Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15 (original) (raw)

Abstract

We have examined the role of phosphorylation in the regulation of human cyclin-dependent kinase-2 (CDK2), a protein closely related to the cell cycle regulatory kinase CDC2. We find that CDK2 from HeLa cells contains three major tryptic phosphopeptides. Analysis of site-directed mutant proteins, expressed by transient transfection of COS cells, demonstrates that the two major phosphorylation sites are Tyr15 (Y15) and Thr160 (T160). Additional phosphorylation probably occurs on Thr14 (T14). Replacement of T160 with alanine abolishes the kinase activity of CDK2, indicating that phosphorylation at this site (as in CDC2) is required for kinase activity. Mutation of Y15 and T14 stimulates kinase activity, demonstrating that phosphorylation at these sites (as in CDC2) is inhibitory. Similarly, CDK2 is activated in vitro by dephosphorylation of Y15 and T14 by the phosphatase CDC25. Analysis of HeLa cells synchronized at various cell cycle stages indicates that CDK2 phosphorylation on T160 increases during S phase and G2, when CDK2 is most active. Phosphorylation on the inhibitory sites T14 and Y15 is also maximal during S phase and G2. Thus, the activity of a subpopulation of CDK2 molecules is inhibited at a time in the cell cycle when overall CDK2 activity is increased.

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  1. Amon A., Surana U., Muroff I., Nasmyth K. Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S. cerevisiae. Nature. 1992 Jan 23;355(6358):368–371. doi: 10.1038/355368a0. [DOI] [PubMed] [Google Scholar]
  2. Booher R., Beach D. Site-specific mutagenesis of cdc2+, a cell cycle control gene of the fission yeast Schizosaccharomyces pombe. Mol Cell Biol. 1986 Oct;6(10):3523–3530. doi: 10.1128/mcb.6.10.3523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  4. Cooper J. A., King C. S. Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src. Mol Cell Biol. 1986 Dec;6(12):4467–4477. doi: 10.1128/mcb.6.12.4467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Desai D., Gu Y., Morgan D. O. Activation of human cyclin-dependent kinases in vitro. Mol Biol Cell. 1992 May;3(5):571–582. doi: 10.1091/mbc.3.5.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Draetta G., Beach D. Activation of cdc2 protein kinase during mitosis in human cells: cell cycle-dependent phosphorylation and subunit rearrangement. Cell. 1988 Jul 1;54(1):17–26. doi: 10.1016/0092-8674(88)90175-4. [DOI] [PubMed] [Google Scholar]
  7. Draetta G. Cell cycle control in eukaryotes: molecular mechanisms of cdc2 activation. Trends Biochem Sci. 1990 Oct;15(10):378–383. doi: 10.1016/0968-0004(90)90235-4. [DOI] [PubMed] [Google Scholar]
  8. Draetta G., Piwnica-Worms H., Morrison D., Druker B., Roberts T., Beach D. Human cdc2 protein kinase is a major cell-cycle regulated tyrosine kinase substrate. Nature. 1988 Dec 22;336(6201):738–744. doi: 10.1038/336738a0. [DOI] [PubMed] [Google Scholar]
  9. Ducommun B., Brambilla P., Félix M. A., Franza B. R., Jr, Karsenti E., Draetta G. cdc2 phosphorylation is required for its interaction with cyclin. EMBO J. 1991 Nov;10(11):3311–3319. doi: 10.1002/j.1460-2075.1991.tb04895.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dunphy W. G., Kumagai A. The cdc25 protein contains an intrinsic phosphatase activity. Cell. 1991 Oct 4;67(1):189–196. doi: 10.1016/0092-8674(91)90582-j. [DOI] [PubMed] [Google Scholar]
  11. Elledge S. J., Richman R., Hall F. L., Williams R. T., Lodgson N., Harper J. W. CDK2 encodes a 33-kDa cyclin A-associated protein kinase and is expressed before CDC2 in the cell cycle. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2907–2911. doi: 10.1073/pnas.89.7.2907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Elledge S. J., Spottswood M. R. A new human p34 protein kinase, CDK2, identified by complementation of a cdc28 mutation in Saccharomyces cerevisiae, is a homolog of Xenopus Eg1. EMBO J. 1991 Sep;10(9):2653–2659. doi: 10.1002/j.1460-2075.1991.tb07808.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fang F., Newport J. W. Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes. Cell. 1991 Aug 23;66(4):731–742. doi: 10.1016/0092-8674(91)90117-h. [DOI] [PubMed] [Google Scholar]
  14. Forsburg S. L., Nurse P. Cell cycle regulation in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Annu Rev Cell Biol. 1991;7:227–256. doi: 10.1146/annurev.cb.07.110191.001303. [DOI] [PubMed] [Google Scholar]
  15. Galaktionov K., Beach D. Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins. Cell. 1991 Dec 20;67(6):1181–1194. doi: 10.1016/0092-8674(91)90294-9. [DOI] [PubMed] [Google Scholar]
  16. Gautier J., Solomon M. J., Booher R. N., Bazan J. F., Kirschner M. W. cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell. 1991 Oct 4;67(1):197–211. doi: 10.1016/0092-8674(91)90583-k. [DOI] [PubMed] [Google Scholar]
  17. Girard F., Strausfeld U., Fernandez A., Lamb N. J. Cyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell. 1991 Dec 20;67(6):1169–1179. doi: 10.1016/0092-8674(91)90293-8. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Gu Y., Franco A., Jr, Gardner P. D., Lansman J. B., Forsayeth J. R., Hall Z. W. Properties of embryonic and adult muscle acetylcholine receptors transiently expressed in COS cells. Neuron. 1990 Aug;5(2):147–157. doi: 10.1016/0896-6273(90)90305-y. [DOI] [PubMed] [Google Scholar]
  21. Heintz N., Sive H. L., Roeder R. G. Regulation of human histone gene expression: kinetics of accumulation and changes in the rate of synthesis and in the half-lives of individual histone mRNAs during the HeLa cell cycle. Mol Cell Biol. 1983 Apr;3(4):539–550. doi: 10.1128/mcb.3.4.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hunt T. Maturation promoting factor, cyclin and the control of M-phase. Curr Opin Cell Biol. 1989 Apr;1(2):268–274. doi: 10.1016/0955-0674(89)90099-9. [DOI] [PubMed] [Google Scholar]
  23. Kamps M. P. Determination of phosphoamino acid composition by acid hydrolysis of protein blotted to Immobilon. Methods Enzymol. 1991;201:21–27. doi: 10.1016/0076-6879(91)01005-m. [DOI] [PubMed] [Google Scholar]
  24. Krek W., Nigg E. A. Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites. EMBO J. 1991 Feb;10(2):305–316. doi: 10.1002/j.1460-2075.1991.tb07951.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Krek W., Nigg E. A. Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates. EMBO J. 1991 Nov;10(11):3331–3341. doi: 10.1002/j.1460-2075.1991.tb04897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kumagai A., Dunphy W. G. Regulation of the cdc25 protein during the cell cycle in Xenopus extracts. Cell. 1992 Jul 10;70(1):139–151. doi: 10.1016/0092-8674(92)90540-s. [DOI] [PubMed] [Google Scholar]
  27. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee M. G., Nurse P. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature. 1987 May 7;327(6117):31–35. doi: 10.1038/327031a0. [DOI] [PubMed] [Google Scholar]
  29. Lew D. J., Dulić V., Reed S. I. Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast. Cell. 1991 Sep 20;66(6):1197–1206. doi: 10.1016/0092-8674(91)90042-w. [DOI] [PubMed] [Google Scholar]
  30. Luo K. X., Hurley T. R., Sefton B. M. Cyanogen bromide cleavage and proteolytic peptide mapping of proteins immobilized to membranes. Methods Enzymol. 1991;201:149–152. doi: 10.1016/0076-6879(91)01014-s. [DOI] [PubMed] [Google Scholar]
  31. Marraccino R. L., Firpo E. J., Roberts J. M. Activation of the p34 CDC2 protein kinase at the start of S phase in the human cell cycle. Mol Biol Cell. 1992 Apr;3(4):389–401. doi: 10.1091/mbc.3.4.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Matsushime H., Roussel M. F., Ashmun R. A., Sherr C. J. Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell. 1991 May 17;65(4):701–713. doi: 10.1016/0092-8674(91)90101-4. [DOI] [PubMed] [Google Scholar]
  33. Meyerson M., Enders G. H., Wu C. L., Su L. K., Gorka C., Nelson C., Harlow E., Tsai L. H. A family of human cdc2-related protein kinases. EMBO J. 1992 Aug;11(8):2909–2917. doi: 10.1002/j.1460-2075.1992.tb05360.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Morgan D. O. Cell cycle control in normal and neoplastic cells. Curr Opin Genet Dev. 1992 Feb;2(1):33–37. doi: 10.1016/s0959-437x(05)80318-1. [DOI] [PubMed] [Google Scholar]
  35. Morla A. O., Draetta G., Beach D., Wang J. Y. Reversible tyrosine phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis. Cell. 1989 Jul 14;58(1):193–203. doi: 10.1016/0092-8674(89)90415-7. [DOI] [PubMed] [Google Scholar]
  36. Motokura T., Bloom T., Kim H. G., Jüppner H., Ruderman J. V., Kronenberg H. M., Arnold A. A novel cyclin encoded by a bcl1-linked candidate oncogene. Nature. 1991 Apr 11;350(6318):512–515. doi: 10.1038/350512a0. [DOI] [PubMed] [Google Scholar]
  37. Murray A. W., Kirschner M. W. Dominoes and clocks: the union of two views of the cell cycle. Science. 1989 Nov 3;246(4930):614–621. doi: 10.1126/science.2683077. [DOI] [PubMed] [Google Scholar]
  38. Ninomiya-Tsuji J., Nomoto S., Yasuda H., Reed S. I., Matsumoto K. Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9006–9010. doi: 10.1073/pnas.88.20.9006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Norbury C., Blow J., Nurse P. Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates. EMBO J. 1991 Nov;10(11):3321–3329. doi: 10.1002/j.1460-2075.1991.tb04896.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pagano M., Pepperkok R., Verde F., Ansorge W., Draetta G. Cyclin A is required at two points in the human cell cycle. EMBO J. 1992 Mar;11(3):961–971. doi: 10.1002/j.1460-2075.1992.tb05135.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Parker L. L., Atherton-Fessler S., Piwnica-Worms H. p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2917–2921. doi: 10.1073/pnas.89.7.2917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pines J., Hunter T. Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B. Nature. 1990 Aug 23;346(6286):760–763. doi: 10.1038/346760a0. [DOI] [PubMed] [Google Scholar]
  43. Pines J., Hunter T. Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2. Cell. 1989 Sep 8;58(5):833–846. doi: 10.1016/0092-8674(89)90936-7. [DOI] [PubMed] [Google Scholar]
  44. Pines J., Hunter T. p34cdc2: the S and M kinase? New Biol. 1990 May;2(5):389–401. [PubMed] [Google Scholar]
  45. Riabowol K., Draetta G., Brizuela L., Vandre D., Beach D. The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells. Cell. 1989 May 5;57(3):393–401. doi: 10.1016/0092-8674(89)90914-8. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Solomon M. J., Glotzer M., Lee T. H., Philippe M., Kirschner M. W. Cyclin activation of p34cdc2. Cell. 1990 Nov 30;63(5):1013–1024. doi: 10.1016/0092-8674(90)90504-8. [DOI] [PubMed] [Google Scholar]
  48. Solomon M. J., Lee T., Kirschner M. W. Role of phosphorylation in p34cdc2 activation: identification of an activating kinase. Mol Biol Cell. 1992 Jan;3(1):13–27. doi: 10.1091/mbc.3.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sorger P. K., Murray A. W. S-phase feedback control in budding yeast independent of tyrosine phosphorylation of p34cdc28. Nature. 1992 Jan 23;355(6358):365–368. doi: 10.1038/355365a0. [DOI] [PubMed] [Google Scholar]
  50. Strausfeld U., Labbé J. C., Fesquet D., Cavadore J. C., Picard A., Sadhu K., Russell P., Dorée M. Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein. Nature. 1991 May 16;351(6323):242–245. doi: 10.1038/351242a0. [DOI] [PubMed] [Google Scholar]
  51. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Th'ng J. P., Wright P. S., Hamaguchi J., Lee M. G., Norbury C. J., Nurse P., Bradbury E. M. The FT210 cell line is a mouse G2 phase mutant with a temperature-sensitive CDC2 gene product. Cell. 1990 Oct 19;63(2):313–324. doi: 10.1016/0092-8674(90)90164-a. [DOI] [PubMed] [Google Scholar]
  53. Tsai L. H., Harlow E., Meyerson M. Isolation of the human cdk2 gene that encodes the cyclin A- and adenovirus E1A-associated p33 kinase. Nature. 1991 Sep 12;353(6340):174–177. doi: 10.1038/353174a0. [DOI] [PubMed] [Google Scholar]
  54. Xiong Y., Connolly T., Futcher B., Beach D. Human D-type cyclin. Cell. 1991 May 17;65(4):691–699. doi: 10.1016/0092-8674(91)90100-d. [DOI] [PubMed] [Google Scholar]