Skip to main content
PMC home page
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1992 Jan;3(1):13–27. doi: 10.1091/mbc.3.1.13

Role of phosphorylation in p34cdc2 activation: identification of an activating kinase.

M J Solomon 1, T Lee 1, M W Kirschner 1
PMCID: PMC275499  PMID: 1532335

Abstract

Phosphorylation of p34cdc2 can both positively and negatively regulate its kinase activity. We have mapped two phosphorylation sites in Xenopus p34cdc2 to Thr-14 and Tyr-15 within the putative ATP-binding region of p34cdc2. Mutation of these sites to Ala-14 and Phe-15 has no effect on the final histone H1 kinase activity of the cyclin/p34cdc2 complex. Phosphopeptide analysis shows that there is at least one more site of phosphorylation on p34cdc2. When Thr-161 is changed to Ala, two phosphopeptide spots disappear and it is no longer possible to activate the H1 kinase activity of p34cdc2. We suggest that Thr-161 is a third site of phosphorylation, which is required for kinase activity. All three phosphorylations are induced by cyclin. None of the phosphorylations appears to be required for binding to cyclin, as indicated by the ability of the triple mutant, Ala-14, Phe-15, Ala-161, to bind cyclin. The activating phosphorylation that requires Thr- or Ser-161 occurs even in a catalytically inactive K33R mutant of p34cdc2 and hence does not appear to be the result of intramolecular autophosphorylation. We have detected an activity in Xenopus extracts required for activation of p34cdc2 and present evidence that this is a p34cdc2 activating kinase which, in a cyclin-dependent manner, probably directly phosphorylates Thr-161.

Full text

PDF
13

Images in this article

15Image
on p.15
16Image
on p.16
17Image
on p.17
17Image
on p.17
18Image
on p.18
19Image
on p.19
20Image
on p.20
21Image
on p.21
22Image
on p.22
23Image
on p.23
24Image
on p.24
25Image
on p.25

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. Brizuela L., Draetta G., Beach D. p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase. EMBO J. 1987 Nov;6(11):3507–3514. doi: 10.1002/j.1460-2075.1987.tb02676.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cyert M. S., Kirschner M. W. Regulation of MPF activity in vitro. Cell. 1988 Apr 22;53(2):185–195. doi: 10.1016/0092-8674(88)90380-7. [DOI] [PubMed] [Google Scholar]
  4. Dunphy W. G., Brizuela L., Beach D., Newport J. The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell. 1988 Jul 29;54(3):423–431. doi: 10.1016/0092-8674(88)90205-x. [DOI] [PubMed] [Google Scholar]
  5. Featherstone C., Russell P. Fission yeast p107wee1 mitotic inhibitor is a tyrosine/serine kinase. Nature. 1991 Feb 28;349(6312):808–811. doi: 10.1038/349808a0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Knighton D. R., Zheng J. H., Ten Eyck L. F., Xuong N. H., Taylor S. S., Sowadski J. M. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):414–420. doi: 10.1126/science.1862343. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Kumagai A., Dunphy W. G. The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system. Cell. 1991 Mar 8;64(5):903–914. doi: 10.1016/0092-8674(91)90315-p. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Lee T. H., Solomon M. J., Mumby M. C., Kirschner M. W. INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell. 1991 Jan 25;64(2):415–423. doi: 10.1016/0092-8674(91)90649-j. [DOI] [PubMed] [Google Scholar]
  16. Lundgren K., Walworth N., Booher R., Dembski M., Kirschner M., Beach D. mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2. Cell. 1991 Mar 22;64(6):1111–1122. doi: 10.1016/0092-8674(91)90266-2. [DOI] [PubMed] [Google Scholar]
  17. Meijer L., Azzi L., Wang J. Y. Cyclin B targets p34cdc2 for tyrosine phosphorylation. EMBO J. 1991 Jun;10(6):1545–1554. doi: 10.1002/j.1460-2075.1991.tb07674.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Minshull J., Blow J. J., Hunt T. Translation of cyclin mRNA is necessary for extracts of activated xenopus eggs to enter mitosis. Cell. 1989 Mar 24;56(6):947–956. doi: 10.1016/0092-8674(89)90628-4. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. 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]
  24. Ward G. E., Kirschner M. W. Identification of cell cycle-regulated phosphorylation sites on nuclear lamin C. Cell. 1990 May 18;61(4):561–577. doi: 10.1016/0092-8674(90)90469-u. [DOI] [PubMed] [Google Scholar]
  25. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
  26. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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

RESOURCES