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. 1991 Dec;11(12):6177–6184. doi: 10.1128/mcb.11.12.6177

Mutations at sites involved in Suc1 binding inactivate Cdc2.

B Ducommun 1, P Brambilla 1, G Draetta 1
PMCID: PMC361799  PMID: 1944283

Abstract

suc1+ encodes an essential cell cycle regulator of the fission yeast Schizosaccharomyces pombe. Its product, a 13-kDa protein, interacts with the Cdc2 protein kinase. Both positive and negative effects on cell cycle progression have been attributed to Suc1. To date, the exact mechanisms and the physiological role of the interaction between Suc1 and Cdc2 remain unclear. Here we have studied the molecular basis of this association. We show that Cdc2 can bind Suc1 or its mammalian homolog directly in the absence of any additional protein component. Using an alanine scanning mutagenesis method, we analyzed the interaction between Cdc2 and Suc1. We show that the integrity of several domains on the Cdc2 protein, including sites directly involved in catalytic activity, is required for binding to Suc1. Furthermore, Cdc2 mutant proteins unable to bind Suc1 (but able to bind cyclins) are nonfunctional when overexpressed in S. pombe, indicating that a specific interaction with Suc1 is required for Cdc2 function.

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Selected References

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  1. Alfa C. E., Ducommun B., Beach D., Hyams J. S. Distinct nuclear and spindle pole body population of cyclin-cdc2 in fission yeast. Nature. 1990 Oct 18;347(6294):680–682. doi: 10.1038/347680a0. [DOI] [PubMed] [Google Scholar]
  2. Booher R. N., Alfa C. E., Hyams J. S., Beach D. H. The fission yeast cdc2/cdc13/suc1 protein kinase: regulation of catalytic activity and nuclear localization. Cell. 1989 Aug 11;58(3):485–497. doi: 10.1016/0092-8674(89)90429-7. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Carr A. M., MacNeill S. A., Hayles J., Nurse P. Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protein kinase gene: implications for cdc2+ protein structure and function. Mol Gen Genet. 1989 Jul;218(1):41–49. doi: 10.1007/BF00330563. [DOI] [PubMed] [Google Scholar]
  6. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Draetta G., Brizuela L., Potashkin J., Beach D. Identification of p34 and p13, human homologs of the cell cycle regulators of fission yeast encoded by cdc2+ and suc1+. Cell. 1987 Jul 17;50(2):319–325. doi: 10.1016/0092-8674(87)90227-3. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Gautier J., Minshull J., Lohka M., Glotzer M., Hunt T., Maller J. L. Cyclin is a component of maturation-promoting factor from Xenopus. Cell. 1990 Feb 9;60(3):487–494. doi: 10.1016/0092-8674(90)90599-a. [DOI] [PubMed] [Google Scholar]
  13. Gibbs C. S., Zoller M. J. Rational scanning mutagenesis of a protein kinase identifies functional regions involved in catalysis and substrate interactions. J Biol Chem. 1991 May 15;266(14):8923–8931. [PubMed] [Google Scholar]
  14. Glotzer M., Murray A. W., Kirschner M. W. Cyclin is degraded by the ubiquitin pathway. Nature. 1991 Jan 10;349(6305):132–138. doi: 10.1038/349132a0. [DOI] [PubMed] [Google Scholar]
  15. Gould K. L., Moreno S., Tonks N. K., Nurse P. Complementation of the mitotic activator, p80cdc25, by a human protein-tyrosine phosphatase. Science. 1990 Dec 14;250(4987):1573–1576. doi: 10.1126/science.1703321. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Hayles J., Aves S., Nurse P. suc1 is an essential gene involved in both the cell cycle and growth in fission yeast. EMBO J. 1986 Dec 1;5(12):3373–3379. doi: 10.1002/j.1460-2075.1986.tb04653.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hayles J., Beach D., Durkacz B., Nurse P. The fission yeast cell cycle control gene cdc2: isolation of a sequence suc1 that suppresses cdc2 mutant function. Mol Gen Genet. 1986 Feb;202(2):291–293. doi: 10.1007/BF00331653. [DOI] [PubMed] [Google Scholar]
  21. Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. doi: 10.1038/329219a0. [DOI] [PubMed] [Google Scholar]
  22. Hindley J., Phear G., Stein M., Beach D. Sucl+ encodes a predicted 13-kilodalton protein that is essential for cell viability and is directly involved in the division cycle of Schizosaccharomyces pombe. Mol Cell Biol. 1987 Jan;7(1):504–511. doi: 10.1128/mcb.7.1.504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Klee C. B., Crouch T. H., Krinks M. H. Calcineurin: a calcium- and calmodulin-binding protein of the nervous system. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6270–6273. doi: 10.1073/pnas.76.12.6270. [DOI] [PMC free article] [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. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  26. Kuret J., Johnson K. E., Nicolette C., Zoller M. J. Mutagenesis of the regulatory subunit of yeast cAMP-dependent protein kinase. Isolation of site-directed mutants with altered binding affinity for catalytic subunit. J Biol Chem. 1988 Jul 5;263(19):9149–9154. [PubMed] [Google Scholar]
  27. Labbe J. C., Picard A., Peaucellier G., Cavadore J. C., Nurse P., Doree M. Purification of MPF from starfish: identification as the H1 histone kinase p34cdc2 and a possible mechanism for its periodic activation. Cell. 1989 Apr 21;57(2):253–263. doi: 10.1016/0092-8674(89)90963-x. [DOI] [PubMed] [Google Scholar]
  28. Lehner C. F., O'Farrell P. H. Drosophila cdc2 homologs: a functional homolog is coexpressed with a cognate variant. EMBO J. 1990 Nov;9(11):3573–3581. doi: 10.1002/j.1460-2075.1990.tb07568.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Maundrell K. nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem. 1990 Jul 5;265(19):10857–10864. [PubMed] [Google Scholar]
  30. Moreno S., Hayles J., Nurse P. Regulation of p34cdc2 protein kinase during mitosis. Cell. 1989 Jul 28;58(2):361–372. doi: 10.1016/0092-8674(89)90850-7. [DOI] [PubMed] [Google Scholar]
  31. Norbury C., Nurse P. Cyclins and cell cycle control. Curr Biol. 1991 Feb;1(1):23–24. doi: 10.1016/0960-9822(91)90116-e. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Pondaven P., Meijer L., Beach D. Activation of M-phase-specific histone H1 kinase by modification of the phosphorylation of its p34cdc2 and cyclin components. Genes Dev. 1990 Jan;4(1):9–17. doi: 10.1101/gad.4.1.9. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Richardson H. E., Stueland C. S., Thomas J., Russell P., Reed S. I. Human cDNAs encoding homologs of the small p34Cdc28/Cdc2-associated protein of Saccharomyces cerevisiae and Schizosaccharomyces pombe. Genes Dev. 1990 Aug;4(8):1332–1344. doi: 10.1101/gad.4.8.1332. [DOI] [PubMed] [Google Scholar]
  36. Russell P., Nurse P. Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog. Cell. 1987 May 22;49(4):559–567. doi: 10.1016/0092-8674(87)90458-2. [DOI] [PubMed] [Google Scholar]
  37. Stach R. W., Shooter E. M. The biological activity of cross-linked beta nerve growth factor protein. J Biol Chem. 1974 Oct 25;249(20):6668–6674. [PubMed] [Google Scholar]
  38. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  39. Wetzel R. Structure, function, nomenclature. Protein Eng. 1988 Apr;2(1):1–3. doi: 10.1093/protein/2.1.1. [DOI] [PubMed] [Google Scholar]
  40. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. Methods Enzymol. 1987;154:329–350. doi: 10.1016/0076-6879(87)54083-6. [DOI] [PubMed] [Google Scholar]

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