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. 2000 Oct 1;351(Pt 1):143–150. doi: 10.1042/0264-6021:3510143

Phosphorylation of Cdc28 and regulation of cell size by the protein kinase CKII in Saccharomyces cerevisiae.

G L Russo 1, C van den Bos 1, A Sutton 1, P Coccetti 1, M D Baroni 1, L Alberghina 1, D R Marshak 1
PMCID: PMC1221344  PMID: 10998356

Abstract

The CDK (cyclin-dependent kinase) family of enzymes is required for the G(1)-to-S-phase and G(2)-to-M-phase transitions during the cell-division cycle of eukaryotes. We have shown previously that the protein kinase CKII catalyses the phosphorylation of Ser-39 in Cdc2 during the G(1) phase of the HeLa cell-division cycle [Russo, Vandenberg, Yu, Bae, Franza and Marshak (1992) J. Biol. Chem. 267, 20317-20325]. To identify a functional role for this phosphorylation, we have studied the homologous enzymes in the budding yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of Cdc2, Cdc28, contains a consensus CKII site (Ser-46), which is homologous with that of human Cdc2. Using in vitro kinase assays, metabolic labelling, peptide mapping and phosphoamino acid analysis, we demonstrate that this site is phosphorylated in Cdc28 in vivo as well in vitro. In addition, S. cerevisiae cells in which Ser-46 has been mutated to alanine show a decrease in both cell volume and protein content of 33%, and this effect is most pronounced in the stationary phase. Because cell size in S. cerevisiae is regulated primarily at the G(1) stage, we suggest that CKII contributes to the regulation of the cell cycle in budding yeast by phosphorylation of Cdc28 as a checkpoint for G(1) progression.

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Selected References

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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. Baroni M. D., Martegani E., Monti P., Alberghina L. Cell size modulation by CDC25 and RAS2 genes in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Jun;9(6):2715–2723. doi: 10.1128/mcb.9.6.2715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baroni M. D., Monti P., Alberghina L. Repression of growth-regulated G1 cyclin expression by cyclic AMP in budding yeast. Nature. 1994 Sep 22;371(6495):339–342. doi: 10.1038/371339a0. [DOI] [PubMed] [Google Scholar]
  4. Baroni M. D., Monti P., Marconi G., Alberghina L. cAMP-mediated increase in the critical cell size required for the G1 to S transition in Saccharomyces cerevisiae. Exp Cell Res. 1992 Aug;201(2):299–306. doi: 10.1016/0014-4827(92)90277-f. [DOI] [PubMed] [Google Scholar]
  5. Beach D., Durkacz B., Nurse P. Functionally homologous cell cycle control genes in budding and fission yeast. Nature. 1982 Dec 23;300(5894):706–709. doi: 10.1038/300706a0. [DOI] [PubMed] [Google Scholar]
  6. Berry L. D., Gould K. L. Regulation of Cdc2 activity by phosphorylation at T14/Y15. Prog Cell Cycle Res. 1996;2:99–105. doi: 10.1007/978-1-4615-5873-6_10. [DOI] [PubMed] [Google Scholar]
  7. Bidwai A. P., Reed J. C., Glover C. V. Casein kinase II of Saccharomyces cerevisiae contains two distinct regulatory subunits, beta and beta'. Arch Biochem Biophys. 1994 Mar;309(2):348–355. doi: 10.1006/abbi.1994.1123. [DOI] [PubMed] [Google Scholar]
  8. Bidwai A. P., Reed J. C., Glover C. V. Cloning and disruption of CKB1, the gene encoding the 38-kDa beta subunit of Saccharomyces cerevisiae casein kinase II (CKII). Deletion of CKII regulatory subunits elicits a salt-sensitive phenotype. J Biol Chem. 1995 May 5;270(18):10395–10404. doi: 10.1074/jbc.270.18.10395. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Brizuela L., Draetta G., Beach D. Activation of human CDC2 protein as a histone H1 kinase is associated with complex formation with the p62 subunit. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4362–4366. doi: 10.1073/pnas.86.12.4362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chester N., Yu I. J., Marshak D. R. Identification and characterization of protein kinase CKII isoforms in HeLa cells. Isoform-specific differences in rates of assembly from catalytic and regulatory subunits. J Biol Chem. 1995 Mar 31;270(13):7501–7514. doi: 10.1074/jbc.270.13.7501. [DOI] [PubMed] [Google Scholar]
  12. Cross F., Roberts J., Weintraub H. Simple and complex cell cycles. Annu Rev Cell Biol. 1989;5:341–396. doi: 10.1146/annurev.cb.05.110189.002013. [DOI] [PubMed] [Google Scholar]
  13. Deshaies R. J. Phosphorylation and proteolysis: partners in the regulation of cell division in budding yeast. Curr Opin Genet Dev. 1997 Feb;7(1):7–16. doi: 10.1016/s0959-437x(97)80103-7. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Elledge S. J. Cell cycle checkpoints: preventing an identity crisis. Science. 1996 Dec 6;274(5293):1664–1672. doi: 10.1126/science.274.5293.1664. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Glover C. V., 3rd On the physiological role of casein kinase II in Saccharomyces cerevisiae. Prog Nucleic Acid Res Mol Biol. 1998;59:95–133. doi: 10.1016/s0079-6603(08)61030-2. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Haase S. B., Reed S. I. Evidence that a free-running oscillator drives G1 events in the budding yeast cell cycle. Nature. 1999 Sep 23;401(6751):394–397. doi: 10.1038/43927. [DOI] [PubMed] [Google Scholar]
  20. Hadwiger J. A., Reed S. I. Invariant phosphorylation of the Saccharomyces cerevisiae Cdc28 protein kinase. Mol Cell Biol. 1988 Jul;8(7):2976–2979. doi: 10.1128/mcb.8.7.2976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hall D. D., Markwardt D. D., Parviz F., Heideman W. Regulation of the Cln3-Cdc28 kinase by cAMP in Saccharomyces cerevisiae. EMBO J. 1998 Aug 3;17(15):4370–4378. doi: 10.1093/emboj/17.15.4370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hanna D. E., Rethinaswamy A., Glover C. V. Casein kinase II is required for cell cycle progression during G1 and G2/M in Saccharomyces cerevisiae. J Biol Chem. 1995 Oct 27;270(43):25905–25914. doi: 10.1074/jbc.270.43.25905. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Issinger O. G. Casein kinases: pleiotropic mediators of cellular regulation. Pharmacol Ther. 1993;59(1):1–30. doi: 10.1016/0163-7258(93)90039-g. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Lörincz A. T., Reed S. I. Primary structure homology between the product of yeast cell division control gene CDC28 and vertebrate oncogenes. Nature. 1984 Jan 12;307(5947):183–185. doi: 10.1038/307183a0. [DOI] [PubMed] [Google Scholar]
  28. Marcote M. J., Knighton D. R., Basi G., Sowadski J. M., Brambilla P., Draetta G., Taylor S. S. A three-dimensional model of the Cdc2 protein kinase: localization of cyclin- and Suc1-binding regions and phosphorylation sites. Mol Cell Biol. 1993 Aug;13(8):5122–5131. doi: 10.1128/mcb.13.8.5122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Marshak D. R., Carroll D. Synthetic peptide substrates for casein kinase II. Methods Enzymol. 1991;200:134–156. doi: 10.1016/0076-6879(91)00135-j. [DOI] [PubMed] [Google Scholar]
  30. Marshak D. R., Vandenberg M. T., Bae Y. S., Yu I. J. Characterization of synthetic peptide substrates for p34cdc2 protein kinase. J Cell Biochem. 1991 Apr;45(4):391–400. doi: 10.1002/jcb.240450413. [DOI] [PubMed] [Google Scholar]
  31. McMillan J. N., Sia R. A., Bardes E. S., Lew D. J. Phosphorylation-independent inhibition of Cdc28p by the tyrosine kinase Swe1p in the morphogenesis checkpoint. Mol Cell Biol. 1999 Sep;19(9):5981–5990. doi: 10.1128/mcb.19.9.5981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Meggio F., Donella Deana A., Brunati A. M., Pinna L. A. Inhibition of rat liver cytosol casein kinases by heparin. FEBS Lett. 1982 May 17;141(2):257–262. doi: 10.1016/0014-5793(82)80061-6. [DOI] [PubMed] [Google Scholar]
  33. Mendenhall M. D., Jones C. A., Reed S. I. Dual regulation of the yeast CDC28-p40 protein kinase complex: cell cycle, pheromone, and nutrient limitation effects. Cell. 1987 Sep 11;50(6):927–935. doi: 10.1016/0092-8674(87)90519-8. [DOI] [PubMed] [Google Scholar]
  34. Nasmyth K. At the heart of the budding yeast cell cycle. Trends Genet. 1996 Oct;12(10):405–412. doi: 10.1016/0168-9525(96)10041-x. [DOI] [PubMed] [Google Scholar]
  35. Nasmyth K. Viewpoint: putting the cell cycle in order. Science. 1996 Dec 6;274(5293):1643–1645. doi: 10.1126/science.274.5293.1643. [DOI] [PubMed] [Google Scholar]
  36. Nurse P. Ordering S phase and M phase in the cell cycle. Cell. 1994 Nov 18;79(4):547–550. doi: 10.1016/0092-8674(94)90539-8. [DOI] [PubMed] [Google Scholar]
  37. Padmanabha R., Chen-Wu J. L., Hanna D. E., Glover C. V. Isolation, sequencing, and disruption of the yeast CKA2 gene: casein kinase II is essential for viability in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Aug;10(8):4089–4099. doi: 10.1128/mcb.10.8.4089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pinna L. A., Meggio F. Protein kinase CK2 ("casein kinase-2") and its implication in cell division and proliferation. Prog Cell Cycle Res. 1997;3:77–97. doi: 10.1007/978-1-4615-5371-7_7. [DOI] [PubMed] [Google Scholar]
  39. Reed S. I. The role of p34 kinases in the G1 to S-phase transition. Annu Rev Cell Biol. 1992;8:529–561. doi: 10.1146/annurev.cb.08.110192.002525. [DOI] [PubMed] [Google Scholar]
  40. Rethinaswamy A., Birnbaum M. J., Glover C. V. Temperature-sensitive mutations of the CKA1 gene reveal a role for casein kinase II in maintenance of cell polarity in Saccharomyces cerevisiae. J Biol Chem. 1998 Mar 6;273(10):5869–5877. doi: 10.1074/jbc.273.10.5869. [DOI] [PubMed] [Google Scholar]
  41. Russo G. L., Vandenberg M. T., Yu I. J., Bae Y. S., Franza B. R., Jr, Marshak D. R. Casein kinase II phosphorylates p34cdc2 kinase in G1 phase of the HeLa cell division cycle. J Biol Chem. 1992 Oct 5;267(28):20317–20325. [PubMed] [Google Scholar]
  42. Sikorski R. S., Boeke J. D. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 1991;194:302–318. doi: 10.1016/0076-6879(91)94023-6. [DOI] [PubMed] [Google Scholar]
  43. Snell V., Nurse P. Genetic analysis of cell morphogenesis in fission yeast--a role for casein kinase II in the establishment of polarized growth. EMBO J. 1994 May 1;13(9):2066–2074. doi: 10.1002/j.1460-2075.1994.tb06481.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Sutton A., Immanuel D., Arndt K. T. The SIT4 protein phosphatase functions in late G1 for progression into S phase. Mol Cell Biol. 1991 Apr;11(4):2133–2148. doi: 10.1128/mcb.11.4.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Toczyski D. P., Galgoczy D. J., Hartwell L. H. CDC5 and CKII control adaptation to the yeast DNA damage checkpoint. Cell. 1997 Sep 19;90(6):1097–1106. doi: 10.1016/s0092-8674(00)80375-x. [DOI] [PubMed] [Google Scholar]
  47. Tokiwa G., Tyers M., Volpe T., Futcher B. Inhibition of G1 cyclin activity by the Ras/cAMP pathway in yeast. Nature. 1994 Sep 22;371(6495):342–345. doi: 10.1038/371342a0. [DOI] [PubMed] [Google Scholar]
  48. Yu I. J., Spector D. L., Bae Y. S., Marshak D. R. Immunocytochemical localization of casein kinase II during interphase and mitosis. J Cell Biol. 1991 Sep;114(6):1217–1232. doi: 10.1083/jcb.114.6.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. de Nadal E., Calero F., Ramos J., Ariño J. Biochemical and genetic analyses of the role of yeast casein kinase 2 in salt tolerance. J Bacteriol. 1999 Oct;181(20):6456–6462. doi: 10.1128/jb.181.20.6456-6462.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]

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