EP0972011A1 - Proteine kinase cyclino-dependante - Google Patents

Proteine kinase cyclino-dependante

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Publication number
EP0972011A1
EP0972011A1 EP98906202A EP98906202A EP0972011A1 EP 0972011 A1 EP0972011 A1 EP 0972011A1 EP 98906202 A EP98906202 A EP 98906202A EP 98906202 A EP98906202 A EP 98906202A EP 0972011 A1 EP0972011 A1 EP 0972011A1
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EP
European Patent Office
Prior art keywords
leu
pro
ala
gly
glu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98906202A
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German (de)
English (en)
Other versions
EP0972011A4 (fr
Inventor
David L. Gerhold
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
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Merck and Co Inc
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Publication date
Priority claimed from GBGB9707491.8A external-priority patent/GB9707491D0/en
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP0972011A1 publication Critical patent/EP0972011A1/fr
Publication of EP0972011A4 publication Critical patent/EP0972011A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes a novel human cyclin-dependent kinase (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.
  • CDK human cyclin-dependent kinase
  • the present invention also relates to associated human CDK proteins and human CDK mutant proteins.
  • the cell cycle consists of two major events separated by two central gap phases. DNA synthesis and replication occur during the S phase while mitosis occurs during the M phase. A first gap phase, called Gi, which occurs between the M phase and the S phase, allows 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).
  • CDKs protein kinases
  • CDK1 - CDK6 phosophorylation of threonine 160/161 (Thrl60/161).
  • 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 Gi cyclins or mitotic cyclins, depending on peak expression levels.
  • a CDK may bind a subset of cyclins.
  • CDK4 is known to bind cyclin Dl or cyclin D3
  • CDK2 is known to bind cyclin A, cyclin Bl, 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.
  • CAK CDK- Activating Kinase
  • CDK/cyclin complexes are thought to occur via phosphorylation at threonine 14 (Thrl4) and/or tyrosine 15 (Tyrl ⁇ ) of the CDK subunit.
  • Thrl4 threonine 14
  • Tyrl ⁇ tyrosine 15
  • the Weel kinase has been suggested as either a Thrl4 kinase or as a Thrl4 and Tyrl ⁇ kinase.
  • CDC25 is thought to be a dual kinase targeting both Thrl4 and/or Tyrl ⁇ (Morgan, 1995, Nature 374: 131-134).
  • 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 CDK10 cDNA which encodes CDK- 10 or an active mutant thereof will also be useful for the recombinant production of large quantities of respective protein.
  • 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 or a mutant such as the exemplified mutant disclosed herein, A therapeutic agent comprised of CDK10 protein would be useful in the treatment of cell cycle and/or CDK ) related diseases or conditions which are CDK10 responsive.
  • the present invention addresses and meets this need.
  • the present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes a novel human cyclin- dependent kinase.
  • This CDK comprises a novel cyclin binding domain signature sequence (Pro-Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID NO:l), lacks Thrl4 and/or Tyrl ⁇ , and also lacks the T-loop domain containing the conserved Thrl60/161 residue.
  • the present invention relates to biologically active fragments or mutants of a novel isolated nucleic acid molecule which encodes mRNA expressing 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:l), which lacks Thrl4 and/or Tyrl ⁇ as well as a T-loop domain containing the conserved Thrl60/161 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.
  • DNA deoxyribonucleic acid molecule
  • cDNA complementary DNA
  • 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).
  • SEQ ID NO:ll 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; SEQ ID NO:l), lacks Thrl4 and Tyrl ⁇ which make up the conserved ATP binding motif of several known CKDs, and also lacks the T-loop domain containing the conserved Thrl60/161 residue.
  • a novel cyclin binding domain signature sequence Pro-Asn-Gln-Ala-Leu-Arg-Glu
  • 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 Tyrl ⁇ which make up the conserved ATP binding motif of known CKDs, and also lacks the T-loop domain containing the conserved Thrl60/161 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 SEQ ID NO: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 NO:2.
  • SEQ ID NO:ll Another preferred aspect of the present invention is disclosed in SEQ ID NO:ll, wherein nucleotide ⁇ 88 of the wild-type form (SEQ ID NO: 2) is mutated from “G" to "A".
  • mutant protein (CDK10-D127N), wherein nucleotide ⁇ 88 of SEQ ID NO:ll is mutated from “G” to "A”, as compared to the wild-type form (SEQ ID NO:2), which results in a change of Aspl27 to Asnl27 as compared to the wild-type amino acid sequence (SEQ ID NO:3), disclosed as SEQ ID NO: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
  • 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.
  • modulators identified in this process are useful as therapeutic agents for controlling cell growth or immune cell proliferation commonly associated with cancer.
  • Figure 2 shows the amino acid sequence (SEQ ID NO:3) of human CDK10.
  • Figure 3 shows the strategy utilized to generate a full- length DNA fragment encoding human CDK10.
  • Figure 4 shows northern blot analysis of human tissue
  • Figure ⁇ shows northern blot analysis of human tissue
  • the present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes a novel cyclin-dependent kinase which comprises a novel human cyclin binding domain (Pro- Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID NO:l), lacks Thrl4 and/or Tyrl ⁇ which make up the conserved ATP binding motif of known CDKs, and also lacks the T-loop domain containing the conserved Thrl60/161 residue.
  • polynucleotide which encodes a novel cyclin-dependent kinase which comprises a novel human cyclin binding domain (Pro- Asn-Gln-Ala-Leu-Arg-Glu; SEQ ID NO:l)
  • Thrl4 and/or Tyrl ⁇ which make up the conserved ATP binding motif of known CDKs
  • T-loop domain containing the conserved Thrl60/161 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.
  • 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:l), lacks Thrl4 and/or Tyrl ⁇ , and also lack a T-loop domain containing the conserved Thrl60/161 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 which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use.
  • FIG. 1 A preferred aspect of the present invention is disclosed in Figure 1 and SEQ ID NO:2, a human cDNA encoding a novel cyclin- dependent kinase, CDK10, disclosed herein as: 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 GCCCTGCG
  • 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; SEQ ID NO:l), lacks Thrl4 and/or Tyrl ⁇ as well as the T-loop domain containing the conserved Thrl60/161 residue.
  • Any such nucleic acid may be isolated and characterized from a mammalian cell, including but not limited to human, human and rodent.
  • a human form is an especially preferred form, such as the isolated cDNA exemplified herein as set forth in SEQ ID NO:2 and a dominant negative mutant form as set forth in SEQ ID NO: 12.
  • the present invention also relates to biologically active fragments and/or mutants of a novel cyclin-dependent kinase which comprises the novel cyclin binding domain (Pro-Asn-Gln-Ala-Leu- Arg-Glu; SEQ ID NO:l), lacks Thrl4 and/or Tyrl ⁇ which make up the conserved ATP binding motif of known CDKs, and also lacks the T-loop domain containing the conserved Thrl60/161 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.
  • 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 NO: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 NO:2.
  • the amino acid sequence of the novel cyclin-dependent kinase, CDK10 is disclosed herein as:
  • SEQ ID NO:ll Another preferred aspect of the present invention is disclosed in SEQ ID NO:ll, wherein nucleotide ⁇ 88 of the wild-type form (SEQ ID NO: 2) is mutated from “G" to "A".
  • mutant protein (CDK10-D127N), wherein nucleotide ⁇ 88 of SEQ ID NO:ll is mutated from “G” to "A”, as compared to the wild-type form (SEQ ID NO:2), which results in a change of Aspl27 to Asnl27 as compared to the wild-type amino acid sequence (SEQ ID NO:3), disclosed as SEQ ID NO: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 immune cell proliferation associated with human cancers. Additionally, an isolated and purified CDK10 cDNA which encodes CDK-10 or an active mutant thereof will also be useful for the recombinant production of large quantities of respective 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 CDKIO 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 regulatory effects of the mutant kinase.
  • 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.
  • DNA deoxyribonucleic acid molecule
  • cDNA complementary DNA
  • 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).
  • this invention is also directed to those DNA sequences which contain alternative codons which code for the eventual translation of the identical amino acid.
  • a sequence bearing one or more replaced codons will be defined as a degenerate variation.
  • 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. Therefore, this invention is also directed to those DNA sequences which express RNA comprising alternative codons which code for the eventual translation of the identical amino acid, as shown below:
  • 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.
  • 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.
  • 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 CDKIO 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 CDKIO protein.
  • fragment is meant to refer to any polypeptide subset of wild type CDKIO.
  • 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 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 is CDK10-D127N, wherein a single base mutation at nucleotide ⁇ 88 of SEQ ID NO:2 results in a single amino acid substitution at residue 127, from aspartic acid to asparagine. This mutation alters kinase activity of CDK10-D127N as compared to the wild type CDKIO protein.
  • variant is meant to refer to a molecule substantially similar in structure and function to either the entire wild type protein or to a fragment thereof.
  • a molecule is "substantially similar" to a wild type CDKlO-like protein if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially 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 two amino acid sequences are not identical.
  • analog refers to a molecule substantially similar in function to either the entire wild type CDKlO-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 7 ⁇ % of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize to a strand or its complement.
  • 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 is 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 CDKIO. 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 CDKIO 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 CDKIO cDNA following the construction of an CDKlO-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 CDKIO protein; (4) screening a CDKlO-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the CDK10 protein.
  • EST expressed sequence tag
  • 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; ( ⁇ ) screening an CDKlO-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 ⁇ ' and 3' gene specific oligonucleotides using SEQ ID NO: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 CDKIO.
  • CDKlO-encoding DNA or a CDKIO 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 CDKlO-encoding DNA. Additionally a CDKIO gene may be isolated by oligonucleotide- or polynucleotide- based hybridization screening 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.
  • cDNA libraries may be prepared from cells or cell lines which have CDKIO activity.
  • the selection of cells or cell lines for use in preparing a cDNA library to isolate a CDKIO cDNA may be done by first measuring cell associated CDKIO activity using any known assay for CDK activity.
  • 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, Molecular 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 CDKIO may also be isolated from a suitable genomic DNA library. 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.
  • the amino acid sequence or DNA sequence of CDKIO or a homologous protein may be necessary.
  • the CDKIO 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 CDKIO DNA fragment. Once suitable amino acid sequences have been identified, the DNA sequences capable of encoding them are synthesized.
  • 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 CDKIO sequence but others in the set will be capable of hybridizing to CDKIO DNA even in the presence of DNA oligonucleotides with mismatches. The mismatched DNA oligonucleotides may still sufficiently hybridize to the CDKIO DNA to permit identification and isolation of CDKIO 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.
  • the appropriate nucleotide sequence for use in a PCR-based method may be obtained from SEQ ID NO:2, either for the purpose of isolating overlapping ⁇ ' and 3' RACE products for generation of a full-length sequence coding for CDKIO, or to isolate a portion of the nucleotide sequence coding for CDKIO for use as a probe to screen one or more cDNA- or genomic-based libraries to isolate a full-length sequence encoding CDKIO or CDKlO-like proteins.
  • the RACE PCR technique the RACE PCR technique
  • PCR amplification was performed using the ElongaseTM. Thermal cycling was completed and a portion 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 the nested gene specific primer PK22L161
  • a DNA fragment 3' to and overlapping the 600 bp 5' fragment was identified by searching public nucleic acid and protein databases.
  • This 3' fragment is an approximately 1.8 Kb cDNA insert available as a Notl-Hindlll fragment in a typical phagemid vector.
  • This cDNA clone is readily identified by Genbank Accession No. H17727, Image Clone ID No. 60484, 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 ⁇ 801 (http://www. resgen.com).
  • CDKIO coding region was assembled in pLITMUS28 (New England Biolabs) as an expression cassette with a BamHI site appended just ⁇ ' to the ATG translational start codon.
  • a BamHI-Xbal fragment bearing CDK10 was recloned into pcDNA3.1 expression vector (Invitrogen) and a BamHI-Ncol fragment bearing CDK10 was recloned into pBlueBacHis2 baculovirus expression vector (Invitrogen).
  • a similar construct was generated which contains dominant-negative single base pair mutation of CDKIO. This mutant was generated from pLITMUS28::CDK10 using the Stratagene "Quik Change" kit and primers 22U-D127N
  • mutant constructions were subcloned into pcDNA3.1 (as a BamHI-Xbal fragment) and pBlueBacHis2 (as a BamHI- Ncol fragment), respectively.
  • the sequence for the ⁇ ' upstream sequences, coding region and 3' untranslated sequences for the human full-length cDNA encoding CDKIO is shown in SEQ ID NO:2.
  • the deduced amino acid sequence of CDKIO from the cloned cDNA is shown in SEQ ID NO:3. Inspection of the determined cDNA sequence reveals the presence of a single open reading frame that encodes a 32 ⁇ amino acid protein.
  • the open reading frame of the CDKIO coding region runs from nucleotide 210 to nucleotide 1182 of SEQ ID NO:2.
  • nucleotide sequence which encodes a preferred mutant form is disclosed as SEQ ID NO:ll.
  • a variety of mammalian expression vectors may be used to express recombinant CDKIO 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
  • I* vectors modified cloning vectors, specifically designed plasmids or viruses.
  • mammalian expression vectors which may be suitable for recombinant CDKIO expression, include but are not limited to, pcDNA3.1 (Invitrogen), pBlueBacHis2 (Invitrogen), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG ⁇ (Stratagene), EBO-pSV2-neo (ATCC 37 ⁇ 93) pBPV- 1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC
  • bacterial expression vectors may be used to express recombinant CDKIO in bacterial cells.
  • Commercially available bacterial expression vectors which may be suitable for recombinant CDKIO expression include, but are not limited to pCR2.1 (Invitrogen), pETlla (Novagen), lambda gtll (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia).
  • fungal cell expression vectors may be used to express recombinant CDK10 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), Pichia expression vector (Invitrogen).
  • insect cell expression vectors may be used to express recombinant receptor in insect cells.
  • Commercially available insect cell expression 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-CDKlO antibodies.
  • CDKIO DNA may also be performed using in vitro produced synthetic mRNA or native mRNA.
  • Synthetic mRNA or mRNA isolated from CDKIO 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 CDKlO- like protein may be used for expression of CDKIO 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.
  • L cells L-M(TK") ATCC CCL 1.3
  • L cells L-M ATCC CCL 1.2
  • Saos-2 ATCC HTB-8 ⁇
  • 293 ATCC CRL l ⁇ 73
  • Raji ATCC CCL 86
  • CV-1 ATCC CCL 70
  • COS-1 ATCC CRL 16 ⁇ 0
  • COS-7 ATCC CRL 16 ⁇ l
  • CHO-K1 ATCC CCL 61
  • 3T3 ATCC CCL 92
  • NIH/3T3 ATCC CRL 16 ⁇ 8
  • HeLa ATCC CCL 2
  • C127I ATCC CRL 1616
  • BS-C-1 ATCC CCL 26
  • MRC- ⁇ 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 CDKIO protein. Identification of CDKIO expressing cells may be done by several means, including but not limited to immunological reactivity with anti-CDKlO 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, pCR2.1, 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.
  • an expression vector such as pcDNA3.1, pCR2.1, pBlueBacHis2 and pLITMUS28
  • Techniques for such manipulations can be found described in Sambrook, et al., supra , are discussed at length in the Example section and are well known and easily available to the artisan of ordinary skill in the art.
  • CDKIO DNA may also be performed using in vitro produced synthetic mRNA.
  • Synthetic mRNA 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.
  • CDKIO cDNA molecules including but not limited to the following can be constructed: the full-length open reading frame of the CDKIO cDNA and various constructs containing portions of the cDNA encoding only specific domains of the protein or rearranged domains of the protein.
  • CDKIO activity and levels of protein expression can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells.
  • this CDKIO 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 CDKIO protein in host cells is quantified by a variety of techniques including, but not limited to, immunoaffinity and/or ligand affinity techniques.
  • CDKlO-specific affinity beads or CDKlO-specific antibodies are used to isolate 3 ⁇ S-methionine labeled or unlabelled CDKIO protein. Labeled CDKIO protein is analyzed by SDS- PAGE. Unlabelled CDKIO protein is detected by Western blotting, ELISA or RIA assays employing CDKIO specific antibodies.
  • CDKIO protein may be recovered to provide CDKIO in active form.
  • CDKIO purification procedures are available and suitable for use.
  • Recombinant CDKIO 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 exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.
  • recombinant CDKIO can be separated from other cellular proteins by use of an immuno- affinity column made with monoclonal or polyclonal antibodies specific for full length CDKIO, or polypeptide fragments of CDKIO.
  • polyclonal or monoclonal antibodies may be raised against a synthetic peptide (usually from about 9 to about 2 ⁇ amino acids in length) from a portion of the protein as disclosed in SEQ ID NO:3.
  • Monospecific antibodies to CDKIO are purified from mammalian antisera containing antibodies reactive against CDKIO or are prepared as monoclonal antibodies reactive with CDKIO using the technique of Kohler and Milstein (197 ⁇ , Nature 2 ⁇ 6: 49 ⁇ -497).
  • Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for CDKIO.
  • Homogenous binding refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the CDKIO, as described above.
  • CDKIO specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with an appropriate concentration of CDKIO or CDKIO 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.
  • acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum 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.
  • SC subcutaneously
  • IP intraperitoneally
  • 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 CDKIO are prepared by immunizing inbred mice, preferably Balb/c, with CDKIO.
  • the mice are immunized by the IP or SC route with about 1 ⁇ g to about 100 ⁇ g, preferably about 10 ⁇ g, of CDKIO 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 day 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 the 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 cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas.
  • Fusion partners may include, but are not limited to: mouse myelomas P3/NSl/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.
  • DMEM Dulbecco's Modified Eagles Medium
  • 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.
  • SPIRA solid phase immunoradioassay
  • the 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 agar technique of MacPherson, 1973, Soft Agar Techniques, in Tissue Culture Methods and Applications, 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 10" to about 6 x 10" hybridoma cells about 4 days after priming.
  • X and the monoclonal antibodies are purified by techniques known in the art.
  • In vitro production of anti-CDKlO mAb is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufficient 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, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of CDKIO in body fluids or tissue and cell extracts.
  • CDKIO 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 HCl (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 CDKIO or CDKIO fragments are slowly passed through the column.
  • the column is then washed with phosphate buffered saline until the optical density (A280) 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 CDKIO activity include agonists, antagonists, inhibitors, activators, and compounds which directly or indirectly affect regulation of the CDKIO activity and/or the CDKlO/cyclin association.
  • the CDKIO protein kinase of the present invention may be obtained from both native and recombinant sources for use in an assay procedure to identify CDKIO modulators.
  • an assay procedure to identify CDKIO modulators will contain the CDKlO-protein of the present invention, native cyclin protein which will form a CDKlO/cyclin complex, and a test compound or sample which contains a putative CDKIO modulator.
  • the test compounds or samples may be tested directly on, for example, purified CDKIO protein whether native or recombinant, subcellular fractions of CDKlO-producing cells whether native or recombinant, and/or whole cells expressing the CDKIO whether native or recombinant.
  • the test compound or sample may be added to the CDKIO in the presence or absence of a known CDKIO modulator.
  • the modulating 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 CDKIO protein, activate the protein, inhibit CDKIO activity, inhibit or enhance the binding of other compounds to the CDKIO protein, modifying receptor regulation, or modifying an intracellular activity.
  • modulators of CDKIO activity are useful in treating disease states involving the cell cycle will be useful in controlling cell growth 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 CDKIO protein results in either cellular proliferation, cell death, nonproliferation, induction of cellular neoplastic transformations or metastatic tumor growth 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 protein 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 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 DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of CDKIO 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 CDKIO.
  • 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 CDKIO protein or anti-CDKlO antibodies suitable for detecting CDKIO.
  • the carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.
  • compositions comprising modulators of CDKIO 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.
  • a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of the protein, DNA, RNA, or modified CDKIO.
  • 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.
  • compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
  • 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 toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
  • 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.
  • 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.
  • 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.
  • compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • 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.
  • CDKIO This involves a consideration of the distribution, equilibrium, and elimination of a drug Isolated and purified CDKIO is also be useful for the recombinant production of large quantities of CDKIO protein.
  • the ability to produce large quantities of the protein would be useful for the production of a therapeutic agent comprising the CDKIO protein.
  • a therapeutic agent comprised of CDKIO protein would be useful in the treatment of cell cycle and/or CDKIO related diseases or conditions which are CDKIO responsive.
  • This new cDNA fragment encodes a novel cyclin-dependent kinase which comprises a novel cyclin binding domain signature sequence, lacks Thrl4 and/or Tyrl ⁇ within the conserved ATP binding motif of known CDKs, and also lacks the T-loop domain containing the conserved Thrl60/161 residue.
  • 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 ⁇ 801 (http://www. resgen.com). The ⁇ ' portion of the gene was isolated by performing ⁇ ' RACE (Frohman, et al., 1988, Proc. Natl. Acad. Sci.85 8998-9002) using MarathonTM-ready human placenta cDNA available from Clontech (Protocol #PTll ⁇ 6-l, 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 API (5'-CCATCCTAATACGACTCACTATAGGGC-3' SEQ ID NO: ⁇ ).
  • PCR amplification was performed using the ElongaseTM long- PCR enzyme mix (stored in 20mM Tris-HCl (pH 8.0 at 2 ⁇ °C), O.lmM EDTA, ImM DTT, stabilizers and ⁇ 0%(v/v) glycerol) and PCR reaction buffer obtained from Gibco-BRL.
  • the buffer comprised 300mM Tris-S ⁇ 4 (pH 9.1 at 2 ⁇ °C), 90mM (NH4) 2 S04 and l. ⁇ mM MgS04- Two microliters of Marathon placenta cDNA template and 10 pmoles each of PK22L234 and API were added to the reaction mix and brought to a total volume of 20ml with sterile water. Thermal cycling was (1) 94°C/30sec, 68°C/6min for ⁇ cycles; (2) 94°C/30sec, 64°C/30sec, 68°C/4min for ⁇ cycles; and, (3) 1 94°C/30sec, 62°C/30sec and 68°C/4min for 30 cycles.
  • This PCR reaction also differed from the first PCR reaction in that nested primers PK22L161 ( ⁇ '-GCCGTCTGGGGAAAAGA-3'; SEQ ID NO:6) and AP2
  • the MarathonTM -ready human placenta cDNA available from Clontech is enhanced by ligation of a double-stranded, ⁇ ' 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 API oligo will hybridize. Therefore, API does not hybridize and extend any of the original cDNA template molecules, instead beginning extension and amplification in the second round of PCR.
  • the 3' portion of a DNA fragment which encodes CDK10 is contained within a DNA plasmid vector, pH17727.
  • This insert contains a ⁇ ' Xhol site unique to the insert and a Ncol site in the 3' unstranslated region unique to the insert.
  • This Xhol-Ncol fragment was isolated and subcloned into Xhol-Ncol digested pLITMUS28 plasmid DNA (New England Biolabs), resulting in pLITMUS28:H17727.
  • the 600 bp PCR fragments obtained from ⁇ ' RACE were cloned into pCR2.1 (Invitrogen) using the Invitrogen TA-cloning kit as described by the manufacturer.
  • a Pmll restriction site is located at approximately the midpoint of the 600 bp PCR product. The Pmll site was used to construct a wild type form of the 600 bp ⁇ ' fragment from 2 independent ⁇ ' RACE PCR clones, pPK22bo4 and pPK22do4.
  • a BamHI-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 Xbal.
  • the resulting construct, pcDNA3.1:CDK10 contains a portion of the CMV promoter and a T7 primer site upstream of the CDKIO ATG translational start codon as well as the BGH polyA region downstream of the translational termination codon.
  • other components to allow growth in E. coli and mammalian cells are present in this vector.
  • a BamHI-Ncol fragment from pLITMUS28: CDKIO containing the CKDIO coding region was cloned into the baculovirus expression vector, pBlueBacHis2 (Invitrogen), which was previously digested with BamHI and Ncol.
  • the resulting construct, pBBH:CDK10 may be used to express recombinant CDKIO from insect cells by
  • 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:
  • the dominant-negative mutation changes the codon GAC (at nucleotides ⁇ 88- ⁇ 90 of SEQ ID NO:2) to AAC (at nucleotides ⁇ 88- ⁇ 90 of SEQ ID NO: 11), thus deletion essential amino acid Aspl27 to Asnl27 (see SEQ ID NO:12), which inactivates kinase activity (see Example 7 and van den Heuvel & Harlow, 1993, Science 262:20 ⁇ 0-20 ⁇ 4).
  • a CDK10-D127N construction was subcloned into pcDNA3.1 (as a BamHI-Xbal fragment), resulting in pcDNA3.1:CDK10-dl27N.
  • a CDK10-D127N construction was also subcloned into pBlueBacHis2 (as a BamHI-Ncol fragment), resulting in P BBH:CDK10-dl27N.
  • 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 Notl-Hindlll insert from pH17727 using the "Universal"
  • Figure 4 and Figure ⁇ show Northern data indicating the presence of CDKIO transcripts in a variety of adult human tissue ( Figure 4) as well as in specific regions of the adult and fetal human brain ( Figure ⁇ ). 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 lobes and the cerebral cortex.
  • Human osteosarcoma cell line Saos2 (ATCC HBT-8 ⁇ ) was grown in DMEM high glucose medium + glutamine +10% fetal calf serum (in concentrations as recommended by Gibco-BRL). Two replicates of the experiment were performed sequentially. Cells were split 1:6 into 10 cm culture dishes two days prior to transfection. Transfection was performed using the CaP04 method according to Chen and Okayama (1987, Mol. and Cell. Biol. 7:-274 ⁇ -27 ⁇ 2). Ten ug of each plasmid DNA (pcDNA3.1, pcDNA3:CDK10, pcDNA3:CDK10-D127N) was transfected into ⁇ 60% confluent cells in each 10 cm dish.
  • HeLa cervical carcinoma cells were treated for 48 hours with a control adenovirus deleted for the El 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 CDKIO protein (amino acid 301 - amino acid 325 of SEQ ID NO: 3).
  • the cell line transfected with Ad/CDKIO- D127N expressed CDK10-D127N at a ⁇ O-fold higher level than endogenous, wild type CDKIO.
  • 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).
  • Table 2 the genes which were suppressed at the mRNA level by CDK10-D127N are summarized in Table 2.
  • a therapeutic agent comprising the CDKIO protein would be useful in the treatment of cell cycle and/or CDKIO related diseases or conditions which are CDKIO responsive as well as showing a potential use for a dominant-negative mutant such as CDK10-D127N, which may be useful in the treatment of cell cycle diseases or conditions which are responsive to the mtuant proteins ability to regulate a phase or phases of the cell cycle.
  • CTTCACCCTC CAAGGCCTCC CCATGGCCAC AGTGGGCCCA CACCACACCC TGCCCCTTAG 1320
  • CTTCACCCTC CAAGGCCTCC CCATGGCCAC AGTGGGCCCA CACCACACCC TGCCCCTTAG 1320

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Abstract

L'invention concerne une molécule d'acide nucléique isolée qui code une nouvelle kinase cyclino-dépendante (CDK = cyclin-dependent kinase) humaine qui comprend une nouvelle séquence de signature du domaine de liaison de la cycline et ne présente pas plusieurs résidus d'acide aminé, qui étaient conservés jusqu'à maintenant, impliqués dans la régulation du complexe cdk/cycline. L'invention concerne des protéines et des formes mutantes biologiquement actives associées.
EP98906202A 1997-02-07 1998-02-06 Proteine kinase cyclino-dependante Withdrawn EP0972011A4 (fr)

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Title
DATABASE EMBL [Online] EBI; 1 July 1995 (1995-07-01) Database accession no. H17727 XP002229224 *
DATABASE SWISS-PROT [Online] EBI; 1 April 1993 (1993-04-01) Database accession no. P29620 XP002229225 -& HATA S.: "complementary DNA cloning of a novel CDC2-positive -CDC28-related protein kinase from rice" FEBS (FEDERATION OF EUROPEAN BIOECHEMICAL SOCIETIES) LETTERS, vol. 279, no. 1, 1991, pages 149-152, XP002229244 *
GRANA X ET AL: "PITALRE, A NUCLEAR CDC2-RELATED PROTEIN KINASE THAT PHOSPHORYLATES THE RETINOBLASTOMA PROTEIN IN VITRO" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 91, April 1994 (1994-04), pages 3834-3838, XP000919000 ISSN: 0027-8424 *
MEYERSON M ET AL: "A family of human cdc2-related protein kinases" EMBO JOURNAL, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 11, no. 8, 1992, pages 2909-2917, XP002128265 ISSN: 0261-4189 -& DATABASE EMBL [Online] EBI; 3 May 1993 (1993-05-03) Database accession no. x66362 XP002229247 -& DATABASE EMBL [Online] EBI; 15 June 1992 (1992-06-15) Database accession no. x66357 XP002229248 *
See also references of WO9835015A1 *

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