCCCTG
GTCCCGTCTG CCTGCTCCTC AGGACCACTC AGTCCACCTG TTCCTCTGCC ACCTGCCTGG
CTTCACCCTC CAAGGCCTCC CCATGGCCAC AGTGGGCCCA CACCACACCC TGCCCCTTAG
CCCTTGCGAG GGTTGGTCTC GAGGCAGAGG TCATGTTCCC AGCCAAGAGT ATGAGAACAT
CCAGTCGAGC AGAGGAGATT CATGGCCTGT GCTCGGTGAG CCTTACCTTC TGTGTGCTAC
TGACGTACCC ATCAGGRCAG TGAGCTCTGC TGCCAGTCAA GGCCTGCATA TGCAGAATGA
CGATGCCTGC CTTGGTGCTG CTTCCCCGAG TGCTGCCTCC TGGTCAAGGA GAAGTGCAGA
GAGTAAGGTG TCCTTATGTT GGAAACTCAA GTGGAAGGAA GATTTGGTTT GGTTTTATTC
TCAGAGCCAT TAAACACTAG TTCAGTATGT GAGATATAGA TTCTAAAAAC CTCAGGTGGC
TCTGCCTTAT GTCTGTTCCT CCTTCATTTC TCTCAAGGGA AATGGCTAAG GTGGCATTGT
CTCATGGCTC TCGTTTTTGG GGTCATGGGG AGGGTAGCAC CAGGCATAGC CACTTTTGCC
CTGAGGGACT CCTGTGTGCT TCACATCACT GAGCACTCAT TTAGAAGTGA GGGAGACAGA
AGTCTAGGCC CAGGGATGGC TCCAGTTGGG GATCCAGCAG GAGACCCTCT GCACATGAGG
CTGGTTTACC AACATCTACT CCCTCAGGAT GAGCGTGAGC CAGAAGCAGC TGTGTATTTA
AGGAAACAAG CGTTCCTGGA ATTAATTTAT AAATTTAATA AATCCCAATA TAATCCCAAA
AAAAAAAAAA AAAAAATTCC TGCGGCCGCA AGGA, set forth as SEQ ID NO:11.

12. A purified DNA molecule encoding a human cyclin dependent kinase wherein. said DNA molecule encodes a protein comprising the amino acid sequence as follows:
Met Asp Gln Tyr Cys Ile Leu Gly Arg Ile Gly Glu Gly Ala His Gly Ile Val Phe Lys Ala Lys His Val Glu Thr Gly Glu Ile Val Ala Leu Lys Lys Val Ala Leu Arg Arg Leu Glu Asp Gly Phe Pro Asn Gln Ala Leu Arg Glu Ile Lys Ala Leu Gln Glu Met Glu Asp Asn Gln Tyr Val Val Gln Leu Lys Ala Val Phe Pro His Gly Gly Gly Phe Val Leu Ala Phe Glu Phe Met Leu Ser Asp Leu Ala Glu Val Val Arg His Ala Gln Arg Pro Leu Ala Gln Ala Gln Val Lys Ser Tyr Leu Gln Met Leu Leu Lys Gly Val Ala Phe Cys His Ala Asn Asn Ile Val His Arg Asn Leu Lys Pro Ala Asn Leu Leu Ile Ser Ala Ser Gly Gln Leu Lys Ile Ala Asp Phe Gly Leu Ala Arg Val Phe Ser Pro Asp Gly Ser Arg Leu Tyr Thr His Gln Val Ala Thr Arg Ser Val Gly Cys Ile Met Gly Glu Leu Leu Asn Gly Ser Pro Leu Phe Pro Gly Lys Asn Asp Ile Glu Gln Leu Cys Tyr Val Leu Arg Ile Leu Gly Thr Pro Asn Pro Gln Val Trp Pro Glu Leu Thr Glu Leu Pro Asp Tyr Asn Lys Ile Ser Phe Lys Glu Gln Val Pro Met Pro Leu Glu Glu Val Leu Pro Asp Val Ser Pro Gln Ala Leu Asp Leu Leu Gly Gln Phe Leu Leu Tyr Pro Pro His Gln Arg Ile Ala Ala Ser Lys Ala Leu Leu His Gln Tyr Phe Phe Thr Ala Pro Leu Pro Ala His Pro Ser Glu Leu Pro Ile Pro Gln Arg Leu Gly Gly Pro Ala Pro Lys Ala His Pro Gly Pro Pro His Ile His Asp Phe His Val Asp Arg Pro Leu Glu Glu Ser Leu Leu Asn Pro Glu Leu Ile Arg Pro Phe Ile Leu Glu Gly, set forth as SEQ ID NO:12.

13. A process for the expression of a human cyclin dependent kinase protein in a recombinant host cell, comprising:
(a) transfecting the expression vector of Ciaim 5 into a suitable host cell; and, (b) culturing the host cells of step (a) under conditions which allow expression of the human cyclin dependent kinase protein from the expression vector.

14. An expression vector for the expression of a human cyclin dependent kinase in a recombinant host cell wherein said expression vector comprises the DNA molecule of Claim 11.

15. The expression vector of Claim 14 which is selected from the group consisting of pcDNA3.1:CDK10-D127N and pBBH:CDK10-D127N.

16. A purified antibody raised against a protein comprising the amino acid sequence as set forth in SEQ ID NO:3.

17. A purified antibody of claim 16 raised against a protein consisting of the amino acid sequence as set forth in SEQ ID
NO:3.

18. A purified antibody of claim 16 raised against a protein consisting of the amino acid sequence consisiting of SEQ ID
NO:12.

19. A purified antibody of claim 16 raised against a peptide fragment comprising; from about amino acid 301 to amino acid 325 of SEQ ID NO:3.

20. A purified antibody of claim 19 raised against a peptide fragment consisting of amino acid 301 to amino acid 325 of SEQ
ID NO:3.

21. A purified human cyclin dependent kinase protein which comprises the amino acid sequence as set forth in SEQ ID NO:3.

22. A purified human cyclin dependent kinase protein which consists of the amino acid sequence as set forth in SEQ ID NO:3.

23. A purified human cyclin dependent kinase protein produced by the method of claim 13.

24. A purified human cyclin dependent kinase protein which comprises the ;amino acid sequence as set forth in SEQ ID NO:12.

25. A purified human cyclin dependent kinase protein which consists of the amino acid sequence as set forth in SEQ ID NO:12.

26. A method for determining whether a substance is capable of binding to cyclin dependent kinase 10 protein comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of cyclin dependent kinase in the cells;
(b) exposing the test cells to the substance;
(c) measuring the amount of binding of the substance to cyclin dependent kinase 10;
(d) comparing the amount of binding of the substance to cyclin dependent kinase 10 in the test cells with the amount of binding of the substance to control cells that have not been transfected with cyclin dependent kinase 10.

27. The method of claim 26 wherein the cyclin dependent kinase 10 comprises an amino acid sequence as set forth in SEQ ID
NO:3.

28. The method of claim 26 wherein the cyclin dependent kinase 10 comprises an amino acid sequence as set forth in SEQ ID
NO:12.