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. 1993 Aug;13(8):5112–5121. doi: 10.1128/mcb.13.8.5112

Expression of p60v-src in Saccharomyces cerevisiae results in elevation of p34CDC28 kinase activity and release of the dependence of DNA replication on mitosis.

F Boschelli 1
PMCID: PMC360166  PMID: 7687746

Abstract

Expression of the oncogenic protein tyrosine kinase p60v-src in the yeast Saccharomyces cerevisiae has been shown to result in rapid cell death (J. S. Brugge, G. Jarosik, J. Andersen, A. Queral-Lustig, M. Fedor-Chaiken, and J. R. Broach, Mol. Cell. Biol. 7:2180-2187, 1987). Work described here demonstrates that v-Src expression results in accumulation of large-budded cells and a nuclear division block without blocking cytokinesis. Flow-cytometric analysis indicates that the DNA content of these cells is elevated beyond the G2 DNA content, and genetic studies indicate that v-Src expression causes aneuploidy. The activity of Cdc28 kinase, which controls the G1/S and G2/M transitions in S. cerevisiae, increases during galactose induction in a Src+ strain but not in an isogenic Src- strain. These observations indicate that v-Src expression disrupts p34CDC28 kinase regulation, allowing DNA replication to proceed in the absence of a prior mitotic event.

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Selected References

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

  1. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Bagrodia S., Chackalaparampil I., Kmiecik T. E., Shalloway D. Altered tyrosine 527 phosphorylation and mitotic activation of p60c-src. Nature. 1991 Jan 10;349(6305):172–175. doi: 10.1038/349172a0. [DOI] [PubMed] [Google Scholar]
  5. Baum P., Yip C., Goetsch L., Byers B. A yeast gene essential for regulation of spindle pole duplication. Mol Cell Biol. 1988 Dec;8(12):5386–5397. doi: 10.1128/mcb.8.12.5386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boeke J. D. One and two codon insertion mutants of bacteriophage f1. Mol Gen Genet. 1981;181(3):288–291. doi: 10.1007/BF00425599. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Broek D., Bartlett R., Crawford K., Nurse P. Involvement of p34cdc2 in establishing the dependency of S phase on mitosis. Nature. 1991 Jan 31;349(6308):388–393. doi: 10.1038/349388a0. [DOI] [PubMed] [Google Scholar]
  9. Brugge J. S., Jarosik G., Andersen J., Queral-Lustig A., Fedor-Chaiken M., Broach J. R. Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells. Mol Cell Biol. 1987 Jun;7(6):2180–2187. doi: 10.1128/mcb.7.6.2180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Burr J. G., Dreyfuss G., Penman S., Buchanan J. M. Association of the src gene product of Rous sarcoma virus with cytoskeletal structures of chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3484–3488. doi: 10.1073/pnas.77.6.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooper J. A., Runge K. Avian pp60c-src is more active when expressed in yeast than in vertebrate fibroblasts. Oncogene Res. 1987 Sep-Oct;1(4):297–310. [PubMed] [Google Scholar]
  12. David-Pfeuty T., Nouvian-Dooghe Y. Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells. J Cell Biol. 1990 Dec;111(6 Pt 2):3097–3116. doi: 10.1083/jcb.111.6.3097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Hamaguchi M., Hanafusa H. Association of p60src with Triton X-100-resistant cellular structure correlates with morphological transformation. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2312–2316. doi: 10.1073/pnas.84.8.2312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hartwell L. H., Culotti J., Pringle J. R., Reid B. J. Genetic control of the cell division cycle in yeast. Science. 1974 Jan 11;183(4120):46–51. doi: 10.1126/science.183.4120.46. [DOI] [PubMed] [Google Scholar]
  17. Hartwell L. H., Mortimer R. K., Culotti J., Culotti M. Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants. Genetics. 1973 Jun;74(2):267–286. doi: 10.1093/genetics/74.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hartwell L. H., Smith D. Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S. cerevisiae. Genetics. 1985 Jul;110(3):381–395. doi: 10.1093/genetics/110.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Hirano T., Funahashi S., Uemura T., Yanagida M. Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis. EMBO J. 1986 Nov;5(11):2973–2979. doi: 10.1002/j.1460-2075.1986.tb04594.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hoyt M. A., Totis L., Roberts B. T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell. 1991 Aug 9;66(3):507–517. doi: 10.1016/0092-8674(81)90014-3. [DOI] [PubMed] [Google Scholar]
  22. Hutter K. J., Eipel H. E. Microbial determinations by flow cytometry. J Gen Microbiol. 1979 Aug;113(2):369–375. doi: 10.1099/00221287-113-2-369. [DOI] [PubMed] [Google Scholar]
  23. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jove R., Hanafusa H. Cell transformation by the viral src oncogene. Annu Rev Cell Biol. 1987;3:31–56. doi: 10.1146/annurev.cb.03.110187.000335. [DOI] [PubMed] [Google Scholar]
  25. Kaplan J. M., Varmus H. E., Bishop J. M. The src protein contains multiple domains for specific attachment to membranes. Mol Cell Biol. 1990 Mar;10(3):1000–1009. doi: 10.1128/mcb.10.3.1000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kornbluth S., Jove R., Hanafusa H. Characterization of avian and viral p60src proteins expressed in yeast. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4455–4459. doi: 10.1073/pnas.84.13.4455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lamb N. J., Fernandez A., Watrin A., Labbé J. C., Cavadore J. C. Microinjection of p34cdc2 kinase induces marked changes in cell shape, cytoskeletal organization, and chromatin structure in mammalian fibroblasts. Cell. 1990 Jan 12;60(1):151–165. doi: 10.1016/0092-8674(90)90725-t. [DOI] [PubMed] [Google Scholar]
  28. Lee M. G., Nurse P. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature. 1987 May 7;327(6117):31–35. doi: 10.1038/327031a0. [DOI] [PubMed] [Google Scholar]
  29. Li R., Murray A. W. Feedback control of mitosis in budding yeast. Cell. 1991 Aug 9;66(3):519–531. doi: 10.1016/0092-8674(81)90015-5. [DOI] [PubMed] [Google Scholar]
  30. Morgan D. O., Kaplan J. M., Bishop J. M., Varmus H. E. Mitosis-specific phosphorylation of p60c-src by p34cdc2-associated protein kinase. Cell. 1989 Jun 2;57(5):775–786. doi: 10.1016/0092-8674(89)90792-7. [DOI] [PubMed] [Google Scholar]
  31. Murray A. W. Creative blocks: cell-cycle checkpoints and feedback controls. Nature. 1992 Oct 15;359(6396):599–604. doi: 10.1038/359599a0. [DOI] [PubMed] [Google Scholar]
  32. Nurse P., Bissett Y. Gene required in G1 for commitment to cell cycle and in G2 for control of mitosis in fission yeast. Nature. 1981 Aug 6;292(5823):558–560. doi: 10.1038/292558a0. [DOI] [PubMed] [Google Scholar]
  33. Ohtsubo M., Okazaki H., Nishimoto T. The RCC1 protein, a regulator for the onset of chromosome condensation locates in the nucleus and binds to DNA. J Cell Biol. 1989 Oct;109(4 Pt 1):1389–1397. doi: 10.1083/jcb.109.4.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Resh M. D., Erikson R. L. Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules. J Cell Biol. 1985 Feb;100(2):409–417. doi: 10.1083/jcb.100.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  37. Schlegel R., Pardee A. B. Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells. Science. 1986 Jun 6;232(4755):1264–1266. doi: 10.1126/science.2422760. [DOI] [PubMed] [Google Scholar]
  38. Shenoy S., Chackalaparampil I., Bagrodia S., Lin P. H., Shalloway D. Role of p34cdc2-mediated phosphorylations in two-step activation of pp60c-src during mitosis. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7237–7241. doi: 10.1073/pnas.89.15.7237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shenoy S., Choi J. K., Bagrodia S., Copeland T. D., Maller J. L., Shalloway D. Purified maturation promoting factor phosphorylates pp60c-src at the sites phosphorylated during fibroblast mitosis. Cell. 1989 Jun 2;57(5):763–774. doi: 10.1016/0092-8674(89)90791-5. [DOI] [PubMed] [Google Scholar]
  40. Smith M. R., DeGudicibus S. J., Stacey D. W. Requirement for c-ras proteins during viral oncogene transformation. Nature. 1986 Apr 10;320(6062):540–543. doi: 10.1038/320540a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Smythe C., Newport J. W. Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2. Cell. 1992 Feb 21;68(4):787–797. doi: 10.1016/0092-8674(92)90153-4. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Thomas J. H., Botstein D. A gene required for the separation of chromosomes on the spindle apparatus in yeast. Cell. 1986 Jan 17;44(1):65–76. doi: 10.1016/0092-8674(86)90485-x. [DOI] [PubMed] [Google Scholar]
  44. Uzawa S., Samejima I., Hirano T., Tanaka K., Yanagida M. The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene. Cell. 1990 Sep 7;62(5):913–925. doi: 10.1016/0092-8674(90)90266-h. [DOI] [PubMed] [Google Scholar]
  45. Vallen E. A., Scherson T. Y., Roberts T., van Zee K., Rose M. D. Asymmetric mitotic segregation of the yeast spindle pole body. Cell. 1992 May 1;69(3):505–515. doi: 10.1016/0092-8674(92)90451-h. [DOI] [PubMed] [Google Scholar]
  46. Weinert T. A., Hartwell L. H. The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science. 1988 Jul 15;241(4863):317–322. doi: 10.1126/science.3291120. [DOI] [PubMed] [Google Scholar]
  47. Wendler P. A., Boschelli F. Src homology 2 domain deletion mutants of p60v-src do not phosphorylate cellular proteins of 120-150 kDa. Oncogene. 1989 Feb;4(2):231–236. [PubMed] [Google Scholar]
  48. 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]
  49. Wittenberg C., Richardson S. L., Reed S. I. Subcellular localization of a protein kinase required for cell cycle initiation in Saccharomyces cerevisiae: evidence for an association between the CDC28 gene product and the insoluble cytoplasmic matrix. J Cell Biol. 1987 Oct;105(4):1527–1538. doi: 10.1083/jcb.105.4.1527. [DOI] [PMC free article] [PubMed] [Google Scholar]

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