Molecular Genetics and Genomics

, Volume 269, Issue 5, pp 672–684 | Cite as

Mutations in the yeast cyclin-dependent kinase Cdc28 reveal a role in the spindle assembly checkpoint

Original Paper


Anaphase onset and mitotic exit are regulated by the spindle assembly or kinetochore checkpoint, which inhibits the anaphase-promoting complex (APC), preventing the degradation of anaphase inhibitors and mitotic cyclins. As a result, cells arrest with high cyclin-dependent kinase (CDK) activity due to the accumulation of cyclins. Aside from this, a clear-cut demonstration of a direct role for CDKs in the spindle checkpoint response has been elusive. Cdc28 is the main CDK driving the cell cycle in budding yeast. In this report, mutations in cdc28 are described that confer specific checkpoint defects, supersensitivity towards microtubule poisons and chromosome loss. Two alleles encode single mutations in the N and C terminal regions, respectively (R10G and R288G), and one allele specifies two mutations near the C terminus (F245L, I284T). These cdc28 mutants are unable to arrest or efficiently prevent sister chromatid separation during treatment with nocodazole. Genetic interactions with checkpoint and apc mutants suggest Cdc28 may regulate checkpoint arrest downstream of the MAD2 and BUB2 pathways. These studies identify a C-terminal domain of Cdc28 required for checkpoint arrest upon spindle damage that mediates chromosome stability during vegetative growth, suggesting that it has an essential surveillance function in the unperturbed cell cycle.


Anaphase Cell cycle Chromosome segregation Mitosis 


  1. Agarwal R, Cohen-Fix O (2002) Phosphorylation of the mitotic regulator Pds1/securin by Cdc28 is required for efficient nuclear localization of Esp1/separase. Genes Dev 16:1371–1382CrossRefPubMedGoogle Scholar
  2. Ahn S, Tobe BT, Fitz Gerald JN, Anderson SL, Acurio A, Kron SJ (2001) Enhanced cell polarity in mutants of the yeast cyclin-dependent kinase Cdc28p. Mol Biol Cell 12:3589–3600PubMedGoogle Scholar
  3. Amon A (1997) Regulation of B-type cyclin proteolysis by Cdc28-associated kinases in budding yeast. EMBO J 16:2693–2702CrossRefPubMedGoogle Scholar
  4. Amon A (1999) The spindle checkpoint. Curr Opin Genet Dev 9:69–75PubMedGoogle Scholar
  5. Andrews B, Measday V (1998) The cyclin family of budding yeast: abundant use of a good idea. Trends Genet 14:66–72CrossRefPubMedGoogle Scholar
  6. Bardin AJ, Visintin R, Amon A (2000) A mechanism for coupling exit from mitosis to partitioning of the nucleus. Cell 102:21–31PubMedGoogle Scholar
  7. Baumer M, Braus GH, Irniger S (2000) Two different modes of cyclin Clb2 proteolysis during mitosis in Saccharomyces cerevisiae. FEBS Lett 468:14–28Google Scholar
  8. Burke DJ (2000) Complexity in the spindle checkpoint. Curr Opin Genet Dev 10:26–31PubMedGoogle Scholar
  9. Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, Kinzler KW, Vogelstein B (1998) Mutations of mitotic checkpoint genes in human cancers. Nature 392:300–303Google Scholar
  10. Cerutti L, Simanis V (2000) Controlling the end of the cell cycle. Curr Opin Genet Dev 10:65–69PubMedGoogle Scholar
  11. Chial HJ, Giddings TH Jr, Siewert EA, Hoyt MA, Winey M (1999) Altered dosage of the Saccharomyces cerevisiae spindle pole body duplication gene, NDC1 , leads to aneuploidy and polyploidy. Proc Natl Acad Sci USA 96:10200–10205CrossRefPubMedGoogle Scholar
  12. Cohen-Fix O, Peters JM, Kirschner MW, Koshland D (1996) Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev 10:3081–3093PubMedGoogle Scholar
  13. Donaldson AD, Kilmartin JV (1996) Spc42p: a phosphorylated component of the S. cerevisiae spindle pole body (SPB) with an essential function during SPB duplication. J Cell Biol 132:887–901PubMedGoogle Scholar
  14. Farr KA, Hoyt MA (1998) Bub1p kinase activates the Saccharomyces cerevisiae spindle assembly checkpoint. Mol Cell Biol 18:2738–2747PubMedGoogle Scholar
  15. Hardwick KG, Murray AW (1995) Mad1p, a phospho-protein component of the spindle assembly checkpoint in budding yeast. J Cell Biol 131:709–720PubMedGoogle Scholar
  16. Hardwick KG, Weiss E, Luca FC, Winey M, Murray AW (1996) Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption. Science 273:953–956PubMedGoogle Scholar
  17. Hardwick KG, Li R, Mistrot C, Chen RH, Dann P, Rudner A, Murray AW (1999) Lesions in many different spindle components activate the spindle checkpoint in the budding yeast Saccharomyces cerevisiae. Genetics 152:509–518PubMedGoogle Scholar
  18. Heo SJ, Tatebayashi K, Ikeda H (1999) The budding yeast cohesin gene SCC1/MCD1/RHC21 genetically interacts with PKA, CDK and APC. Curr Genet 36:329–338CrossRefPubMedGoogle Scholar
  19. Hoyt MA (2000) Exit from mitosis: spindle pole power. Cell 102:267–270PubMedGoogle Scholar
  20. Hoyt MA, Totis L, Roberts BT (1991) S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66:507–517PubMedGoogle Scholar
  21. Irniger S (2002) Cyclin destruction in mitosis: a crucial task of Cdc20. FEBS Lett 532:7–11CrossRefPubMedGoogle Scholar
  22. Jaspersen SL, Charles JF, Morgan DO (1999) Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14. Curr Biol 9:227–236CrossRefPubMedGoogle Scholar
  23. Jensen S, Geymonat M, Johnson AL, Segal M, Johnston LH (2002) Spatial regulation of the guanine nucleotide exchange factor Lte1 in Saccharomyces cerevisiae. J Cell Sci 115:4977–4991CrossRefPubMedGoogle Scholar
  24. Juang YL, Huang J, Peters JM, McLaughlin ME, Tai CY, Pellman D (1997) APC-mediated proteolysis of Ase1 and the morphogenesis of the mitotic spindle. Science 275:1311–1314CrossRefPubMedGoogle Scholar
  25. Kaiser P, Moncollin V, Clarke DJ, Watson MH, Bertolaet BL, Reed SI, Bailly E (1999) Cyclin-dependent kinase and Cks/Suc1 interact with the proteasome in yeast to control proteolysis of M-phase targets. Genes Dev 13:1190–1202PubMedGoogle Scholar
  26. Kitazono AA, Kron SJ (2002) An essential function of yeast cyclin-dependent kinase Cdc28 maintains chromosome stability. J Biol Chem 277:48627–48634CrossRefPubMedGoogle Scholar
  27. Kitazono AA, Tobe BTD, Kalton H, Diamant N, Kron SJ (2001) Marker-fusion PCR for one-step mutagenesis of essential genes in yeast. Yeast 19:141–149CrossRefGoogle Scholar
  28. Koshland D, Hieter P (1987) Visual assay for chromosome ploidy. Methods Enzymol 155:351–372PubMedGoogle Scholar
  29. Lamb JR, Michaud WA, Sikorski RS, Hieter PA (1994) Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis. EMBO J 13:4321–4328PubMedGoogle Scholar
  30. Li R, Murray AW (1991) Feedback control of mitosis in budding yeast. Cell 66:519–531PubMedGoogle Scholar
  31. Li X, Cai M (1997) Inactivation of the cyclin-dependent kinase Cdc28 abrogates cell cycle arrest induced by DNA damage and disassembly of mitotic spindles in Saccharomyces cerevisiae. Mol Cell Biol 17:2723–2734PubMedGoogle Scholar
  32. Longtine MS, McKenzie A 3rd, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14:953–961PubMedGoogle Scholar
  33. Mendenhall MD, Hodge AE (1998) Regulation of Cdc28 cyclin-dependent protein kinase activity during the cell cycle of the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev 62:1191–1243PubMedGoogle Scholar
  34. Morgan DO (1997) Cyclin-dependent kinases: engines, clocks, and microprocessors. Annu Rev Cell Dev Biol 13:261–291Google Scholar
  35. Morgan DO (1999) Regulation of the APC and the exit from mitosis. Nat Cell Biol 1: E47–53Google Scholar
  36. Musacchio A, Hardwick KG (2002) The spindle checkpoint: structural insights into dynamic signalling. Nat Rev Mol Cell Biol 3:731–741CrossRefPubMedGoogle Scholar
  37. Nasmyth K, Peters JM, Uhlmann F (2000) Splitting the chromosome: cutting the ties that bind sister chromatids. Science 288:1379–1385CrossRefPubMedGoogle Scholar
  38. Page AM, Hieter P (1999) The anaphase-promoting complex: new subunits and regulators. Annu Rev Biochem 68:583–609CrossRefPubMedGoogle Scholar
  39. Pangilinan F, Spencer F (1996) Abnormal kinetochore structure activates the spindle assembly checkpoint in budding yeast. Mol Biol Cell 7:1195–1208PubMedGoogle Scholar
  40. Pavletich NP (1999) Mechanisms of cyclin-dependent kinase regulation: structures of Cdks, their cyclin activators, and Cip and INK4 inhibitors. J Mol Biol 287:821–828CrossRefPubMedGoogle Scholar
  41. Pereira G, Hofken T, Grindlay J, Manson C, Schiebel E (2000) The Bub2p spindle checkpoint links nuclear migration with mitotic exit. Mol Cell 6:1–10PubMedGoogle Scholar
  42. Peters JM (1999) Subunits and substrates of the anaphase-promoting complex. Exp Cell Res 248:339–349CrossRefPubMedGoogle Scholar
  43. Pines J (1999) Four-dimensional control of the cell cycle. Nat Cell Biol 1: E73–79CrossRefPubMedGoogle Scholar
  44. Reed SI (1980) The selection of S. cerevisiae mutants defective in the start event of cell division. Genetics 95:561–577PubMedGoogle Scholar
  45. Rudner AD, Murray AW (2000) Phosphorylation by Cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex. J Cell Biol 149:1377–1390CrossRefPubMedGoogle Scholar
  46. Rudner AD, Hardwick KG, Murray AW (2000) Cdc28 activates exit from mitosis in budding yeast. J Cell Biol 149:1361–1376CrossRefPubMedGoogle Scholar
  47. Segal M, Clarke DJ, Maddox P, Salmon ED, Bloom K, Reed SI (2000) Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast. J Cell Biol 148:441–452CrossRefPubMedGoogle Scholar
  48. Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19–27PubMedGoogle Scholar
  49. Straight AF, Murray AW (1997) The spindle assembly checkpoint in budding yeast. Methods Enzymol 283:425–440PubMedGoogle Scholar
  50. Sundberg HA, Davis TN (1997) A mutational analysis identifies three functional regions of the spindle pole component Spc110p in Saccharomyces cerevisiae. Mol Biol Cell 8:2575–2590PubMedGoogle Scholar
  51. Tennyson CMV, Andrews BJ (1998) In vitro assay for cyclin-dependent kinase activity in yeast. Technical Tips Online 1:P01174Google Scholar
  52. Thomas BJ, Rothstein R (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56:619–630PubMedGoogle Scholar
  53. Uhlmann F, Wernic D, Poupart MA, Koonin EV, Nasmyth K (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103:375–386PubMedGoogle Scholar
  54. Wang Y, Burke DJ (1995) Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae. Mol Cell Biol 15:6838–6844PubMedGoogle Scholar
  55. Wasch R, Cross FR (2002) APC-dependent proteolysis of the mitotic cyclin Clb2 is essential for mitotic exit. Nature 418:556–562CrossRefPubMedGoogle Scholar
  56. Wassmann K, Benezra R (2001) Mitotic checkpoints: from yeast to cancer. Curr Opin Genet Dev 11:83–90CrossRefPubMedGoogle Scholar
  57. Weiss E, Winey M (1996) The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint. J Cell Biol 132:111–123PubMedGoogle Scholar
  58. Yamaguchi S, Decottignies A, Nurse P (2003) Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint. EMBO J 22:1075–1087CrossRefPubMedGoogle Scholar
  59. Yeong FM, Lim HH, Padmashree CG, Surana U (2000) Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20. Mol Cell 5:501–511PubMedGoogle Scholar
  60. Zachariae W, Nasmyth K (1999) Whose end is destruction: cell division and the anaphase-promoting complex. Genes Dev 13:2039–2058PubMedGoogle Scholar
  61. Zachariae W, Schwab M, Nasmyth K, Seufert W (1998) Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex. Science 282:1721–1724CrossRefPubMedGoogle Scholar
  62. Zhou J, Yao J, Joshi H (2002) Attachment and tension in the spindle assembly checkpoint. J Cell Sci 115:3547–3555CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Center for Molecular Oncology and Dept. of Molecular Genetics and Cell BiologyUniversity of ChicagoChicagoUSA

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