Skip to main content
PMC home page
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1993 Feb;4(2):195–208. doi: 10.1091/mbc.4.2.195

Regulation of Saccharomyces cerevisiae CDC7 function during the cell cycle.

H J Yoon 1, S Loo 1, J L Campbell 1
PMCID: PMC300915  PMID: 8382976

Abstract

The yeast Cdc7 function is required for the G1/S transition and is dependent on passage through START, a point controlled by the Cdc28/cdc2/p34 protein kinase. CDC7 encodes a protein kinase activity, and we now show that this kinase activity varies in the cell cycle but that protein levels appear to remain constant. We present several lines of evidence that periodic activation of CDC7 kinase is at least in part through phosphorylation. First, the kinase activity of the Cdc7 protein is destroyed by dephosphorylation of the protein in vitro with phosphatase. Second, Cdc7 protein is hypophosphorylated and inactive as a kinase in extracts of cells arrested at START but becomes active and maximally phosphorylated subsequent to passage through START. The phosphorylation pattern of Cdc7 protein is complex. Phosphopeptide mapping reveals four phosphopeptides in Cdc7 prepared from asynchronous yeast cells. Both autophosphorylation and phosphorylation in trans appear to contribute to this pattern. Autophosphorylation is shown to occur by using a thermolabile Cdc7 protein. A protein in yeast extracts can phosphorylate and activate Cdc7 protein made in Escherichia coli, and phosphorylation is thermolabile in cdc28 mutant extracts. Cdc7 protein carrying a serine to alanine change in the consensus recognition site for Cdc28 kinase shows an altered phosphopeptide map, suggesting that this site is important in determining the overall Cdc7 phosphorylation pattern.

Full text

PDF
207

Images in this article

199Image
on p.199
199Image
on p.199
200Image
on p.200
201Image
on p.201
201Image
on p.201
202Image
on p.202
202Image
on p.202
204Image
on p.204

Selected References

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

  1. Axelrod A., Rine J. A role for CDC7 in repression of transcription at the silent mating-type locus HMR in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Feb;11(2):1080–1091. doi: 10.1128/mcb.11.2.1080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bahman M., Buck V., White A., Rosamond J. Characterisation of the CDC7 gene product of Saccharomyces cerevisiae as a protein kinase needed for the initiation of mitotic DNA synthesis. Biochim Biophys Acta. 1988 Dec 20;951(2-3):335–343. doi: 10.1016/0167-4781(88)90104-2. [DOI] [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. Buck V., White A., Rosamond J. CDC7 protein kinase activity is required for mitosis and meiosis in Saccharomyces cerevisiae. Mol Gen Genet. 1991 Jul;227(3):452–457. doi: 10.1007/BF00273937. [DOI] [PubMed] [Google Scholar]
  5. Chapman J. W., Johnston L. H. The yeast gene, DBF4, essential for entry into S phase is cell cycle regulated. Exp Cell Res. 1989 Feb;180(2):419–428. doi: 10.1016/0014-4827(89)90068-2. [DOI] [PubMed] [Google Scholar]
  6. Cross F. R. DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4675–4684. doi: 10.1128/mcb.8.11.4675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cross F. R., Tinkelenberg A. H. A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle. Cell. 1991 May 31;65(5):875–883. doi: 10.1016/0092-8674(91)90394-e. [DOI] [PubMed] [Google Scholar]
  8. D'Urso G., Marraccino R. L., Marshak D. R., Roberts J. M. Cell cycle control of DNA replication by a homologue from human cells of the p34cdc2 protein kinase. Science. 1990 Nov 9;250(4982):786–791. doi: 10.1126/science.2173140. [DOI] [PubMed] [Google Scholar]
  9. Din S., Brill S. J., Fairman M. P., Stillman B. Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev. 1990 Jun;4(6):968–977. doi: 10.1101/gad.4.6.968. [DOI] [PubMed] [Google Scholar]
  10. Dirick L., Nasmyth K. Positive feedback in the activation of G1 cyclins in yeast. Nature. 1991 Jun 27;351(6329):754–757. doi: 10.1038/351754a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Dunphy W. G., Newport J. W. Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase. Cell. 1989 Jul 14;58(1):181–191. doi: 10.1016/0092-8674(89)90414-5. [DOI] [PubMed] [Google Scholar]
  13. Dunphy W. G., Newport J. W. Unraveling of mitotic control mechanisms. Cell. 1988 Dec 23;55(6):925–928. doi: 10.1016/0092-8674(88)90234-6. [DOI] [PubMed] [Google Scholar]
  14. Fairman M. P., Stillman B. Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J. 1988 Apr;7(4):1211–1218. doi: 10.1002/j.1460-2075.1988.tb02933.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Fong H. K., Hurley J. B., Hopkins R. S., Miake-Lye R., Johnson M. S., Doolittle R. F., Simon M. I. Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2162–2166. doi: 10.1073/pnas.83.7.2162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ghiara J. B., Richardson H. E., Sugimoto K., Henze M., Lew D. J., Wittenberg C., Reed S. I. A cyclin B homolog in S. cerevisiae: chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis. Cell. 1991 Apr 5;65(1):163–174. doi: 10.1016/0092-8674(91)90417-w. [DOI] [PubMed] [Google Scholar]
  18. Goebl M. G., Yochem J., Jentsch S., McGrath J. P., Varshavsky A., Byers B. The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme. Science. 1988 Sep 9;241(4871):1331–1335. doi: 10.1126/science.2842867. [DOI] [PubMed] [Google Scholar]
  19. Hadwiger J. A., Wittenberg C., Mendenhall M. D., Reed S. I. The Saccharomyces cerevisiae CKS1 gene, a homolog of the Schizosaccharomyces pombe suc1+ gene, encodes a subunit of the Cdc28 protein kinase complex. Mol Cell Biol. 1989 May;9(5):2034–2041. doi: 10.1128/mcb.9.5.2034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hadwiger J. A., Wittenberg C., Richardson H. E., de Barros Lopes M., Reed S. I. A family of cyclin homologs that control the G1 phase in yeast. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6255–6259. doi: 10.1073/pnas.86.16.6255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Hartwell L. H. Sequential function of gene products relative to DNA synthesis in the yeast cell cycle. J Mol Biol. 1976 Jul 15;104(4):803–817. doi: 10.1016/0022-2836(76)90183-2. [DOI] [PubMed] [Google Scholar]
  23. Hartwell L. H. Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae. J Bacteriol. 1973 Sep;115(3):966–974. doi: 10.1128/jb.115.3.966-974.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hartwell L. H., Weinert T. A. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989 Nov 3;246(4930):629–634. doi: 10.1126/science.2683079. [DOI] [PubMed] [Google Scholar]
  25. Hereford L. M., Hartwell L. H. Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis. J Mol Biol. 1974 Apr 15;84(3):445–461. doi: 10.1016/0022-2836(74)90451-3. [DOI] [PubMed] [Google Scholar]
  26. Hollingsworth R. E., Jr, Ostroff R. M., Klein M. B., Niswander L. A., Sclafani R. A. Molecular genetic studies of the Cdc7 protein kinase and induced mutagenesis in yeast. Genetics. 1992 Sep;132(1):53–62. doi: 10.1093/genetics/132.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hollingsworth R. E., Jr, Sclafani R. A. DNA metabolism gene CDC7 from yeast encodes a serine (threonine) protein kinase. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6272–6276. doi: 10.1073/pnas.87.16.6272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jazwinski S. M. CDC7-dependent protein kinase activity in yeast replicative-complex preparations. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2101–2105. doi: 10.1073/pnas.85.7.2101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Johnston L. H., Eberly S. L., Chapman J. W., Araki H., Sugino A. The product of the Saccharomyces cerevisiae cell cycle gene DBF2 has homology with protein kinases and is periodically expressed in the cell cycle. Mol Cell Biol. 1990 Apr;10(4):1358–1366. doi: 10.1128/mcb.10.4.1358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Johnston L. H., Thomas A. P. A further two mutants defective in initiation of the S phase in the yeast Saccharomyces cerevisiae. Mol Gen Genet. 1982;186(3):445–448. doi: 10.1007/BF00729467. [DOI] [PubMed] [Google Scholar]
  31. Kenny M. K., Lee S. H., Hurwitz J. Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9757–9761. doi: 10.1073/pnas.86.24.9757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kenny M. K., Schlegel U., Furneaux H., Hurwitz J. The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. J Biol Chem. 1990 May 5;265(13):7693–7700. [PubMed] [Google Scholar]
  33. Kitada K., Johnston L. H., Sugino T., Sugino A. Temperature-sensitive cdc7 mutations of Saccharomyces cerevisiae are suppressed by the DBF4 gene, which is required for the G1/S cell cycle transition. Genetics. 1992 May;131(1):21–29. doi: 10.1093/genetics/131.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Lewin B. Driving the cell cycle: M phase kinase, its partners, and substrates. Cell. 1990 Jun 1;61(5):743–752. doi: 10.1016/0092-8674(90)90181-d. [DOI] [PubMed] [Google Scholar]
  36. Moll T., Tebb G., Surana U., Robitsch H., Nasmyth K. The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5. Cell. 1991 Aug 23;66(4):743–758. doi: 10.1016/0092-8674(91)90118-i. [DOI] [PubMed] [Google Scholar]
  37. Moreno S., Nurse P. Substrates for p34cdc2: in vivo veritas? Cell. 1990 May 18;61(4):549–551. doi: 10.1016/0092-8674(90)90463-o. [DOI] [PubMed] [Google Scholar]
  38. Nash R., Tokiwa G., Anand S., Erickson K., Futcher A. B. The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog. EMBO J. 1988 Dec 20;7(13):4335–4346. doi: 10.1002/j.1460-2075.1988.tb03332.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nasmyth K. A. FAR-reaching discoveries about the regulation of START. Cell. 1990 Dec 21;63(6):1117–1120. doi: 10.1016/0092-8674(90)90404-3. [DOI] [PubMed] [Google Scholar]
  40. Njagi G. D., Kilbey B. J. cdc7-1 a temperature sensitive cell-cycle mutant which interferes with induced mutagenesis in Saccharomyces cerevisiae. Mol Gen Genet. 1982;186(4):478–481. doi: 10.1007/BF00337951. [DOI] [PubMed] [Google Scholar]
  41. Pardee A. B. G1 events and regulation of cell proliferation. Science. 1989 Nov 3;246(4930):603–608. doi: 10.1126/science.2683075. [DOI] [PubMed] [Google Scholar]
  42. Patterson M., Sclafani R. A., Fangman W. L., Rosamond J. Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae. Mol Cell Biol. 1986 May;6(5):1590–1598. doi: 10.1128/mcb.6.5.1590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Peterson T. A., Yochem J., Byers B., Nunn M. F., Duesberg P. H., Doolittle R. F., Reed S. I. A relationship between the yeast cell cycle genes CDC4 and CDC36 and the ets sequence of oncogenic virus E26. Nature. 1984 Jun 7;309(5968):556–558. doi: 10.1038/309556a0. [DOI] [PubMed] [Google Scholar]
  44. Piggott J. R., Rai R., Carter B. L. A bifunctional gene product involved in two phases of the yeast cell cycle. Nature. 1982 Jul 22;298(5872):391–393. doi: 10.1038/298391a0. [DOI] [PubMed] [Google Scholar]
  45. Pines J., Hunter T. p34cdc2: the S and M kinase? New Biol. 1990 May;2(5):389–401. [PubMed] [Google Scholar]
  46. Reed S. I., Hadwiger J. A., Lörincz A. T. Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4055–4059. doi: 10.1073/pnas.82.12.4055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Reed S. I. The selection of S. cerevisiae mutants defective in the start event of cell division. Genetics. 1980 Jul;95(3):561–577. doi: 10.1093/genetics/95.3.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Reed S. I., Wittenberg C. Mitotic role for the Cdc28 protein kinase of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5697–5701. doi: 10.1073/pnas.87.15.5697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Reneke J. E., Blumer K. J., Courchesne W. E., Thorner J. The carboxy-terminal segment of the yeast alpha-factor receptor is a regulatory domain. Cell. 1988 Oct 21;55(2):221–234. doi: 10.1016/0092-8674(88)90045-1. [DOI] [PubMed] [Google Scholar]
  50. Richardson H. E., Wittenberg C., Cross F., Reed S. I. An essential G1 function for cyclin-like proteins in yeast. Cell. 1989 Dec 22;59(6):1127–1133. doi: 10.1016/0092-8674(89)90768-x. [DOI] [PubMed] [Google Scholar]
  51. Roach P. J. Multisite and hierarchal protein phosphorylation. J Biol Chem. 1991 Aug 5;266(22):14139–14142. [PubMed] [Google Scholar]
  52. Schild D., Byers B. Meiotic effects of DNA-defective cell division cycle mutations of Saccharomyces cerevisiae. Chromosoma. 1978 Dec 21;70(1):109–130. doi: 10.1007/BF00292220. [DOI] [PubMed] [Google Scholar]
  53. Schultz M. C., Choe S. Y., Reeder R. H. Specific initiation by RNA polymerase I in a whole-cell extract from yeast. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):1004–1008. doi: 10.1073/pnas.88.3.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sclafani R. A., Patterson M., Rosamond J., Fangman W. L. Differential regulation of the yeast CDC7 gene during mitosis and meiosis. Mol Cell Biol. 1988 Jan;8(1):293–300. doi: 10.1128/mcb.8.1.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  57. Sudbery P. E., Goodey A. R., Carter B. L. Genes which control cell proliferation in the yeast Saccharomyces cerevisiae. Nature. 1980 Nov 27;288(5789):401–404. doi: 10.1038/288401a0. [DOI] [PubMed] [Google Scholar]
  58. Surana U., Robitsch H., Price C., Schuster T., Fitch I., Futcher A. B., Nasmyth K. The role of CDC28 and cyclins during mitosis in the budding yeast S. cerevisiae. Cell. 1991 Apr 5;65(1):145–161. doi: 10.1016/0092-8674(91)90416-v. [DOI] [PubMed] [Google Scholar]
  59. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wittenberg C., Reed S. I. Control of the yeast cell cycle is associated with assembly/disassembly of the Cdc28 protein kinase complex. Cell. 1988 Sep 23;54(7):1061–1072. doi: 10.1016/0092-8674(88)90121-3. [DOI] [PubMed] [Google Scholar]
  61. Wittenberg C., Sugimoto K., Reed S. I. G1-specific cyclins of S. cerevisiae: cell cycle periodicity, regulation by mating pheromone, and association with the p34CDC28 protein kinase. Cell. 1990 Jul 27;62(2):225–237. doi: 10.1016/0092-8674(90)90361-h. [DOI] [PubMed] [Google Scholar]
  62. Wobbe C. R., Weissbach L., Borowiec J. A., Dean F. B., Murakami Y., Bullock P., Hurwitz J. Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1834–1838. doi: 10.1073/pnas.84.7.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wold M. S., Kelly T. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2523–2527. doi: 10.1073/pnas.85.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yoon H. J., Campbell J. L. The CDC7 protein of Saccharomyces cerevisiae is a phosphoprotein that contains protein kinase activity. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3574–3578. doi: 10.1073/pnas.88.9.3574. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES