Abstract
A cyclin-dependent kinase (cdk)-activating kinase (CAK) has been shown previously to catalyze T-loop phosphorylation of cdks in most eukaryotic cells. This enzyme exists in either of two forms: the major one contains cdk7, cyclin H and an assembly factor called MAT-1, whilst the minor one lacks MAT-1. Cdk7 is unusual among cdks because it contains not one but two residues (S170 and T176 in Xenopus cdk7) in its T-loop that are phosphorylated in vivo. We have investigated the role of S170 and T176 phosphorylation in the assembly and activity of cyclin H-cdk7 dimers. In the absence of MAT-1, phosphorylation of the T-loop appears to be required for cdk7 to bind cyclin H. Phosphorylation of both residues does not require cyclin H binding in vitro. Phosphorylation of S170 is sufficient for cdk7 to bind cyclin H with low affinity, but high affinity binding requires T176 phosphorylation. By mutational analysis, we demonstrate that in addition to its role in promotion of cyclin H binding, S170 phosphorylation plays a direct role in the control of CAK activity. Finally, we show that dual phosphorylation of S170 and T176, or substitution of both phosphorylatable residues by aspartic residues, is sufficient to generate CAK activity to one-third of its maximal value in vitro, even in the absence of cyclin H and MAT-1, and may thus provide further clues as to how cyclins activate cdk subunits.
Full Text
The Full Text of this article is available as a PDF (552.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Buck V., Russell P., Millar J. B. Identification of a cdk-activating kinase in fission yeast. EMBO J. 1995 Dec 15;14(24):6173–6183. doi: 10.1002/j.1460-2075.1995.tb00308.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Connell-Crowley L., Solomon M. J., Wei N., Harper J. W. Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro. Mol Biol Cell. 1993 Jan;4(1):79–92. doi: 10.1091/mbc.4.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Damagnez V., Mäkelä T. P., Cottarel G. Schizosaccharomyces pombe Mop1-Mcs2 is related to mammalian CAK. EMBO J. 1995 Dec 15;14(24):6164–6172. doi: 10.1002/j.1460-2075.1995.tb00307.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Bondt H. L., Rosenblatt J., Jancarik J., Jones H. D., Morgan D. O., Kim S. H. Crystal structure of cyclin-dependent kinase 2. Nature. 1993 Jun 17;363(6430):595–602. doi: 10.1038/363595a0. [DOI] [PubMed] [Google Scholar]
- Desai D., Wessling H. C., Fisher R. P., Morgan D. O. Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2. Mol Cell Biol. 1995 Jan;15(1):345–350. doi: 10.1128/mcb.15.1.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Devault A., Martinez A. M., Fesquet D., Labbé J. C., Morin N., Tassan J. P., Nigg E. A., Cavadore J. C., Dorée M. MAT1 ('menage à trois') a new RING finger protein subunit stabilizing cyclin H-cdk7 complexes in starfish and Xenopus CAK. EMBO J. 1995 Oct 16;14(20):5027–5036. doi: 10.1002/j.1460-2075.1995.tb00185.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Drapkin R., Reinberg D. The multifunctional TFIIH complex and transcriptional control. Trends Biochem Sci. 1994 Nov;19(11):504–508. doi: 10.1016/0968-0004(94)90139-2. [DOI] [PubMed] [Google Scholar]
- Feaver W. J., Svejstrup J. Q., Henry N. L., Kornberg R. D. Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK. Cell. 1994 Dec 16;79(6):1103–1109. doi: 10.1016/0092-8674(94)90040-x. [DOI] [PubMed] [Google Scholar]
- Fesquet D., Labbé J. C., Derancourt J., Capony J. P., Galas S., Girard F., Lorca T., Shuttleworth J., Dorée M., Cavadore J. C. The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues. EMBO J. 1993 Aug;12(8):3111–3121. doi: 10.1002/j.1460-2075.1993.tb05980.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fischer L., Gerard M., Chalut C., Lutz Y., Humbert S., Kanno M., Chambon P., Egly J. M. Cloning of the 62-kilodalton component of basic transcription factor BTF2. Science. 1992 Sep 4;257(5075):1392–1395. doi: 10.1126/science.1529339. [DOI] [PubMed] [Google Scholar]
- Fisher R. P., Jin P., Chamberlin H. M., Morgan D. O. Alternative mechanisms of CAK assembly require an assembly factor or an activating kinase. Cell. 1995 Oct 6;83(1):47–57. doi: 10.1016/0092-8674(95)90233-3. [DOI] [PubMed] [Google Scholar]
- Fisher R. P., Morgan D. O. A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase. Cell. 1994 Aug 26;78(4):713–724. doi: 10.1016/0092-8674(94)90535-5. [DOI] [PubMed] [Google Scholar]
- Gerber M. R., Farrell A., Deshaies R. J., Herskowitz I., Morgan D. O. Cdc37 is required for association of the protein kinase Cdc28 with G1 and mitotic cyclins. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4651–4655. doi: 10.1073/pnas.92.10.4651. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gould K. L., Moreno S., Owen D. J., Sazer S., Nurse P. Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function. EMBO J. 1991 Nov;10(11):3297–3309. doi: 10.1002/j.1460-2075.1991.tb04894.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gould K. L., Nurse P. Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature. 1989 Nov 2;342(6245):39–45. doi: 10.1038/342039a0. [DOI] [PubMed] [Google Scholar]
- Hanks S. K., Hunter T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 1995 May;9(8):576–596. [PubMed] [Google Scholar]
- Humbert S., van Vuuren H., Lutz Y., Hoeijmakers J. H., Egly J. M., Moncollin V. p44 and p34 subunits of the BTF2/TFIIH transcription factor have homologies with SSL1, a yeast protein involved in DNA repair. EMBO J. 1994 May 15;13(10):2393–2398. doi: 10.1002/j.1460-2075.1994.tb06523.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jeffrey P. D., Russo A. A., Polyak K., Gibbs E., Hurwitz J., Massagué J., Pavletich N. P. Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex. Nature. 1995 Jul 27;376(6538):313–320. doi: 10.1038/376313a0. [DOI] [PubMed] [Google Scholar]
- Kaldis P., Sutton A., Solomon M. J. The Cdk-activating kinase (CAK) from budding yeast. Cell. 1996 Aug 23;86(4):553–564. doi: 10.1016/s0092-8674(00)80129-4. [DOI] [PubMed] [Google Scholar]
- Kato J. Y., Matsuoka M., Strom D. K., Sherr C. J. Regulation of cyclin D-dependent kinase 4 (cdk4) by cdk4-activating kinase. Mol Cell Biol. 1994 Apr;14(4):2713–2721. doi: 10.1128/mcb.14.4.2713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Knighton D. R., Zheng J. H., Ten Eyck L. F., Ashford V. A., Xuong N. H., Taylor S. S., Sowadski J. M. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. doi: 10.1126/science.1862342. [DOI] [PubMed] [Google Scholar]
- Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- Labbé J. C., Cavadore J. C., Dorée M. M phase-specific cdc2 kinase: preparation from starfish oocytes and properties. Methods Enzymol. 1991;200:291–301. doi: 10.1016/0076-6879(91)00147-o. [DOI] [PubMed] [Google Scholar]
- Labbé J. C., Martinez A. M., Fesquet D., Capony J. P., Darbon J. M., Derancourt J., Devault A., Morin N., Cavadore J. C., Dorée M. p40MO15 associates with a p36 subunit and requires both nuclear translocation and Thr176 phosphorylation to generate cdk-activating kinase activity in Xenopus oocytes. EMBO J. 1994 Nov 1;13(21):5155–5164. doi: 10.1002/j.1460-2075.1994.tb06845.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee T. H., Turck C., Kirschner M. W. Inhibition of cdc2 activation by INH/PP2A. Mol Biol Cell. 1994 Mar;5(3):323–338. doi: 10.1091/mbc.5.3.323. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lorca T., Labbé J. C., Devault A., Fesquet D., Capony J. P., Cavadore J. C., Le Bouffant F., Dorée M. Dephosphorylation of cdc2 on threonine 161 is required for cdc2 kinase inactivation and normal anaphase. EMBO J. 1992 Jul;11(7):2381–2390. doi: 10.1002/j.1460-2075.1992.tb05302.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morgan D. O. Principles of CDK regulation. Nature. 1995 Mar 9;374(6518):131–134. doi: 10.1038/374131a0. [DOI] [PubMed] [Google Scholar]
- Mäkelä T. P., Tassan J. P., Nigg E. A., Frutiger S., Hughes G. J., Weinberg R. A. A cyclin associated with the CDK-activating kinase MO15. Nature. 1994 Sep 15;371(6494):254–257. doi: 10.1038/371254a0. [DOI] [PubMed] [Google Scholar]
- Poon R. Y., Yamashita K., Adamczewski J. P., Hunt T., Shuttleworth J. The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2. EMBO J. 1993 Aug;12(8):3123–3132. doi: 10.1002/j.1460-2075.1993.tb05981.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Poon R. Y., Yamashita K., Howell M., Ershler M. A., Belyavsky A., Hunt T. Cell cycle regulation of the p34cdc2/p33cdk2-activating kinase p40MO15. J Cell Sci. 1994 Oct;107(Pt 10):2789–2799. doi: 10.1242/jcs.107.10.2789. [DOI] [PubMed] [Google Scholar]
- Rosenblatt J., Gu Y., Morgan D. O. Human cyclin-dependent kinase 2 is activated during the S and G2 phases of the cell cycle and associates with cyclin A. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2824–2828. doi: 10.1073/pnas.89.7.2824. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sali A., Blundell T. L. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993 Dec 5;234(3):779–815. doi: 10.1006/jmbi.1993.1626. [DOI] [PubMed] [Google Scholar]
- Seroz T., Hwang J. R., Moncollin V., Egly J. M. TFIIH: a link between transcription, DNA repair and cell cycle regulation. Curr Opin Genet Dev. 1995 Apr;5(2):217–221. doi: 10.1016/0959-437x(95)80011-5. [DOI] [PubMed] [Google Scholar]
- Shuttleworth J., Godfrey R., Colman A. p40MO15, a cdc2-related protein kinase involved in negative regulation of meiotic maturation of Xenopus oocytes. EMBO J. 1990 Oct;9(10):3233–3240. doi: 10.1002/j.1460-2075.1990.tb07522.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Solomon M. J., Harper J. W., Shuttleworth J. CAK, the p34cdc2 activating kinase, contains a protein identical or closely related to p40MO15. EMBO J. 1993 Aug;12(8):3133–3142. doi: 10.1002/j.1460-2075.1993.tb05982.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Solomon M. J. The function(s) of CAK, the p34cdc2-activating kinase. Trends Biochem Sci. 1994 Nov;19(11):496–500. doi: 10.1016/0968-0004(94)90137-6. [DOI] [PubMed] [Google Scholar]
- Tassan J. P., Jaquenoud M., Fry A. M., Frutiger S., Hughes G. J., Nigg E. A. In vitro assembly of a functional human CDK7-cyclin H complex requires MAT1, a novel 36 kDa RING finger protein. EMBO J. 1995 Nov 15;14(22):5608–5617. doi: 10.1002/j.1460-2075.1995.tb00248.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thuret J. Y., Valay J. G., Faye G., Mann C. Civ1 (CAK in vivo), a novel Cdk-activating kinase. Cell. 1996 Aug 23;86(4):565–576. doi: 10.1016/s0092-8674(00)80130-0. [DOI] [PubMed] [Google Scholar]
- Valay J. G., Simon M., Dubois M. F., Bensaude O., Facca C., Faye G. The KIN28 gene is required both for RNA polymerase II mediated transcription and phosphorylation of the Rpb1p CTD. J Mol Biol. 1995 Jun 9;249(3):535–544. doi: 10.1006/jmbi.1995.0316. [DOI] [PubMed] [Google Scholar]