Skip to main content
PMC home page
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1992 May;3(5):571–582. doi: 10.1091/mbc.3.5.571

Activation of human cyclin-dependent kinases in vitro.

D Desai 1, Y Gu 1, D O Morgan 1
PMCID: PMC275609  PMID: 1535244

Abstract

We have analyzed the activation of human cyclin-dependent kinases in a cell-free system. Human CDC2, cyclin-dependent kinase 2 (CDK2), cyclin A, and cyclin B1 were produced in insect cells by infection with recombinant baculoviruses. CDC2 or CDK2 monomers in lysates of infected cells could be activated by the addition of lysates containing cyclin A or B1. CDC2 activation by cyclin B1, as well as CDK2 activation by cyclins A and B1, was accompanied by the formation of high molecular weight complexes. In contrast, CDC2 did not bind effectively to cyclin A. CDC2 activation by cyclin B1 was studied in detail and was found to be accompanied by phosphorylation of CDC2 on Threonine 161. The binding of CDC2 to cyclin B1 also occurred under conditions where CDC2 phosphorylation was prevented, resulting in an inactive complex that could then be phosphorylated and activated on addition of cell extract. Highly purified CDC2 and cyclin B1 also formed inactive complexes that could be activated in an ATP-dependent fashion by unidentified components in crude cell extracts. These data suggest that the CDC2 activation process begins with cyclin binding, after which CDC2 phosphorylation, catalyzed by a separate enzyme, leads to activation.

Full text

PDF
571

Images in this article

574Image
on p.574
576Image
on p.576
577Image
on p.577
578Image
on p.578
579Image
on p.579

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. 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]
  3. Brodsky M. H., Warton M., Myers R. M., Littman D. R. Analysis of the site in CD4 that binds to the HIV envelope glycoprotein. J Immunol. 1990 Apr 15;144(8):3078–3086. [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. Field J., Nikawa J., Broek D., MacDonald B., Rodgers L., Wilson I. A., Lerner R. A., Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. doi: 10.1128/mcb.8.5.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. Izumi T., Maller J. L. Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions. Mol Cell Biol. 1991 Aug;11(8):3860–3867. doi: 10.1128/mcb.11.8.3860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. Luckow V. A., Summers M. D. High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors. Virology. 1989 May;170(1):31–39. doi: 10.1016/0042-6822(89)90348-6. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Morgan D. O., Kaplan J. M., Bishop J. M., Varmus H. E. Mitosis-specific phosphorylation of p60c-src by p34cdc2-associated protein kinase. Cell. 1989 Jun 2;57(5):775–786. doi: 10.1016/0092-8674(89)90792-7. [DOI] [PubMed] [Google Scholar]
  26. Morgan D. O., Kaplan J. M., Bishop J. M., Varmus H. E. Production of p60c-src by baculovirus expression and immunoaffinity purification. Methods Enzymol. 1991;200:645–660. doi: 10.1016/0076-6879(91)00177-x. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. 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]
  31. Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990 Apr 5;344(6266):503–508. doi: 10.1038/344503a0. [DOI] [PubMed] [Google Scholar]
  32. Paris J., Le Guellec R., Couturier A., Le Guellec K., Omilli F., Camonis J., MacNeill S., Philippe M. Cloning by differential screening of a Xenopus cDNA coding for a protein highly homologous to cdc2. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):1039–1043. doi: 10.1073/pnas.88.3.1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Parker L. L., Atherton-Fessler S., Lee M. S., Ogg S., Falk J. L., Swenson K. I., Piwnica-Worms H. Cyclin promotes the tyrosine phosphorylation of p34cdc2 in a wee1+ dependent manner. EMBO J. 1991 May;10(5):1255–1263. doi: 10.1002/j.1460-2075.1991.tb08067.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. 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]
  39. 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]
  40. 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]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology

RESOURCES