Phosphatase 2A Negatively Regulates Mitotic Exit in<i>Saccharomyces cerevisiae</i>

Molecular Biology of the Cell - Tập 17 Số 1 - Trang 80-89 - 2006
Yanchang Wang1, Tuen-Yung Ng2
1Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306, USA
2Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306.

Tóm tắt

In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway.

Từ khóa


Tài liệu tham khảo

Alexandru, G., Uhlmann, F., Mechtler, K., Poupart, M. A., and Nasmyth, K. (2001). Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast.Cell105, 459–472.

Alexandru, G., Zachariae, W., Schleiffer, A., and Nasmyth, K. (1999). Sister chromatid separation and chromosome re-duplication are regulated by different mechanisms in response to spindle damage.EMBO J.18, 2707–2721.

Bardin, A. J., Visintin, R., and Amon, A. (2000). A mechanism for coupling exit from mitosis to partitioning of the nucleus.Cell102, 21–31.

Booher, R. N., Deshaies, R. J., and Kirschner, M. W. (1993). Properties ofSaccharomyces cerevisiaewee1 and its differential regulation of p34CDC28 in response to G1 and G2 cyclins.EMBO J.12, 3417–3426.

Charles, J. F., Jaspersen, S. L., Tinker-Kulberg, R. L., Hwang, L., Szidon, A., and Morgan, D. O. (1998). The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery inS. cerevisiae.Curr. Biol.8, 497–507.

Evans, D. R., and Stark, M. J. (1997). Mutations in theSaccharomyces cerevisiaetype 2A protein phosphatase catalytic subunit reveal roles in cell wall integrity, actin cytoskeleton organization and mitosis.Genetics145, 227–241.

Geymonat, M., Spanos, A., Smith, S. J., Wheatley, E., Rittinger, K., Johnston, L. H., and Sedgwick, S. G. (2002). Control of mitotic exit in budding yeast. In vitro regulation of Tem1 GTPase by Bub2 and Bfa1.J. Biol. Chem.277, 28439–28445.

Geymonat, M., Spanos, A., Walker, P. A., Johnston, L. H., and Sedgwick, S. G. (2003). In vitro regulation of budding yeast Bfa1/Bub2 GAP activity by Cdc5J. Biol. Chem.278, 14591–14594.

Healy, A. M., Zolnierowicz, S., Stapleton, A. E., Goebl, M., DePaoli-Roach, A. A., and Pringle, J. R. (1991). CDC55, aSaccharomyces cerevisiaegene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase.Mol. Cell. Biol.11, 5767–5780.

Hoyt, M. A., Totis, L., and Roberts, B. T. (1991).S. cerevisiaegenes required for cell cycle arrest in response to loss of microtubule function.Cell66, 507–517.

Hu, F., Wang, Y., Liu, D., Li, Y., Qin, J., and Elledge, S. J. (2001). Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints.Cell107, 655–665.

Hwang, L. H., Lau, L. F., Smith, D. L., Mistrot, C. A., Hardwick, K. G., Hwang, E. S., Amon, A., and Murray, A. W. (1998). Budding yeast Cdc20, a target of the spindle checkpoint.Science279, 1041–1044.

10.1091/mbc.9.10.2803

Komarnitsky, S. I., Chiang, Y. C., Luca, F. C., Chen, J., Toyn, J. H., Winey, M., Johnston, L. H., and Denis, C. L. (1998). DBF2 protein kinase binds to and acts through the cell cycle-regulated MOB1 protein.Mol. Cell. Biol.18, 2100–2107.

Lee, S. E., Frenz, L. M., Wells, N. J., Johnson, A. L., and Johnston, L. H. (2001a). Order of function of the budding-yeast mitotic exit-network proteins Tem1, Cdc15, Mob1, Dbf2, and Cdc5.Curr. Biol.11, 784–788.

Lee, S. E., Jensen, S., Frenz, L. M., Johnson, A. L., Fesquet, D., and Johnston, L. H. (2001b). The Bub2-dependent mitotic pathway in yeast acts every cell cycle and regulates cytokinesis.J. Cell Sci.114, 2345–2354.

Lengronne, A., and Schwob, E. (2002). The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G(1).Mol. Cell9, 1067–1078.

Li, R. (1999). Bifurcation of the mitotic checkpoint pathway in budding yeast.Proc. Natl. Acad. Sci. USA96, 4989–4994.

Liu, H., Krizek, J., and Bretscher, A. (1992). Construction of a GAL1-regulated yeast cDNA expression library and its application to the identification of genes whose overexpression causes lethality in yeast.Genetics132, 665–673.

Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification inSaccharomyces cerevisiae.Yeast14, 953–961.

10.1091/mbc.9.1.29

Mah, A. S., Jang, J., and Deshaies, R. J. (2001). Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex.Proc. Natl. Acad. Sci. USA98, 7325–7330.

Michaelis, C., Ciosk, R., and Nasmyth, K. (1997). Cohesins: chromosomal proteins that prevent premature separation of sister chromatids.Cell91, 35–45.

Millward, T. A., Zolnierowicz, S., and Hemmings, B. A. (1999). Regulation of protein kinase cascades by protein phosphatase 2A.Trends Biochem. Sci.24, 186–191.

Minshull, J., Straight, A., Rudner, A. D., Dernburg, A. F., Belmont, A., and Murray, A. W. (1996). Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast.Curr. Biol.6, 1609–1620.

Morgan, D. O. (1999). Regulation of the APC and the exit from mitosis.Nat. Cell Biology1, E47–E53.

Noton, E., and Diffley, J. F. (2000). CDK inactivation is the only essential function of the APC/C and the mitotic exit network proteins for origin resetting during mitosis.Mol. Cell5, 85–95.

Pereira, G., Hofken, T., Grindlay, J., Manson, C., and Schiebel, E. (2000). The Bub2p spindle checkpoint links nuclear migration with mitotic exit.Mol. Cell6, 1–10.

Ronne, H., Carlberg, M., Hu, G. Z., and Nehlin, J. O. (1991). Protein phosphatase 2A inSaccharomyces cerevisiae: effects on cell growth and bud morphogenesis.Mol. Cell. Biol.11, 4876–4884.

Schwab, M., Lutum, A. S., and Seufert, W. (1997). Yeast Hct1 is a regulator of Clb2 cyclin proteolysis.Cell90, 683–693.

Shirayama, M., Matsui, Y., and Toh, E. A. (1994). The yeast TEM1 gene, which encodes a GTP-binding protein, is involved in termination of M phase.Mol. Cell. Biol.14, 7476–7482.

Shirayama, M., Zachariae, W., Ciosk, R., and Nasmyth, K. (1998). The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex inSaccharomyces cerevisiae.EMBO J.17, 1336–1349.

Shou, W., Seol, J. H., Shevchenko, A., Baskerville, C., Moazed, D., Chen, Z. W., Jang, J., Charbonneau, H., and Deshaies, R. J. (1999). Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex.Cell97, 233–244.

Stegmeier, F., Visintin, R., and Amon, A. (2002). Separase, polo kinase, the kinetochore protein Slk19, and Spo12 function in a network that controls Cdc14 localization during early anaphase.Cell108, 207–220.

10.1091/mbc.11.2.593

10.1091/mbc.12.10.2961

Visintin, R., Craig, K., Hwang, E. S., Prinz, S., Tyers, M., and Amon, A. (1998). The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation.Mol. Cell2, 709–718.

Visintin, R., Hwang, E. S., and Amon, A. (1999). Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus [comment].Nature398, 818–823.

Wang, Y., and Burke, D. J. (1997). Cdc55p, the B-type regulatory subunit of protein phosphatase 2A, has multiple functions in mitosis and is required for the kinetochore/spindle checkpoint inSaccharomyces cerevisiae.Mol. Cell. Biol.17, 620–626.

Wang, Y., Shirogane, T., Liu, D., Harper, J. W., and Elledge, S. J. (2003). Exit from exit: resetting the cell cycle through Amn1 inhibition of G protein signaling.Cell112, 697–709.

Yang, H., Jiang, W., Gentry, M., and Hallberg, R. L. (2000). Loss of a protein phosphatase 2A regulatory subunit (Cdc55p) elicits improper regulation of Swe1p degradation.Mol. Cell. Biol.20, 8143–8156.