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WO1997009432A1 - Kinase cyclinodependante - Google Patents

Kinase cyclinodependante Download PDF

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Publication number
WO1997009432A1
WO1997009432A1 PCT/EP1995/003439 EP9503439W WO9709432A1 WO 1997009432 A1 WO1997009432 A1 WO 1997009432A1 EP 9503439 W EP9503439 W EP 9503439W WO 9709432 A1 WO9709432 A1 WO 9709432A1
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WO
WIPO (PCT)
Prior art keywords
cdk8
fragment
nucleic acid
cyclin
protein
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.)
Ceased
Application number
PCT/EP1995/003439
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English (en)
Inventor
Erich Alois Nigg
Jean-Pierre Tassan
Sharon J. Schultz
Malika Jaquenoud
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Novartis AG
Original Assignee
Ciba Geigy AG
Novartis AG
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Ciba Geigy AG, Novartis AG filed Critical Ciba Geigy AG
Priority to PCT/EP1995/003439 priority Critical patent/WO1997009432A1/fr
Priority to AU35200/95A priority patent/AU3520095A/en
Publication of WO1997009432A1 publication Critical patent/WO1997009432A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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)

Definitions

  • This invention relates to a cyclin-dependent kinase (CDK), derivatives of said kinase, antibodies specific for said kinase, and to means and methods for the production thereof.
  • CDK cyclin-dependent kinase
  • the invention is also directed to nucleic acids coding for a kinase of the invention, to a method of obtaining such nucleic acid molecules, and to the expression thereof. Furthermore, the invention is directed to uses of the proteins and nucleic acids of the invention.
  • CDKs cyclin-dependent kinases
  • cyclin regulatory subunits play a pivotal role in ceU cycle regulation in aU eukaryotes (Norbury, C. & Nurse, P. (1992) Ann. Rev. Biochem.61, 441-470).
  • Cyclins are positive regulatory subunits of CDKs and constitute multiprotein families in both yeasts and metazoans (Hunt, T. (1991) Seminars in CeU Biology 2, 213-222; Sherr, C. J. (1993) CeU 73, 1059-1065).
  • Metazoan cyclin C was originaUy isolated from human and Drosophila cDNA Ubraries by virtue of its abiUty to rescue Saccharomyces cerevisiae cells deficient in G 1 cyclin function (Lew, D. J. et al. (1991) CeU 66, 1197-1206; Leopold, P. & O'FarreU, P. H. (1991) CeU 66, 1207-1216; Lahue, E E. et al. (1991) Genes & Development 5, 2166-2175).
  • a CDK partner for this cyclin has not been identified. This lack of information on a potential kinase partner has significantly hampered, for example, progress towards under ⁇ standing the function of cyclin C, and the search for improved anti-proliferative drugs.
  • the present invention has achieved identification, isolation and sequencing of DNA encoding a cyclin C-dependent protein kinase, which is designated CDK 8, thus enabling in vitro production of said kinase, e.g. by recombinant DNA techniques.
  • the present invention relates to a purified or isolated cyclin-dependent kinase, designated CDK8, or a fragment thereof. It is an additional object of the instant invention to provide immunogens for raising antibodies against CDK8 as weU as to obtain antibodies capable of specificaUy binding to CDK8.
  • the present invention relates to isolated nucleic acids (DNA, RNA) coding for CDK8.
  • the invention provides a nucleic acid sequence that is complementary to, or hybridizes under stringent conditions to, a nucleic acid sequence encoding CDK8.
  • the invention also provides a method for amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase (chain) reaction with nucleic acid (DNA or RNA) encoding (or complementary to) CDK8.
  • the nucleic acid is DNA and further comprises a replicable vector comprising the nucleic acid encoding CDK8 operably linked to control sequences recognized by a host transformed by the vector.
  • the invention provides host cells transformed with such vector and a method of using a nucleic acid encoding CDK8 to effect the production of CDK8, comprising expressing CDK8 nucleic acid in a culture of the transformed host cells and, if desired, recovering CDK8 from the host ceU culture.
  • isolated is intended to refer to a molecule of the invention in an enriched or, preferably, substantiaUy pure form obtainable from a natural source or by means of genetic engineering.
  • isolated DNAs, RNAs and proteins of the invention may be useful in ways that the DNAs, RNAs and proteins as they naturally occur are not, such as identification of compounds modulating the activity of CDK8.
  • the present invention relates to an isolated serine/threonine kinase designated CDK8, which specificaUy interacts with cyclin C and which has more than 60 %, preferably more than 90 %, sequence identity with the protein having the amino acid sequence set forth in SEQ ID NO:2, or an immunologicaUy or biologicaUy active fragment thereof comprising at least eight amino acids.
  • CDK8 Full-length CDK8 has a molecular mass of about 50 to 55 kUoDalton (kD), as determinable e.g. by sodium dodecyl sulfate electrophoresis (SDS-PAGE).
  • the catalytical domain of CDK8 has a molecular mass of about 30 to 32 kD.
  • CDK8 naturaUy occurs in metazoan organisms. The equi brium distribution of CDK8 is predominantly nuclear.
  • a preferred protein of the invention is human CDK8, or a fragment thereof, particularly the protein having the amino acid sequence set forth in SEQ ID NO:2, or a biologicaUy active fragment thereof .
  • a biologicaUy active fragment is capable of binding cyclin C, i.e. to comprise the cyclin C-binding domain. Determination that a CDK8 fragment of the invention contains a cyclin C-binding domain may be made by use of any of several methods known to those skilled in the art, including determination of the binding kinetics and affinity of the fragment for cyclin C as weU as inhibition studies using anti-CDK8 antibodies as described hereinbelow.
  • CDK8 of SEQ ID NO:2 is a 53 kD protein consisting of 872 amino acids and containing aU the sequence motifs and 11 subdomains characteristic of a serine threonine kinase (Hanks, S. K., Quinn, A- M. & Hunter, T. (1988) Science 241, 42-52).
  • CDK8 of SEQ ID NO:2 contains phosphorylatable residues corresponding to threonine 14 and tyrosine 15 in subdomain L but lacks a threonine phosphorylation site in the exact same position of threonine 161 in the T-loop between subdomains VH and VTH (Solomon, M J. (1994) Trends Biochem. Sci. 19, 496-500; Morgan, D.O. (1995) Nature 374, 131-134.). Most likely, CDK8 need not be phosphorylated by a CDK-activating kinase for activation.
  • isolated CDK8 means enriched or substantiaUy pure CDK8 which has been identified and is free of one or more components of its natural environment
  • Enriched CDK8 refers to a preparation containing said protein in a concentration higher than natural, e.g. a ceUular extract comprising CDK8.
  • SubstantiaUy pure CDK8 is homogenous CDK8, which is substantiaUy free from other compounds with which it is normaUy associated in vivo, particularly free from naturaUy occurring macromolecules. Homogenicity is determined by reference to purity standards known to those skiUed in the art, e.g. purity sufficient to aUow determination of the N-terminal amino acid sequence.
  • Isolated CDK8 includes CDK8 in recombinant ceU culture.
  • a preferred isolated protein of the invention is a recombinant protein.
  • a protein of the invention is capable of binding cyclin C, i.e. it has a cyclin C-binding domain.
  • the invention also relates to a a composition of matter comprising a protein of the invention bound to cyclin C, and optionaUy further proteins interacting with the CDK8/cyclin C complex.
  • SubstantiaUy pure CDK8 may be obtained from tissue homogenates or ceU lysates, dirough microbial expression, by chemical synthesis, or by purification means known to those skiUed in the art, such as affinity chromatography. Such techniques may also be used to obtain biologicaUy active fragments of CDK8, which contain a binding domain for cyclin C.
  • CDK8 A specific interaction (binding) between CDK8 and cyclin C is found in vitro and in vivo. The interaction is specific in that CDK8 does not interact with any of cyclins A, Bl, C, Dl, E or H. On the other hand, cyclin C does not interact with cyclin-dependent kinases CDC2, CDK2, CDK4 or CDK7. With respect to amino acid sequence similarity, CDK8 is only distantly related to other CDKs, such as CDC2 and budding yeast CDC28 (36% sequence identity), but shows a striking sequence simUarity to SRB10 of S. cerevisiae (Liao, S.-M. et aL (1995) Nature 374, 193-196). The CDK8/cyclin C complex is found to be structuraUy related to SRB10/SRB11, a CDK/cyclin pair recently shown to be part ofthe RNA polymerase II holoenzyme of S. cerevisae.
  • Specific interaction of a protein of the invention with cyclin C may be determined according to methods readUy available to those of skiU in the art, e.g. the methods described herein.
  • the incubation mixture is analyzed for the formation of a CDK8/cyclin C protein complex, e.g. by means of immunoanalytical techniques, such as immunoprecipitation and immunoblotting, or a method suitable for determining complex size or molecular mass of the two proteins, such as gel filtration, gradient centrifugation, e.g.
  • the CDK8/cyclin C complex has a molecular mass of about 85 to 90 kD. SDS-PAGE of a CDK8/cyclin C complex yields bands at about 50 to 55 kD and 30 to 32 kD, representing (fuU-length) CDK8 and cyclin C, respectively ⁇
  • cyclin C the specificity of in vitro interaction between a protein of the invention and cyclin C may be demonstrated involving in vitro transcription-translation of the protein of the invention, cyclin C and anyone or aU of cyclins A, Bl, Dl, E and H., e.g. using suitable ceU extracts , such as (rabbit) reticulocyte lysates.
  • ceU extracts such as (rabbit) reticulocyte lysates.
  • each incubation mixture may be analyzed for the presence of a CDK8/cyclin complex by SDS-PAGE and a suitable detection memod, such as fluorography, and/or by immunoprecipitation with an anti-CDK8 antibody.
  • aU cyclins mentioned above only cyclin C associates with CDK8 and co-immunoprecipitates reproducibly tiierewith.
  • kinase activity of a protein of the invention can be measured according to conventional methods known in the art For example, kinase activity may be determined in immune complexes as decribed by Ewen, M. et aL ((1993), CeU 74, 1009-1020). Briefly, a CDK8/cyclin C complex is immunoprecipitated wim an antibody-protein-A agarose complex specific for CDK8.
  • the immune complexes are incubated with a suitable proteinaceous kinase substrate in the presence of a phosphate donor, such as ATP or GTP, which is detectably labeled, e.g. [ 32 P] ATP.
  • a phosphate donor such as ATP or GTP
  • the incubation mixture is suitably analyzed for presence of the label, e.g. by electrophoresis and subsequent autoradiography.
  • kinase activity may be determined in a kinase assay comprising CDK8 and cyclin C in a soluble form, an abovementioned phosphate donor and a suitable proteinaceous substrate.
  • Soluble CDK8 or cyclin C may be obtained e.g. from suitably transformed yeast cells or insect cells, e.g. from baculovirus-transfected ceU lysates.
  • Phosphorylation of the proteinaceous substrate is analyzed, e.g. by electrophoresis and subsequent autoradiography.
  • CDK8 of the invention also includes amino acid mutants, glycosylation variants and derivatives of the beforementioned proteins.
  • functional amino acid variants include naturaUy occurring aUeUc or interspecies variations of the CDK8 amino acid sequence.
  • Prefened interspecies variants of the protein of SEQ ID NO:2 are mammalian CDK8 proteins.
  • a functional amino acid (sequence) variant of me CDK8 of SEQ ID NO:2 may be substitutional, insertional or deletional.
  • Substimtions, deletions and insertions may be combined to arrive at an amino acid mutant of the invention.
  • Amino acid substimtions are typicaUy of single residues, insertions usuaUy wiU be on the order of from one to about ten amino acid residues, and deletions will usuaUy range from about one to thirty residues.
  • a substitutional amino acid mutant is any polypeptide having an amino acid sequence substantiaUy identical to the sequence set forth in SEQ ID NO:2, in which one or more residues have been conservatively substimted with a functionaUy-similar amino acid residue.
  • Conservative substimtions include e.g. the substimtion of one non-polar (hydrophobic) residue, such as methionine, valine, leucine, isoleucine for another, substimtion of one polar (hydrophUic) residue for another, such as between glycine and serine, between arginine and lysine, and between glutamine and asparagine.
  • Substitutional or deletional mutagenesis may be employed to eliminate O- or N-linked glycosylation sites.
  • Deletions of cysteine or other labUe amino acid residues may also be desirable, for example to increase the oxidative stab ity of a protein of the invention.
  • a derivative of a protein of the invention is a covalent or aggregative conjugate of said protein with another chemical moiety, said derivative displaying essentiaUy the same biological activity as the underivatized protein of the invention.
  • An exemplary covalent conjugate according to the invention is a conjugate of a protein of the invention with another protein or peptide, such as a fusion protein comprising a protein of the invention, e.g. a CDK8 fragment and a carrier protein suitable for enhancing the in vivo antigenicity of said fragment
  • a covalent conjugate of the invention further includes a protein of the invention labeUed with a detectable group, e.g. a protein of the invention which is radiolabeUed, covalently bound to a rare earth chelate or conjugated to a fluorescent moiety.
  • a protein of the invention is obtainable from a natural source, e.g. by isolation from a metazoan organism, particularly human cells including ceU lines, such as HeLa cells or HL60 cells, or human tissue expressing CDK8, or, by chemical synthesis or recombinant DNA techniques.
  • chemical synthesis of a protein of the invention is performed according to conventional methods known in the art In general, those methods comprise the sequential addition of one or more amino acid residues to a growing (poly)peptide chain. If required, potentiaUy reactive groups, e.g. free amino or carboxy groups, are protected by a suitable, selectively removable protecting group. Chemical synthesis may be particularly advantageous for fragments of CDK8 having no more than about 100 to 150 amino acid residues.
  • the invention also provides a method for preparing a protein of the invention, said method being characterized in that suitable host cells producing the protein of the invention are multipUed in vitro or in vivo.
  • the host cells are transformed or transfected with a hybrid vector comprising an expression cassette comprising a promoter and a DNA sequence coding for a protein of the invention which DNA is controUed by said promoter.
  • the protein of the invention may be recovered. Recovery comprises e.g. isolating the protein of the invention from the host cells or isolating the host cells comprising the protein, e.g. from the culture broth.
  • Suitable host cells include eukaryotic cells, e.g. animal cells, plant cells and fungi, and prokaryotic cells, such as gram-positive and gram-negative bacteria, e.g. E. coU.
  • a protein of the invention can be produced directly in recombinant ceU culture or as a fusion with a signal sequence, preferably a host-homologous signal.
  • in vitro means ex vivo, thus including e.g. ceU culture and tissue culture conditions.
  • An amino acid mutant as defined hereinbefore may be produced e.g. from a DNA encoding a protein of SEQ ID NO:2, which DNA has been subjected to site-specific in vitro mutagenesis resulting e.g. in an addition, exchange and or deletion of one or more amino acids. WhUe the site for introducing an amino acid variation is predetermined, the mutation per se need not be predetermined, but random mutagenesis may be performed at the target codon or region.
  • substitutional, deletional and insertional variants are prepared by recombinant methods and screened for cyclin C-binding affinity, kinase activity and/or immuno-crossreactivity with the native forms of the protein of the invention.
  • mutants ofthe invention may be prepared by chemical synthesis using methods routinely employed in the art
  • a protein of the invention may be derivatized in vitro according to conventional methods known in the art
  • a protein of the invention may be used, for example, as immunogen, e.g. to raise CDK8 specific immunoreagents, in a drug or Ugand screening assay, or in a purification method, such as affinity purification of a binding Ugand, such as cyclin C or an anti-CDK8 antibody.
  • a protein of the invention, or a fragment thereof, suitable for in vivo administration and capable of competing with endogenous CDK8 for an endogenous Ugand, e.g. cyclin C, is envisaged as therapeutic agent .
  • the invention also relates to the use of a protein of the invention, or a fragment thereof, for the generation of a monoclonal or polyclonal antibody, which specificaUy binds to CDK8.
  • a protein of the invention or a fragment thereof, for the generation of a monoclonal or polyclonal antibody, which specificaUy binds to CDK8.
  • anti-CDK8 antibody is intended to include immune sera.
  • Particularly useful for this purpose is a protein fragment consisting of at least eight or more, preferably eight to about twenty, consecutive amino acids of CDK8 of SEQ ID NO:2.
  • the invention provides polyclonal and monoclonal antibodies generated against CDK8.
  • Such antibodies may be useful e.g. for immunoassays including immunohistochemistry, as weU as diagnostic and therapeutic appUcations.
  • antibodies specific for the cyclin C binding site, or portions thereof, of CDK8 are suitable for blocking the endogenous CDK8.
  • Particularly useful are antibodies selectively recognizing and binding to CDK8.
  • the antibodies of the invention can be administered to a subject in need thereof, particularly a human, employing standard methods.
  • the antibodies of the invention can be prepared according to methods weU known in the art through immunization of a mammal using as antigen CDK8 (including antigenic fragments thereof and fusion proteins), hereafter referred to as "immunogenic CDK8".
  • Factors to consider in selecting CDK8 fragments as antigens include antigenicity and uniqueness to the protein.
  • Immunogenic CDK8 according to the invention includes e.g. a tagged CDK8 fusion protein comprising a polyamino acid tag and CDK8, or a fragment thereof.
  • a suitable polyamino acid tag is e.g. polyhistidine.
  • the fragment may be a carboxy-terminal fragment of CDK8 comprising e.g.
  • the antigen used for production of anti-CDK8 antibodies may represent an active or inactive form of CDK8.
  • the antibodies as provided by the present invention may be capable of distinguishing between the active or inactive form.
  • the antibodies raised against a protein of the invention may react with a non-glycosylated or glycosylated form of CDK8, or bom.
  • a multiple injection immunization protocol is used for immunizing animals widi immunogenic CDK8.
  • a good antibody response can be obtained in rabbits by intramuscular injection of about 300 ⁇ g immunogenic CDK8 emulsified in complete Freud's adjuvant foUowed several weeks later by one or more boosts of die same antigen in incomplete Freud's adjuvant
  • immunogenic CDK8 molecules used to immnniT the animal may be fused or coupled to a carrier protein by conjugation using techniques which are weU-known in the art
  • Commmonly used carrier proteins which may be chemicaUy coupled to the molecules include key hole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and a bacterial toxoid, e.g. tetanus or diphteria toxoid.
  • Polyclonal antibodies produced by the immunized animals can be further purified by techniques conventionaUy used in immunology arts for the purification and/or concentration of polyclonal, or monoclonal antibodies, such as affinity chromatography.
  • antibodies of the invention may be purified by binding to and elution from a matrix to which the peptide against which the antibodies are raised to is bound.
  • monoclonal antibodies specific for CDK8 are preferred, e.g. for use in detecting CDK8 in analyte samples (e.g. tissue samples and ceU lines).
  • analyte samples e.g. tissue samples and ceU lines.
  • immunization of mouse, rat or goat is preferred.
  • the general method used for the production of hybridomas is weU known (K ⁇ hler and Milstein (1975), Nature 256, 495).
  • the term antibody as used herein is intended to include intact molecules as weU as fragments thereof, such as Fab or F(ab') 2 fragments, which are capable of binding die epitopic determinant
  • Confirmation of CDK8 specificity among antibodies of the invention can be accomplished using routine screening techniques known to be suitable for die determination for the elementary reaction pattern of the antibody of interest such as the enzyme-linked immunosorbent assay (ELISA). For example, it is possible to evaluate the specificity of an antibody of interest without undue experimentation in a competitive binding assay. Such an assay is useful for deterrnining whether the antibody being tested prevents an anti-CDK8 antibody of the invention from binding to CDK8. If the antibody being tested competes with me antibody of the invention, as shown by a decrease in CDK8 binding by me antibody of the invention, then it is likely that me (monoclonal) antibodies bind to the same or a closely related epitope.
  • ELISA enzyme-linked immunosorbent assay
  • the invention is further intended to include chimeric antibodies of the CDK8-specific antibodies described above, or biologicaUy active fragments thereof.
  • die term "chimeric antibody” refers to an antibody in which die variable regions of the antibodies derived from one species are combined wim the constant regions of antibodies derived from a different species, or alternatively refers to CDR grafted antibodies.
  • Chimeric antibodies are constructed by recombinant DNA technology.
  • methods of producing chimeric humanized antibody molecules are known in the art and include combining murine variable regions with human constant regions, or by grafting the murine antibody complementary regions (CDRs) onto the human framework.
  • CDRs are defined as the amino acid sequences on the Ught and heavy chains of an antibody which form the three-dimensional loop structure that contributes to the formation of the antigen binding site. Any ofthe above described antibodies or biologicaUy active fragments can be used to generated chimeric and CDR grafted antibodies.
  • the invention also encompasses ceU lines (including hybridomas and transfectomas) which produce monoclonal antibodies of the invention.
  • ceU lines including hybridomas and transfectomas
  • the isolation of ceU lines producing monoclonal antibodies of the invention can be accomplished using routine screening techniques which permit determination of the elelmentary reaction pattern of the monoclonal antibody of interest
  • anti-idiotypic antibodies which can be used e.g. to screen monoclonal antibodies to identify whether the antibody has the same binding specificity as a monoclonal antibody of the invention.
  • These antibodies can also be used for immunization purposes.
  • anti-CDK8 antibodies may be used diagnosticaUy, e.g. to detect CDK8 expression in a biological ceU or tissue sample or to monitor the level ofits expression.
  • a suitable ceU sample is derived from skin biopsies, sputum specimens, or urinary specimens. CeUs may be obtained from any convenient source, such as skin, blood or hair foUicles.
  • anti-CDK8 antibodies are useful for detection of die CDK8/cyclin C complex, e.g. by co-immunoprecipitation.
  • CDK8 may be detected and or bound using anti-CDK8 antibodies in either Uquid or soUd phase immunoassay formats (i.e. when bound to a carrier).
  • Uquid or soUd phase immunoassay formats examples include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, ceUuloses, polyacrylamides, agaroses and magnetite.
  • Examplary types of immunoassays which can utilized monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format Specific examples of such immunoassays include the radioimmunoassay (RIA) and the sandwich (immunometric) assay.
  • RIA radioimmunoassay
  • sandwich immunometric
  • the anti-CDK8 antibodies of die invention may be unlabeled or detectably labeUed.
  • labeling There are many different Iables and methods of labeling known to those of skill in the art Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, coUoidal metals, chen ⁇ luminescent compounds, and bioluminescent compounds.
  • Another labeling technique which may result in greater sensitivity consists of coupling the antibodies of me invention to low molecular weight haptens, such as biotin. These haptens can then be specificaUy labeled by means of a second reaction.
  • the anti-CDK8 antibodies of the invention may also be useful for in vivo diagnosis, such as to identify a site of deregulated ceU proliferation, or to monitor a particular therapy.
  • the detectably labeled monoclonal antibody is given in a dose which is diagnosticaUy effective, meaning that the amount of detectably labeUed anti-CDK8 antibody is administered in sufficient quantity to enable detection of die site having cells which (over)express CDK8.
  • This invention further covers a nucleic acid (DNA, RNA) comprising a purified, preferably recombinant nucleic acid (DNA, RNA) coding for a protein of the invention, or a fragment of such a nucleic acid.
  • these nucleic acids are useful as probes, thus e.g. readily enabling tiiose skilled in the art to identify and/or isolate nucleic acid encoding CDK8.
  • the nucleic acid may be unlabeled or labeled with a detectable moiety.
  • nucleic acid according to d e invention is useful e.g. in a method for deterrnining the presence of CDK8, said method comprising hybridizing me DNA (or RNA) encoding (or complementary to) CDK8 to test sample nucleic acid and to determine the presence of CDK8.
  • Purified CDK8 encoding nucleic acid of the invention includes nucleic acid that is free from at least one contaminant nucleic acid with which it is ordinarily associated in the natural source of CDK8 nucleic acid. Purified nucleic acid dius is present in other than in the form or setting in which it is found in nature. However, purified CDK8 nucleic acid embraces such nucleic acid in ordinarily CDKS expressing cells where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different DNA sequence than that found in nature. The CDK8 gene maps to human chromosome 13ql2.
  • die invention provides a purified or isolated DNA molecule encoding a CDK8 protein of the invention, or a fragment of such DNA.
  • a DNA comprises a coding single-stranded DNA, a double-stranded DNA consisting of said coding DNA and complementary DNA tiiereto, or this complementary (single stranded) DNA itself.
  • the invention relates to a DNA comprising a DNA coding for die above captioned preferred CDK8, or a fragment diereof, e.g. the DNA widi die nucleotide sequence set forth in SEQ ID NO: 1, or a fragment diereof.
  • the nucleic acid sequences provided herein may be employed to identify DNAs encoding CDK8 amino acid variants, particularly aUeUc or interspecies variants.
  • a method for identifying such DNA comprises contacting metazoan, particularly mammalian DNA with a nucleic acid probe described above and identifying DNA(s) which hybridize to said probe.
  • Exemplary nucleic acids of the invention can alternatively be characterized as tiiose nucleic acids which encode a protein of the invention and hybridize to the DNA having the sequence set forth in SEQ ID NO: 1, or a selected portion (fragment) of said DNA.
  • Preferred are such DNA molecules encoding a protein of the invention which hybridize under stringent conditions to the above-mentioned DNAs.
  • Stringency of hybridization refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skill in die field. As known to those of skiU in the art, die stability of hybrids is reflected in the melting temperature (T m ) of the hybrid which decreases approximately 1 to 1.5°C with every 1% decrease in sequence homology. In general, the stabitity of a hybrid is a function of sodium ion concentration and temperature. TypicaUy, die hybridization reaction is performed under conditions of higher stringency, foUowed by washes of varying stringency. Given the guidance of die present invention, die nucleic acids of the invention are obtainable according to methods weU known in the art The present invention further relates to a process for die preparation of such nucleic acids.
  • a DNA of the invention is obtainable by chemical synthesis, by recombinant DNA technology or by polymerase chain reaction (PCR). Preparation by recombinant DNA technology may involve screening a suitable cDNA or genomic Ubrary.
  • a suitable method for preparing a DNA or of tiie invention may e.g. comprise the syntiiesis of a number of oUgonucleotides, their use for amplification of DNA by PCR methods, and their spUcing to give the desired DNA sequence.
  • Suitable Ubraries are commerciaUy avaUable, e.g. the Ubraries employed in the Examples, or can be prepared from tissue samples.
  • a DNA or RNA comprising substantiaUy die entire coding region of CDK8, or a suitable oUgonucleotide probe based on said DNA.
  • a suitable oUgonucleotide probe (for screening involving hybridization) is a single stranded DNA or RNA d at has a sequence of nucleotides that includes at least about 20 to about 30 contiguous bases diat are die same as (or complementary to) any about 20 or more contiguous bases of the nucleic acid sequence set forth in SEQ ID NO:l.
  • the nucleic acid sequences selected as probes should be of sufficient lengtii and sufficiendy unambiguous so diat false positive results are minimized.
  • Preferred regions from which to construct probes include 5' and/or 3' coding sequences, sequences predicted to encode Ugand binding sites, and me like.
  • either die fuU-length cDNA clone disclosed herein or fragments diereof can be used as probes.
  • nucleic acid probes of tiie invention are labeled wid suitable label means, e.g. a chemical moiety, for ready detection upon hybridization.
  • suitable label means is a radiolabel.
  • the preferred method of labelling a DNA fragment is by incorporating 32 P-labeled ⁇ -dATP widi die Klenow fragment of DNA polymerase in a random priming reaction, as is weU known in the art OUgonucleotides are usuaUy end-labeled witii 32 P-labeled ⁇ -ATP and polynucleotide kinase.
  • other methods e.g. non-radioactive
  • a suitable Ubrary e.g. with a portion of DNA including substantiaUy die entire CDK8-encoding sequence or a suitable oUgonucleotide based on a portion of said DNA
  • positive clones are identified by detecting a hybridization signal; the identified clones are characterized by restriction enzyme mapping and/or DNA sequence analysis, and dien examined, e.g. by comparison witii die sequences set forth herein, to ascertain whetiier they include DNA encoding a complete CDK8 (i.e., if tiiey include translation initiation and termination codons).
  • die selected clones are incomplete, they may be used to rescreen the same or a different Ubrary to obtain overlapping clones. If the Ubrary is genomic, then the overlapping clones may include exons and introns. If die Ubrary is a cDNA Ubrary, then the overlapping clones wiU include an open reading frame. In both instances, complete clones may be identified by comparison with die DNAs and deduced amino acid sequences provided herein.
  • nucleotide sequences of die invention may be used as hybridization probes.
  • antisense-type therapeutic agents may be designed.
  • nucleic acid of die invention can be readUy modified by nucleotide substimtion, nucleotide deletion, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof.
  • modified sequences can be used to produce mutant CDK8s which differ from the proteins found in nature. Mutagenesis may be predetermined (site-specific) or random. A mutation which is not a sUent mutation must not place sequences out of reading frames and preferably wiU not create complementary regions that could hybridize to produce secondary mRNA structures such as loops or hairpins.
  • the cDNA or genomic DNA encoding native or mutant CDK8 of the invention can be incorporated into vectors for further manipulation.
  • the invention concerns a recombinant DNA which is a hybrid vector comprising at least one of die above mentioned DNAs.
  • the hybrid vectors of die invention comprise an origin of repUcation or an autonomously repUcating sequence, one or more dominant marker sequences and, optionaUy, expression control sequences, signal sequences and additional restriction sites.
  • the hybrid vector of the invention comprises an above described nucleic acid insert operably linked to an expression control sequence, in particular tiiose described hereinafter.
  • Vectors typicaUy perform two functions in coUaboration witii compatible host cells. One function is to faciUtate the cloning of the nucleic acid diat encodes a CDK8 protein of die invention, i.e. to produce usable quantities of the nucleic acid (cloning vectors).
  • the odier function is to provide for repUcation and expression of the gene constructs in a suitable host either by maintenance as an extrachromosomal element or by integration into the host chromosome (expression vectors).
  • a cloning vector comprises the DNAs as described above, an origin of repUcation or an autonomously repUcating sequence, selectable marker sequences, and optionaUy, signal sequences and additional restriction sites.
  • An expression vector additionaUy comprises expression control sequences essential for die transcription and translation of d e DNA of d e invention.
  • an expression vector refers to a recombinant DNA construct, such as a plasmid, a phage, recombinant virus or odier vector d at, upon introduction into a suitable host ceU, results in expression of the cloned DNA.
  • Suitable expression vectors are weU known in the art and include those that are repUcable in eukaryotic and/or prokaryotic cells.
  • Most expression vectors are capable of repUcation in at least one class of organisms but can be transfected into anotiier organism for expression.
  • a vector is cloned in E. coU and d en die same vector is transfected into yeast or mammalian cells even though it is not capable of repUcating independentiy of the host ceU chromosome.
  • DNA may also be amplified by insertion into die host genome.
  • the recovery of genomic DNA encoding CDK8 is more complex than that of exogenously repUcated vector because restriction enzyme digestion is required to excise CDK8 DNA, DNA can be amplified by PCR and be directiy transfected into die host cells without any repUcation component
  • expression and cloning vector contain a selection gene also referred to as selectable marker.
  • This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed witii the vector containing the selection gene will not survive in the culture medium.
  • Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not avaUable from complex media.
  • E. coU genetic marker and an JL coU origin of repUcation are advantageously included.
  • E. coU plasmids such as pBR322, Bluescript vector or a pUC plasmid.
  • Suitable selectable markers for mammaUan cells are those diat enable die identification of cells competent to take up CDK8 nucleic acid, such as dihydrofolate reductase (DHFR, metiiotrexate resistance), thymidine kinase, or genes confering resistance to G418 or hygromycin.
  • the mammaUan ceU transfectants are placed under selection pressure which only those transfectants are uniquely adapted to survive which have taken up and are expressing the marker.
  • Expression and cloning vectors usuaUy contain a promoter that is recognized by die host organism and is operably linked to CDK8 nucleic acid. Such promoter may be inducible or constitotive. The promoters are operably linked to DNA encoding CDK8 by removing the promoter from die source DNA by restriction enzyme digestion and inserting die isolated promoter sequence into the vector. Botii the native CDK8 promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of CDK8 DNA, However, heterologous promoters are preferred, because tiiey generaUy aUow for greater transcription and higher yields of expressed CDK8 as compared to native CDK8 promoter.
  • Promoters suitable for use with prokaryotic hosts include, for example, the ⁇ -lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the tac promoter.
  • Their nucleotide sequences have been published, thereby enabling the skilled worker operably to Ugate diem to DNA encoding CDK8, using linkers or adaptors to supply any required restriction sites.
  • Promoters-f or use in bacterial systems will also generaUy contain a Shine-Delgarno sequence operably linked to die DNA encoding CDK8.
  • CDK8 gene transcription from vectors in mammaUan host cells may be controUed by promoters compatible with the host ceU systems, e.g. promoters derived from the genomes of viruses.
  • Transcription of a DNA encoding a protein according to die invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector.
  • the various DNA segments of die vector DNA are operatively linked, i.e. tiiey are contiguous and placed into a functional relationship to each odier employing conventional Ugation techniques. Isolated plasmids or DNA fragments are cleaved, taUored, and reUgated in the form desired to generate the plasmids required.
  • ampUfication and/or expression may be measured in a sample directiy, for example, by conventional Southern blotting, northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), in situ hybridization, using an appropriately labeUed probe based on a sequence provided herein, binding assays, immunodetection and functional assays.
  • Those skiUed in die art will readUy envisage how diese metiiods may be modified, if desired.
  • the invention further provides host cells capable of producing a CDK8 protein of d e invention and including heterologous (foreign) DNA encoding said protein.
  • the nucleic acids of d e invention can be expressed in a wide variety of host cells, e.g. tiiose mentioned above, diat are transformed or transfected widi an appropriate expression vector.
  • a protein of the invention may also be expressed as a fusion protein. Recombinant cells can men be cultured under conditions whereby d e protein (s) encoded by die DNA of die invention is (are) expressed.
  • Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-prositive organisms, such as E. coU, e.g. E. coU K-12 strains, DH5 ⁇ and HB 101, or Bacilli.
  • Further host cells suitable for CDK8 encoding vectors include eukaryotic microbes such as filamentous fungi or yeast e.g. Saccharomyces cerevisiae. Higher eukaryotic cells include insect amphebian and vertebrate cells. In recent years propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.
  • the host cells referred to in tins apphcation comprise cells in in vitro culture as weU as cells that are within a host animaL DNA may be stably incorporated into the cells or may be transiently expressed according to conventional methods.
  • Stably transfected mammaUan cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing die transfected cells under conditions selective for cells expressing the marker gene. To prepare transient transfectants, mammaUan cells are transfected wid a reporter gene to monitor transfection efficiency. To produce such stably or transientiy transfected cells, die cells should be transfected wid a sufficient amount of CDK8-encoding nucleic acid to form CDK8 of die invention. The precise amounts of DNA encoding CDK8 of the invention may be empiricaUy determined and optimized for a particular ceU and assay. A DNA of die invention may also be expressed in non-human transgenic animals, particularly transgenic warm-blooded animals.
  • Mediods for producing transgenic animals including mice, rats, rabbits, sheep and pigs, are known in die art and are disclosed, for example by Hammer et aL ((1985) Nature 315, 680-683).
  • An expression unit including a DNA of die invention coding for a CDK8 together with appropriately positioned expression control sequences, is introduced into pronuclei of fertilized eggs. Introduction may be achieved, e.g. by microinjection. Integration of the injected DNA is detected, e.g. by blot analysis of DNA from suitable tissue samples. It is preferred diat die introduced DNA be incorporated into d e germ line of the animal so tiiat it is passed to die animal's progeny.
  • a knock-out animal may be developed by introducing a mutation in die CDK8 sequence, tiiereby generating an animal which does not express die functional CDK8 gene anymore.
  • Such knock-out animal is useful e.g. for studying the role of the CDK8 or the CDK8/cyclin C complex in metabolism, but in particular for providing a mammalian animal model with a suitable genetic background for introducing and expressing transgenes encoding die homologous human CDK8.
  • Expression of human counterpart CDK8 on a homologous gene knock-out background has the unique advantage of excluding differences in efficacies of drugs on a given protein (in this case CDK8) caused by species-specific sequence differences in said protein.
  • Host cells are transfected or transformed widi die above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Heterologous DNA may be introduced into host cells by any mediod known in the art, such as transfection widi a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique, by electroporation or by Upofectm-mediated. Numerous mediods of transfection are known to the skiUed worker in die field. Successful transfection is generaUy recognized when any indication of the operation of diis vector occurs in the host ceU. Transformation is achieved using standard techniques appropriate to die particular host cells used (see, e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).
  • the DNA provided herein may be expressed in any suitable host ceU, e.g. tiiose referred to above, prefened for expression of DNA encoding functional CDK8 are eukaryotic expression systems, particularly mammaUan expression systems, including commerciaUy avaUable systems and oti er systems known to tiiose of skiU in the art
  • the present invention provides a method for identifying compounds capable of binding to CDK8, said method comprising employing a protein of the invention in a binding assay.
  • a binding assay may be useful for identification of a CDK8 Ugand including a novel endogenous Ugand.
  • a binding assay according to die invention involves exposure of a protein of the invention, e.g. die CDK8 of SEQ ID NO:2, to a Ugand candidate under conditions and for a time sufficient to aUow binding of said potential Ugand to said protein of die invention, and deterrnining quaUtatively and/or quantitatively, whether binding has occurred, e.g.
  • Binding of a Ugand to the protein of the invention may be analyzed according to conventional methods, e.g. methods suitable for detecting the association of two proteins, such as electrophoresis or immunoanalytical methods, e.g. immunoprecipitation witii an anti-CDK8 antibody.
  • a preferred binding assay is a competitive binding assay.
  • the principle underlying a competitive binding assay is generaUy known in die art Briefly, such a binding assay is performed by aUowing a compound to be tested for its capability to compete widi a known, suitably labeled Ugand for die binding site at a target molecule, i.e. a protein of the invention.
  • a suitably labeled Ugand is e.g. a radioactively labeled Ugand or a Ugand which can be detected by its optical properties, such as absorbance or fluorescence. After removing unbound Ugand and test compound die amount of labeled Ugand bound to d e protein of die invention is measured.
  • d e amount of bound labeled Ugand is reduced in die presence of the test compound, said compound is found to bind to the target molecule, Le. die protein of die invention.
  • Compounds binding to die target protein of the invention may modulate a functional property of CDK8 and may thereby be identified as an agonist or antagonist in a functional assay.
  • a competitive binding assay may be performed e.g. with transformed or transfected host cells expressing die protein of the invention, or with a soluble or immobilized protein of the invention.
  • anotiier aspect die present invention relates to a functional assay, which is suitable for detection of a change of a physical-chemical property of CDK8, such as activity, status of phosphorylation, conformation, and die binding affinity of associatable molecules.
  • a functional assay which is suitable for detection of a change of a physical-chemical property of CDK8, such as activity, status of phosphorylation, conformation, and die binding affinity of associatable molecules.
  • Such functional response is the result of the interaction of me compound to be tested widi CDK8 and may affect e.g. d e activity or phosphorylation status of anotiier protein influenced by CDK8 widiin a ceU expressing functional CDK8 (as compared to a negative control).
  • the present invention also provides a method suitable for identifying a component or agent which modulates the biological activity of CDK8, said mediod comprising contacting CDK8 of SEQ ID NO:2, or another suitable protein of the invention, witii at least one component or agent whose abUity to modulate the activity of CDK8 is sought to be investigated, and determining the change of CDK8 activity of said protein of the invention caused by said component or agent
  • the method enables identification of stimulatory or inhibitory components of CDK8 activity, herein referred to as agonists or antagonists of CDK8.
  • An in vitro assay for an agonist or antagonist may require that a protein of the invention is produced in sufficient amounts in a functional form using recombinant DNA methods.
  • An assay is men designed to measure a functional property of CDK8. Production of a protein of the invention is regarded as occurring in sufficient amounts, if CDK8 activity results in a measurable response. It is preferred to perform an assay of the invention in the presence of cyclin C under conditions aUowing formation of functionaUy active CDK8.
  • CDK8 is intended to include a CDK8 protein complex comprising CDK8 and one or more further proteins associated with CDK8.
  • An exemplary CDK8 protein complex is a complex comprising the CDK8 of SEQ ID NO:2 and cyclin C.
  • a component or agent which modulates die activity of CDK8 refers to a compound or signal tiiat is capable of altering the response pathway mediated by functionaUy active CDK8 within a ceU (as compared to the absence of CDK8).
  • Modulation of CDK8 activity particularly refers to modulation of one or more of the foUowing properties of CDK8: cyclin C binding affinity and/or kinetics, catalytic activity, the ability to regulate the ceU cycle during the interphase, particularly at transition from the G j -phase to the S-phase, its regulatory role in transcription and die abUity to integrate ceU growth with transcription events.
  • cyclin C binding affinity and/or kinetics the ability to regulate the ceU cycle during the interphase, particularly at transition from the G j -phase to the S-phase, its regulatory role in transcription and die abUity to integrate ceU growth with transcription events.
  • a change in cyclin C binding activity andor kinetics may be determined using the methods described herein. More specificaUy, the effect of a particular compound or signal on die interaction of CDK8 widi cyclin C may be determined in an assay suitable for dereimining the affinity and/or rate of binding of cyclin C to CDK8, or kinase activity of CDK8.
  • CDK8 and cyclin C are (co-) expressed in an appropriate expression system, such as yeast E. coU, insect cells or mammaUan cells transformed witii suitable expression vectors.
  • CDK8 and cyclin C may be expressed in baculovirus-infected insect cells, either separately or jointly.
  • the proteins are recovered from the cells, and in enriched or purified from, e.g. in a ceU extract exposed to the test compound under conditions aUowing interaction.
  • the host cells may be metaboUcaUy labeled, e.g. [ 35 S]-labeled.
  • a compound inhibiting the interaction of CDK8 with cyclin C (antagonist) corresponding decreases in CDK8/cyclin C complex formation, or the ability of CDK8 to phosphorylate a suitable substrate are detectable.
  • die antagonist associates with CDK8 this inhibition wiU correlate widi the association widi die antagonist and CDK8, as detectable e.g. by immunoprecipitation.
  • Modulation of the catalytic activity may be assessed by analyzing phosphorylation of a suitable CDK8 substrate, e.g. by employing a protein kinase assay as decribed hereinbefore.
  • CatalyticaUy active CDK8 requires association of CDK8 with cyclin C.
  • ceU cycle progression may be analyzed, e.g. in mammalian, particularly human cells, according to methods known in the art, e.g. as described by Tassan, J.P. et al. ((1994), J. CeU Biology 127, 467-478). Briefly, cells, e.g.
  • HeLa cells, containing recombinant CDK8 encoding nucleic acid, and, optionaUy, recombinant cyclin C-encoding nucleic acid are synchronized at d e G 0 or G-. stage, e.g. by centifugal elutriation, using drug arrest-release protocols involving e.g. lovastatin or mimosine, or serum or isoleucine starvation (see e.g. Krek, W. & Nigg, E.A. (1991) EMBO J. 10, 305-31; O'Connor, P.M. & Jackman, J. in "CeU Cycle - Material and Methods" (1995), ed. M.
  • the interaction widi die transcription apparatus or the abUity to integrate ceUular events with ceU cycle progression may be analyzed using in vitro transcription systems or reporter assays involving e.g. chloramphenicol transferase (CAT) or luciferase in vivo.
  • CAT chloramphenicol transferase
  • luciferase in vivo.
  • CDK8 of SEQ ID NO:2 may be used in a soluble, immobilized or ceUular form.
  • the protein of the invention is attached to a soUd suppor To obtain a ceUular form of the protein of the invention, it is produced by a suitably transformed host ceU which is employed in the assay.
  • the protein of the invention is a recombinant protein.
  • cyclin C may be present in a soluble, immobilized or ceUular form, with the provision tiiat it is available in a form capable of associating with the protein of die invention.
  • CeUular cyclin C may be homologous or heterologous to the producing ceU. For production of heterologous cyclin C cells are transformed with a suitable expression vector.
  • the protein of the invention and cyclin C may be produced by (coupled) in vitro transcription-translation, e.g. using a system as described in the Examples.
  • the protein of the invention and cyclin C are obtainable from the same species, e.g. an assay of the invention employing human CDK8 further comprises human cyclin C.
  • an agonist is understood to refer to a molecule that is capable of interacting with CDK8 in such a way diat its biological activity is enhanced.
  • an agonist may be characterized in that it mimics a natural CDK8 Ugand, such as cyclin C, or increases the interaction between CDK8 and a natural Ugand, such as cyclin C.
  • antagonizing molecules are useful.
  • an antagonist is understood to refer to a molecule that is capable of interacting wit ⁇ f CDK8, which interaction results in a decrease of CDK8 activity.
  • antagonists are identified by their abUity to interact with CDK8, thereby reducing the ability of a natural Ugand, e.g. cyclin C, to stimulate CDK8 activity.
  • a natural Ugand e.g. cyclin C
  • an agonist or antagonist which is capable of selectively modulating die activity of CDK8 without substantiaUy affecting the activity of any odier CDK.
  • a CDK8 antagonist may be able to inhibit transition of a eukaryotic ceU from the G j phase into the S phase.
  • the assays of the invention may be useful to identify compounds or signals which are capable of acting as therapeutic agents in a mammal in need diereof, which are effective against a disease or disorder caused by a decrease or increase of ceUular CDK8 activity.
  • the assays described herein render possible e.g. identification of ceU growtii inhibitors, which may be suitable as therapeutic agents against hyperproliferative disorders, such as benign and maUgnant mmors, and psioriasis, e.g. components which are capable of affecting progression of the ceU cyclus, particularly progression from the G ! to S phase.
  • compounds identified by a method according to d e invention may be therapeuticaUy effective in diseases which are caused by inappropriate transcription or dysfunctional DNA repair.
  • the assays provided herein wiU enable identification and design of CDK8-specific compounds, particularly molecules specificaUy binding to CDK8 (tigands), eventuaUy leading to die development of a disease-specific drug. If designed for a very specific interaction with only CDK8 (or a predetermined selection of CDKs including CDK8), such a drug is unlikely to exhibit unwanted side effects.
  • Host cells expressing a nucleic acid coding for a protein of the invention are e.g. useful for drug screening, and the present invention encompasses a mediod for identifying a compound or signal which modulates the biological activity of CDK8, said mediod comprising exposing cells containing heterologous DNA encoding a suitable protein of the invention, wherein said cells produce functionaUy active CDK8, to at least one compound or signal, whose ability to modulate the activity of said CDK8 is sought to be determined, and thereafter monitoring said cells for changes caused by said modulation.
  • the invention covers an assay for identifying compounds which modulate die activity of CDK8, said assay comprising:
  • Preferred such cells are suitably manipulated mammalian cells, particularly human cells, such as HeLa cells, which express CDK8, and optionaUy cyclin C.
  • CeUs producing functionaUy active CDK8 may be employed for d e identification of compounds, particularly low molecular weight molecules including peptides capable of acting as agonists or antagonists of CDK8 and whicfh are bioavaUable in vitro and in vivo.
  • mammalian cells e.g. HEK293 cells, L cells, CHO-K1 cells, LLCPK-1 cells, GH3 cells, CT26 cells, T24 cells, KB85 ceUs and Melanoma A375 cells (avaUable e.g. from the American Tissue Type Culture CoUection (ATCC)) are cultured in an appropriate standard medium, e.g. a commerciaUy avaUable medium.
  • a CDK8 expression plasmid is transiently transfected into die cells, e.g. by calcium-phosphate precipitation (Ausubel, F. M., et al. (1993) Cunent Protocols in Molecular Biology, Greene and Wiley, USA).
  • CeU lines stably expressing CDK8 may be generated e.g. by Upofectm-mediated transfection with CDK8 expression plasmids and a plasmid comprising a selectable marker gene, e.g. pSV2-Neo (Southern and Berg, J. Mol. Appl. Genet 1, 327-341 (1982)), a plasmid vector encoding the G-418 resistence gene.
  • CeUs surviving die selection are isolated and grown in the selection medium.
  • Resistant clonal ceU lines are analyzed, e.g. for immunoreactivity with CDK8-specific antibodies or by assays for CDK8 functional responses foUowing agonist addition.
  • CeUs producing die desired CDK8 are used in a method for detecting compounds binding to die CDK8 or in a method for identifying an agonist or antagonist
  • a prefened metiiod for detecting a CDK8 agonist comprises the steps of (a) exposing a protein of the invention coupled to a CDK8 response pathway, under conditions and for a time sufficient to aUow interaction of the compound witii the protein of the invention and an associated response through the pathway, and (b) detecting an increase or decrease in die stimulation of the response pathway resulting from the interaction of the compound with the protein of the invention, relative to the absence of the tested compound and therefrom deterrnining the presence of a CDK8 agonist
  • a preferred method for identifying a CDK8 antagonist comprises the steps of (a) exposing a compound in the presence of a known CDK8 agonist e.g. cyclin C, to a protein of the invention coupled to a CDK8 response patiiway, under conditions and for a time sufficient to aUow interaction of the agonist with the protein of die invention and an associated response through the pathway, and (b) detecting an inhibition of the stimulation of the response pathway induced by die agonist said inhibition resulting from the interaction of the compound widi me protein of die invention, relative to the stimulation of the response patiiway by the CDK8 agonist alone and tiierefrom deterrnining the presence of a CDK8 antagonist Inhibition may be detected, e.g.
  • test compound competes widi the agonist for the protein of the invention.
  • Compounds which may be screened utilizing such method include blocking antibodies specificaUy binding to a protein of the invention.
  • an assay is useful for the screening for compounds interacting with a CDK8 agonist e.g. cyclin C.
  • the agonistic effect is neutralized or reduced, e.g. by binding of die test compound to die agonist e.g. cyclin C, dius affecting agonist interaction with die protein of die invention.
  • Examples are CDK8 fragments comprising part or aU of the agonist binding domain, e.g. the cyclin C binding domain.
  • interaction of an agonist or antagonist with CDK8 denotes binding of die agonist or antagonist to CDK8.
  • GeneraUy, conditions and times sufficient for interaction of an agonist or antagonist with CDK8 may vary with the source and purity of CDK8, however, conditions generaUy suitable for interaction occur between about 4°C and about 40°C, preferably between about 4°C and about 37°C, in a buffer solution containing between 0 and 2 M NaCl, preferably between 0.1 and 0.9 M NaCl, and widiin a pH range of between 5 and 9, preferably between 6.5 and 8. Sufficient time for the binding and response wiU generaUy be between about 15 min and about 24 h after exposure.
  • die buffer solution comprises magnesium ions (Mg 2"1 ⁇ , added e.g.
  • a magnesium salt such as magnesium acetate or magnesium nitrate
  • calcium ions Ca 2+
  • Suitable conditions are e.g. those existing in a commerciaUy available reticulocyte lysate, such as die lysates used in the Examples.
  • Firtatty also relates to a method of inhibiting activation of CDK8 in a cett, comprising introducing into said ceU an agent which inhibits die binding of CDK8 to cyclin C.
  • agent may be selected from die group consisting of an oUgonucleotide Which binds nucleic acid encoding CDK8, diereby inhibiting e.g.
  • CDK8 an antibody which specificaUy binds CDK8, particularly an antibody which binds to an epitope in die cyclin C binding domain of CDK8; a compound which binds to die cyclin C binding domain of CDK8; a compound which binds cyclin C in such a manner that cyclin C is prevented from binding to CDK8, such as a CDK8 fragment provided by the present invention; a compound diat degrades cyclin C; and a compound tiiat degrades CDK8.
  • the invention particularly relates to the specific embodiments (e.g. die proteins, nucleic acids, methods for die preparation, assays and uses diereof) as described in the Examples which serve to Ulustrate the present invention, but should not be construed as a limitation thereof.
  • the nimA gene encodes a protein-serine/threonine kinase, termed NTMA, that is critical for entry into mitosis in the filamentous fungus AspergiUus nidulans.
  • NTMA protein-serine/threonine kinase
  • a pair of degenerate oUgonucleotides, refened to as oUgonucleotides (69) and (72), is used as primers to amplify cDNAs by polymerase chain reaction (PCR) starting from poly(A) + RNA obtained from the highly proliferative leukemia ceU Une HL-60.
  • the primers which are designed to be specific for conserved amino acid motifs in the catalytic domain of NIMA, are first tested for dieir ability to amplify the expected DNA fragment from tiie nim A cDNA isolated from A. nidulans (Osmani. S.A. et aL (1988), CeU 53, 237-244). As determined by electrophoresis in agarose gels, die chosen primer pair generates HL-60 cDNAs of the predicted sizes in amounts comparable to PCR carried out with the nimA cDNA, Bands representing PCR products of die expected sizes are gel isolated and Ugated with die pGEM-3Zf (-) vector to create a PCR Ubrary.
  • the Ubrary is initiaUy screened by blue-white colony color selection to identify recombinants containing inserts.
  • Primer (72) and sequencing of inserts is used to identify a putative protein kinase sequence on the basis of the kinase domains defined by Hanks et aL ((1988) Science 241, 42-52) .
  • Three clones are found to contain a 216 bp partial human cDNA coding for 72 amino acids.
  • the plasmid of one clone is referred to as HsPK 35.
  • the nucleotide sequence of the cDNA and the deduced amino acid sequence are set forth in SEQ ID NO:5.
  • HL-60 cells Cold-frozen cells (Collins, S J. (1987) Blood 70, 1233-1244) are cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum. For routine passage, cells are grown to a density of 2 x IO 6 cells/ml and transferred every 2 days to fresh culture medium at a density of 2 x 10 s cells/ml.
  • Total RNA is prepared from undifferentiated HL-60 cells by die acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski, P. & and Sacchi, N. (1987 ⁇ Anal. Biochem. 162, 156-159. In brief, 1.5 x IO 7 exponentiaUy growing cells are peUeted and resuspended in 2 ml of guanidinium thiocyanate (GSCN), 0.1 M 2-mercaptoethanol, 50 mM sodium acetate, ImM EDTA and 0.25 % N-lauroylsarcosine (4 M GSCN solution).
  • GSCN guanidinium thiocyanate
  • 2-mercaptoethanol 50 mM sodium acetate
  • ImM EDTA ImM EDTA
  • 0.25 % N-lauroylsarcosine 4 M GSCN solution.
  • RNA is precipitated at -20° C for 60 min, peUeted, washed with 70% ethanol, and resuspended in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.1 % sodium dodecyl sulfate.
  • Poly (A) + RNA is prepared from total RNA using die PolyATtract mRNA Isolation System (Promega Corp.) according to die manufactorer's instructions.
  • HL-60 cDNA is prepared from 1 ⁇ g of poly(A) + RNA and used in PCR containing 10 ⁇ g/ml each of the degenerate primers designated 69 and 72, respectively: A CA A 5'- GCGCGGTACC A GACCANAT TC - 3' (69) G TG G
  • the oUgonucleotides are synthesized according to standard procedures and used witiiout further purification.
  • a restriction site for Kpnl is present at the 5'te ⁇ ninus of primer 69, whUe a restriction site for PstI is present at the 5' terminus of primer 72.
  • the PCR cycle is 1.5 min at 95°C, 2 min at 37°C, and 3 min at 63°C for 35 cycles (Wilks, A.F. (1989) Proc. Natl. Acad. Sci. USA 86, 1603-1607).
  • One-tenth of die 100 ⁇ l PCR reaction is analyzed by electrophoresis in a 2% agarose gel and the sizes of the PCR products are determined after staining with eti ⁇ dium bromide.
  • PCR products are phenol extracted, precipitated in 0.3 M sodium acetate (pH 5.2) with two volumes of 100 % ethanol at -20°C overnight peUeted at 10,000 x g for 20 min, and resuspended in 5-10 ⁇ l of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA.
  • the PCR products are digested widi Kpnl and PstI and purified by electrophoresis in 2% agarose gels, and the appropriate bands of DNA are cut out and recovered by electroelution (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual (1989) Cold Spring Habor, NY, Cold Spring Habor Laboratory Press).
  • Recovered DNAs are Ugated widi Kpnl- and Pstl-digested pGEM-3Zf (-) vector (Promega Corp.), transformed into E. coU strain DH 5 ⁇ , and transformants are plated onto LB plates containing 100 ⁇ g/ml ampicillin mat had been impregnated with 40 ⁇ l of 2 % 5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside (X-GAL) in N,N-dimethylformamide and 40 ⁇ l of 100 mM isopropyl- 1-thio- ⁇ -D-galactoside to select forrecombinants which contain inserts disrupting die ⁇ -galactoside gene (Maniatis, T.
  • Plasmid DNA is isolated from overnight cultures of white colonies using the Magic Minipreps DNA Purification System (Promega Corp.). Inserts are initiaUy characterized by die dideoxynucleotide chain termination metiiod witii single T-base reactions (T-tracks), and unique inserts are sequenced widi four base reactions.
  • Example 1 To prepare a ⁇ P-labeled probe for hybridization, the human cDNA obtained in Example 1 is excised from d e HsPK35 plasmid by digestion widi Psfl and EcoRI. The fragment is gel isolated and labeled with 32 P dATP using a random-primed DNA labeling kit (Boehringer Mannheim Biochemicals, IndianapoUs, IN).
  • Example 3 Cloning and sequencing of a cDNA coding for CDK8
  • the labeled 216 bp cDNA fragment prepared in Example 2 is used for screening a ⁇ phage cDNA Ubrary. FoUowing plaque purification of phages, inserts are excised and subcloned into Bluescript vectors. Colony hybridization, phage isolation, subcloning and plasmid sequencing are performed as foUows:
  • Plaques 10 ⁇ plaque forming units from a human testis ⁇ gtll cDNA Ubrary (Clontech Laboratories, Palo Alto, CA) are screened by DNA hybridization. Plaques are transfened to nitroceUulose filters that are treated with 1.5 M NaCl, 0.5 M NaOH for 5 min, then with 1.5 M NaCl, 0.5 M Tris-HCl, pH 8.0, and fmaUy with 2X SSC for 5 min (IX SSC is 0.15 M NaCl and 15 mM sodium citrate).
  • FUters are baked at 80°C for 2 hours and prehybridized at 42°C for 4 hours in solution A (0.1 M Pipes, pH 7.0, 0.8 M NaCl, 0.1% N-lauroyl sarcosine, 50% formamide, 5X Denhardt's solution, and 200 ⁇ g/ml salmon sperm DNA (IX Denhardt's solution is 0.02% bovine serum albumin, 0.02% FicoU, and 0.02% polyvinylpynoUdone). FUters are hybridized widi the 32p-labeled probe of Example 2 at 42°C overnight in solution A, 2X Denhardt's solution, 100 ⁇ g/ml salmon sperm DNA, and 10% dextran sulfate.
  • solution A 0.1 M Pipes, pH 7.0, 0.8 M NaCl, 0.1% N-lauroyl sarcosine, 50% formamide, 5X Denhardt's solution, and 200 ⁇ g/ml salmon sperm DNA
  • the longest insert (1772 bp) is sequenced in botii orientations and found to encode the entire CDK8 protein consisting of 464 amino acids.
  • the complete cDNA sequence and deduced protein sequence of CDK8 are set forth in SEQ JD NOs. 1 and 2.
  • a DNA fragment conesponding to CDK8 has been isolated as part of a gene mapping project, indicating diat CDK8 maps to human chromosome 13ql2 (Accession No. L23208). This locus is associated widi several human disease genes, including die breast cancer susceptibUity gene BRCA2 (Wooster, R. et al. (1994) Science 265, 2088-2090).
  • subdomain I from amino acid (aa) 27 to aa 38
  • subdomain JJ from aa 47 to aa 52
  • subdomain HI from aa 59 to aa 68
  • subdomain JN from aa 78 to aa 85
  • subdomain V from aa 98 to aa 106
  • subdomain VI from aa 139 to 149
  • subdomain VJJ from aa 174 to 180
  • subdomain VHI from aa 197 to aa 207
  • subdomain JX from aa 214 to 222
  • subdomain X from aa 257 to aa 265
  • subdomain XI from aa 317 to aa 326.
  • aU domains are defined approximately meaning mat the given positions of amino acids are to be understood as approximate positions.
  • Amino acid residues highly conserved among protein serine/threonine kinases are at amino acid positions 28, 30, 33, 35, 52, 66, 151, 153, 156, 173, 175, 216, 221 and 323 in SEQ JX) ⁇ O:2.
  • the SACRE motif is found at amino acid positions 62-66 (SEQ JD NO:2) , in the region conesponding to PSTAIRE in CDC2 (Meyerson, M. et al. (1992) EMBO J. 11, 2909-2917).
  • the putative initiator ATG codon is in a suitable context for translation initiation, and in vitro translation of this cDNA yields a protein that comigrates exactly with immunoreactive HeLa ceU CDK8 protein.
  • the nucleotide sequences at die 5' and 3' ends of die 216 bp cDNA of Example 1 differ from the nucleotide sequence determined for die CDK8 cDNA that is isolated from die phage Ubrary. These N- and C-terminal sequences conespond in fact to the annealing regions of degenerate oUgonucleotides (72) and (69) tiiat are used as primers in the PCR reaction.
  • a polyhistidine-tagged CDK8 fusion protein is generated using die QIAexpress bacterial expression system (QIAGEN Inc., Chatsworth, CA).
  • a plasmid (termed PQE10-CDK8) is constructed by excising a 1275-bp BamHl fragment from d e Bluescript plasmid containing die human CDK8 1772 bp cDNA (Example 3) and subcloning it into the pQElO vector.
  • the expressed protein contains a poly-His tag fused to the 274 COOH-terminal amino acids of CDK8.
  • the protein is expressed in Escherichia coU strain Ml 5 pREP4 (QIAGEN) using super medium (25g bacto-tryptone, 15g bacto-yeast extract and 5g NaCl/Uter). Recombinant protein is purified under denaturing conditions as described by the manufacturer (Qiagen Inc.). Poly-His-tagged CDK8 protein is finally eluted in buffer E (8 M urea, 0.1 M NaH 2 PO 4 , lOmM Tris-HCl, pH 4.5) and stored at -20°C.
  • buffer E 8 M urea, 0.1 M NaH 2 PO 4 , lOmM Tris-HCl, pH 4.5
  • Rabbit antibodies against bacteriaUy expressed CDK8 protein (Example 4) are produced and affinity-purified according to standard methods (Maridor, G. et al. (1993) J. CeU Sci. 106, 535-544; Harlow, E. & Lane, D. (1988) Antibodies: a laboratory manual, Cold Spring Harbor Laboratory, U.S.A.). Intramuscular injections are carried out every four weeks with 280 ⁇ g of purified recombinant CDK8 protein in buffer E (see Example 3) emulsified in Freund's adjuvant Complete adjuvant is used for the first injection and incomplete adjuvant for aU subsequent injections.
  • CDK8 as weU as human cyclins
  • A, Bl, C, Dl, E and H ( d e sequences are avaUable from data bases, such as
  • GENEBANK are produced by coupled in vitro transcription-translation. CDK8 is then incubated separately with each cyclin, and one part of each sample is resolved by
  • CDK 8 and human cyclins A, Bl, C, Dl, E and H are translated in a rabbit reticulocyte lysate in the presence of [ 35 S]-meti ⁇ onine/cysteine (Express 35 S, Dupont NEN).
  • In vitro transcription-translation experiments are performed using die TNT system (Promega Corp.) primed wid appropriate plasmids. After mixing, translation reactions are incubated for 1 hour at 30°C.
  • poCKSl affinity-beads for assaying die binding of cyclins to endogenous reticulocyte CDKs is described by Maridor et al. (1993) in J. CeU Sci. 106, 535-544.
  • samples are diluted 1:10 in NP40 buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1 % NP40, 1 mM PMSF, and 10 ⁇ g/ml each of leupeptin, aprotinin, and pepstatin) and incubated for 2 hrs at 4°C with anti-CDK8 or anti-CDK4 antibodies.
  • Immune complexes are isolated as described by Tassan, J.P. et al. (1994) J. CeU Biol. 127, 467-478; Krek, W. & Nigg, E.A. (1991) EMBO J. 10, 305-316). Western blotting is performed as described (Maridor, G.
  • exponentiaUy growing HeLa ceUs are labeled for 3.5 hrs with [ 35 S]-meti ⁇ ionine/cysteine, lysed in RTPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 % NP40, 1 % deoxycholate, 0.1% SDS, 1 mM PMSF and 10 ⁇ g/ml each of leupeptin, pepstatin, and aprotinin), and incubated with anti-CDK8 antibodies as described (Tassan, J.P. et al. (1994), supra; Krek, W. & Nigg, E. A. (1991) EMBO J. 10, 305-316).
  • One-dimensional peptide mapping widi V8 protease is carried out according to Cleveland (Cleveland, D. W. (1983) Metii. Enzymol. 96, 222-229).
  • p9 CKS1 affinity-beads are used for isolating complexes formed between [ 35 S]-labeled in vitro-translated cyclins and endogenous CDKs present in the reticulocyte lysate. Consistent with previous results (Maridor, G. et al. (1993) J. CeU Sci. 106, 535-544), cyclins A and Bl can readily be recovered on reflecting their affinities for CDC2 and/or CDK2. In contrast, cyclin C does not bind to p9CKSl fo ⁇ indicating diat cyclin C does not interact with die complex partners of A- or B-type cyclins.
  • CDK4 is tiien immunoprecipitated widi anti-CDK4 antibodies.
  • CDK4 is not labeUed, because it comigrates almost exactiy with cyclin C in the gel system used.
  • cyclin Dl binds to CDK4, as expected (Matsushime, H. et al., supra) , cyclin C does not.
  • CDK8 and cyclin C interact in vivo
  • antibodies raised against recombinant CDK8 are used for coimmunoprecipitation experiments.
  • these antibodies recognize a single protein that comigrates exactiy with in vitro-translated CDK8, whereas a myc-epitope tagged version of CDK8 displays the expected reduced electrophoretic mobiUty.
  • tiiey precipitate a number of ceUular proteins, many of which are not precipitated by die conesponding pre-immune serum.
  • CDK8 displays 36 % sequence identity to botii human CDC2 and budding yeast CDC28, Ulustrating that this novel mammaUan CDK is only distantiy related to the prototypic ceU cycle-regulatory CDKs.
  • CDK8 is, however, closely related to budding yeast SRB10 (Liao, S.-M. et al. (1995) Nature 374, 193-196), a recently discovered CDK. This simUarity is particularly interesting since SRBIO is shown to interact with SRB11, a budding yeast cyclin whose closest known mammaUan relative is cyclin C (Liao, S.-M.
  • CDK8 and SRBIO show d at tiiese two kinases display a considerable degree of overaU structural similarity (48 % identity over subdomains JJI-XI).
  • the structural simUarity between CDK8/cyclin C and SRBIO/SRBI 1 suggests diat die two CDK/cyclin pairs perform related functions. It is assumed diat CDK8/cyclin C interact with the mammaUan transcription apparams and thereby contribute to integrate ceU growth with ceU cycle progression.
  • Example 7 Expression of recombinant CDK8/cvclin C in insect ceUs
  • the CDK8/cycUn C complex is reconstituted by coinfecting SF9 insect cells with recombinant baculoviruses expressing either CDK8 or cyclin C.
  • a EcoRV-NotI fragment spanning the entire coding sequence of CDK8 is cloned into the Smal-NotI sites of the vector pVL1393 (Pharmingen Corp.) and a Pstl-BamHI fragment coding for the fuU-lengdi cyclin C is inserted in die Pstl-BamHI sites ofthe vector pVL1392 (Pharmingen Corp.).
  • the EcoRV site in the CDK8 cDNA, and the PstI and BamHl sites in the cyclin C cDNA are introduced by site-directed mutagenesis, using PCR.
  • CDK8 and cyclin C baculoviruses are generated by cotransfection of pVL1393-CDK8 or pVL1392-cyclin C and baculoGold (Pharmigen Corp.) and subsequent amplification of viral DNA.
  • AU procedures relating to Sf9 insect ceU growth, transfections, infections, plaque assays, and viral amplification are as described by O'ReiUy, D.R., MiUer, L.K., and Lucknow, V.A. (1992) Baculovirus Expression Vectors: A Laboratory Manual. W.H. Freeman & Co, New York. SEQUFNCE LISTING
  • MOLECULE TYPE cDNA to mRNA
  • FEATURE
  • AAG CAT CCA AAC GTC ATT TCT CTT CAA
  • aECTIRED SHEET (RUtf 91) CAT ATA ATC AAG TTT CAC AGA GCT TCT AAA GCA AAC AAG AAG CCA GTT 389 His He He Lys Phe His Arg Ala Ser Lys Ala Asn Lys Lys Pro Val 110 115 120

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Abstract

L'invention concerne une kinase cyclinodépendante (CDK), des dérivés de celle-ci, des anticorps spécifiques de cette kinase, ainsi que des moyens et procédés de production de celle-ci. L'invention concerne également des acides nucléiques codant pour une kinase de l'invention, un procédé d'obtention de telles molécules d'acide nucléique et l'expression de celles-ci. En outre, l'invention a trait à des utilisations des protéines et des acides nucléiques de l'invention.
PCT/EP1995/003439 1995-09-01 1995-09-01 Kinase cyclinodependante Ceased WO1997009432A1 (fr)

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PCT/EP1995/003439 WO1997009432A1 (fr) 1995-09-01 1995-09-01 Kinase cyclinodependante
AU35200/95A AU3520095A (en) 1995-09-01 1995-09-01 Cyclin-dependent kinase

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999025844A1 (fr) * 1997-11-13 1999-05-27 Amgen Inc. Kinase dpk pour la prevention de la mort cellulaire chez les mammiferes
WO2004080287A3 (fr) * 2003-03-10 2005-03-17 Abbott Lab Methode permettant de tuer les cellules cancereuses
US20070054294A1 (en) * 2000-10-25 2007-03-08 Rangwala Tasneem S Cotton event PV-GHGT07(1445) and compositions and methods for detection thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL "Human chromosome-specific mRNA.", XP002001605, retrieved from EBI *
DAVID O. MORGAN: "Principles of CDK regulation", NATURE, vol. 374, no. 6518, 9 March 1995 (1995-03-09), LONDON GB, pages 131 - 134, XP002001603 *
SHA-MEI LIAO ET AL.: "A kinase-cyclin pair in the RNA polymerase II holoenzyme", NATURE, vol. 374, no. 6518, 9 March 1995 (1995-03-09), pages 193 - 196, XP002001609 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999025844A1 (fr) * 1997-11-13 1999-05-27 Amgen Inc. Kinase dpk pour la prevention de la mort cellulaire chez les mammiferes
US5981248A (en) * 1997-11-13 1999-11-09 Amgen Inc. Mammalian cell death preventing kinase, DPK
US20070054294A1 (en) * 2000-10-25 2007-03-08 Rangwala Tasneem S Cotton event PV-GHGT07(1445) and compositions and methods for detection thereof
US7807357B2 (en) * 2000-10-25 2010-10-05 Monsanto Technology Llc Cotton event PV-GHGT07(1445) and compositions and methods for detection thereof
US7820392B2 (en) 2000-10-25 2010-10-26 Monsanto Technology Llc Cotton event PV-GHGT07(1445) and compositions and methods for detection thereof
WO2004080287A3 (fr) * 2003-03-10 2005-03-17 Abbott Lab Methode permettant de tuer les cellules cancereuses

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