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. 1998 Mar 2;17(5):1328–1335. doi: 10.1093/emboj/17.5.1328

The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts.

P Descombes 1, E A Nigg 1
PMCID: PMC1170481  PMID: 9482730

Abstract

Polo-like kinases (Plks), named after the Drosophila gene product polo, have been implicated in the regulation of multiple aspects of mitotic progression, including the activation of the Cdc25 phosphatase, bipolar spindle formation and cytokinesis. Genetic analyses performed in yeast and Drosophila suggest a function for Plks at late stages of mitosis, but biochemical data to support such a function in vertebrate organisms are lacking. Here we have taken advantage of Xenopus egg extracts for exploring the function of Plx1, a Xenopus Plk, during the cell cycle transition from M phase to interphase (I phase). We found that the addition of a catalytically inactive Plx1 mutant to M phase-arrested egg extracts blocked their Ca2+-induced release into interphase. Concomitantly, the proteolytic destruction of several targets of the anaphase-promoting complex and the inactivation of the Cdc2 protein kinase (Cdk1) were prevented. Moreover, the M to I phase transition could be abolished by immunodepletion of Plx1, but was restored upon the addition of recombinant Plx1. These results demonstrate that the exit of egg extracts from M phase arrest requires active Plx1, and they strongly suggest an important role for Plx1 in the activation of the proteolytic machinery that controls the exit from mitosis.

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Selected References

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  1. Abrieu A., Lorca T., Labbé J. C., Morin N., Keyse S., Dorée M. MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts. J Cell Sci. 1996 Jan;109(Pt 1):239–246. doi: 10.1242/jcs.109.1.239. [DOI] [PubMed] [Google Scholar]
  2. Cohen-Fix O., Peters J. M., Kirschner M. W., Koshland D. Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev. 1996 Dec 15;10(24):3081–3093. doi: 10.1101/gad.10.24.3081. [DOI] [PubMed] [Google Scholar]
  3. Deshaies R. J. The self-destructive personality of a cell cycle in transition. Curr Opin Cell Biol. 1995 Dec;7(6):781–789. doi: 10.1016/0955-0674(95)80061-1. [DOI] [PubMed] [Google Scholar]
  4. Ferrell J. E., Jr, Wu M., Gerhart J. C., Martin G. S. Cell cycle tyrosine phosphorylation of p34cdc2 and a microtubule-associated protein kinase homolog in Xenopus oocytes and eggs. Mol Cell Biol. 1991 Apr;11(4):1965–1971. doi: 10.1128/mcb.11.4.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Funabiki H., Yamano H., Kumada K., Nagao K., Hunt T., Yanagida M. Cut2 proteolysis required for sister-chromatid seperation in fission yeast. Nature. 1996 May 30;381(6581):438–441. doi: 10.1038/381438a0. [DOI] [PubMed] [Google Scholar]
  6. Funabiki H., Yamano H., Nagao K., Tanaka H., Yasuda H., Hunt T., Yanagida M. Fission yeast Cut2 required for anaphase has two destruction boxes. EMBO J. 1997 Oct 1;16(19):5977–5987. doi: 10.1093/emboj/16.19.5977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gallant P., Nigg E. A. Cyclin B2 undergoes cell cycle-dependent nuclear translocation and, when expressed as a non-destructible mutant, causes mitotic arrest in HeLa cells. J Cell Biol. 1992 Apr;117(1):213–224. doi: 10.1083/jcb.117.1.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Glover D. M., Ohkura H., Tavares A. Polo kinase: the choreographer of the mitotic stage? J Cell Biol. 1996 Dec;135(6 Pt 2):1681–1684. doi: 10.1083/jcb.135.6.1681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Golsteyn R. M., Mundt K. E., Fry A. M., Nigg E. A. Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function. J Cell Biol. 1995 Jun;129(6):1617–1628. doi: 10.1083/jcb.129.6.1617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grieco D., Porcellini A., Avvedimento E. V., Gottesman M. E. Requirement for cAMP-PKA pathway activation by M phase-promoting factor in the transition from mitosis to interphase. Science. 1996 Mar 22;271(5256):1718–1723. doi: 10.1126/science.271.5256.1718. [DOI] [PubMed] [Google Scholar]
  12. Hamanaka R., Smith M. R., O'Connor P. M., Maloid S., Mihalic K., Spivak J. L., Longo D. L., Ferris D. K. Polo-like kinase is a cell cycle-regulated kinase activated during mitosis. J Biol Chem. 1995 Sep 8;270(36):21086–21091. doi: 10.1074/jbc.270.36.21086. [DOI] [PubMed] [Google Scholar]
  13. Hardy C. F., Pautz A. A novel role for Cdc5p in DNA replication. Mol Cell Biol. 1996 Dec;16(12):6775–6782. doi: 10.1128/mcb.16.12.6775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hartley R. S., Rempel R. E., Maller J. L. In vivo regulation of the early embryonic cell cycle in Xenopus. Dev Biol. 1996 Feb 1;173(2):408–419. doi: 10.1006/dbio.1996.0036. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Hershko A., Ciechanover A. The ubiquitin system for protein degradation. Annu Rev Biochem. 1992;61:761–807. doi: 10.1146/annurev.bi.61.070192.003553. [DOI] [PubMed] [Google Scholar]
  17. Holloway S. L., Glotzer M., King R. W., Murray A. W. Anaphase is initiated by proteolysis rather than by the inactivation of maturation-promoting factor. Cell. 1993 Jul 2;73(7):1393–1402. doi: 10.1016/0092-8674(93)90364-v. [DOI] [PubMed] [Google Scholar]
  18. Hunt T., Luca F. C., Ruderman J. V. The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo. J Cell Biol. 1992 Feb;116(3):707–724. doi: 10.1083/jcb.116.3.707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Irniger S., Piatti S., Michaelis C., Nasmyth K. Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast. Cell. 1995 Apr 21;81(2):269–278. doi: 10.1016/0092-8674(95)90337-2. [DOI] [PubMed] [Google Scholar]
  20. Ishida N., Tanaka K., Tamura T., Nishizawa M., Okazaki K., Sagata N., Ichihara A. Mos is degraded by the 26S proteasome in a ubiquitin-dependent fashion. FEBS Lett. 1993 Jun 21;324(3):345–348. doi: 10.1016/0014-5793(93)80148-n. [DOI] [PubMed] [Google Scholar]
  21. Jentsch S., Schlenker S. Selective protein degradation: a journey's end within the proteasome. Cell. 1995 Sep 22;82(6):881–884. doi: 10.1016/0092-8674(95)90021-7. [DOI] [PubMed] [Google Scholar]
  22. King R. W., Deshaies R. J., Peters J. M., Kirschner M. W. How proteolysis drives the cell cycle. Science. 1996 Dec 6;274(5293):1652–1659. doi: 10.1126/science.274.5293.1652. [DOI] [PubMed] [Google Scholar]
  23. King R. W., Peters J. M., Tugendreich S., Rolfe M., Hieter P., Kirschner M. W. A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B. Cell. 1995 Apr 21;81(2):279–288. doi: 10.1016/0092-8674(95)90338-0. [DOI] [PubMed] [Google Scholar]
  24. Kitada K., Johnson A. L., Johnston L. H., Sugino A. A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5. Mol Cell Biol. 1993 Jul;13(7):4445–4457. doi: 10.1128/mcb.13.7.4445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kumagai A., Dunphy W. G. Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. Science. 1996 Sep 6;273(5280):1377–1380. doi: 10.1126/science.273.5280.1377. [DOI] [PubMed] [Google Scholar]
  26. Lahav-Baratz S., Sudakin V., Ruderman J. V., Hershko A. Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9303–9307. doi: 10.1073/pnas.92.20.9303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lane H. A., Nigg E. A. Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes. J Cell Biol. 1996 Dec;135(6 Pt 2):1701–1713. doi: 10.1083/jcb.135.6.1701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lane H. A., Nigg E. A. Cell-cycle control: POLO-like kinases join the outer circle. Trends Cell Biol. 1997 Feb;7(2):63–68. doi: 10.1016/S0962-8924(96)10051-9. [DOI] [PubMed] [Google Scholar]
  29. Lohka M. J., Maller J. L. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J Cell Biol. 1985 Aug;101(2):518–523. doi: 10.1083/jcb.101.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lorca T., Cruzalegui F. H., Fesquet D., Cavadore J. C., Méry J., Means A., Dorée M. Calmodulin-dependent protein kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs. Nature. 1993 Nov 18;366(6452):270–273. doi: 10.1038/366270a0. [DOI] [PubMed] [Google Scholar]
  31. Lorca T., Galas S., Fesquet D., Devault A., Cavadore J. C., Dorée M. Degradation of the proto-oncogene product p39mos is not necessary for cyclin proteolysis and exit from meiotic metaphase: requirement for a Ca(2+)-calmodulin dependent event. EMBO J. 1991 Aug;10(8):2087–2093. doi: 10.1002/j.1460-2075.1991.tb07741.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Minshull J., Sun H., Tonks N. K., Murray A. W. A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts. Cell. 1994 Nov 4;79(3):475–486. doi: 10.1016/0092-8674(94)90256-9. [DOI] [PubMed] [Google Scholar]
  33. Mundt K. E., Golsteyn R. M., Lane H. A., Nigg E. A. On the regulation and function of human polo-like kinase 1 (PLK1): effects of overexpression on cell cycle progression. Biochem Biophys Res Commun. 1997 Oct 20;239(2):377–385. doi: 10.1006/bbrc.1997.7378. [DOI] [PubMed] [Google Scholar]
  34. Murray A. W. Cell cycle extracts. Methods Cell Biol. 1991;36:581–605. [PubMed] [Google Scholar]
  35. Murray A. W., Solomon M. J., Kirschner M. W. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature. 1989 May 25;339(6222):280–286. doi: 10.1038/339280a0. [DOI] [PubMed] [Google Scholar]
  36. Nigg E. A. Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. Bioessays. 1995 Jun;17(6):471–480. doi: 10.1002/bies.950170603. [DOI] [PubMed] [Google Scholar]
  37. Ohkura H., Hagan I. M., Glover D. M. The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells. Genes Dev. 1995 May 1;9(9):1059–1073. doi: 10.1101/gad.9.9.1059. [DOI] [PubMed] [Google Scholar]
  38. Peters J. M., King R. W., Hög C., Kirschner M. W. Identification of BIME as a subunit of the anaphase-promoting complex. Science. 1996 Nov 15;274(5290):1199–1201. doi: 10.1126/science.274.5290.1199. [DOI] [PubMed] [Google Scholar]
  39. Rempel R. E., Sleight S. B., Maller J. L. Maternal Xenopus Cdk2-cyclin E complexes function during meiotic and early embryonic cell cycles that lack a G1 phase. J Biol Chem. 1995 Mar 24;270(12):6843–6855. doi: 10.1074/jbc.270.12.6843. [DOI] [PubMed] [Google Scholar]
  40. Rudner A. D., Murray A. W. The spindle assembly checkpoint. Curr Opin Cell Biol. 1996 Dec;8(6):773–780. doi: 10.1016/s0955-0674(96)80077-9. [DOI] [PubMed] [Google Scholar]
  41. Sagata N., Watanabe N., Vande Woude G. F., Ikawa Y. The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs. Nature. 1989 Nov 30;342(6249):512–518. doi: 10.1038/342512a0. [DOI] [PubMed] [Google Scholar]
  42. Sagata N. What does Mos do in oocytes and somatic cells? Bioessays. 1997 Jan;19(1):13–21. doi: 10.1002/bies.950190105. [DOI] [PubMed] [Google Scholar]
  43. Schwab M., Lutum A. S., Seufert W. Yeast Hct1 is a regulator of Clb2 cyclin proteolysis. Cell. 1997 Aug 22;90(4):683–693. doi: 10.1016/s0092-8674(00)80529-2. [DOI] [PubMed] [Google Scholar]
  44. Shirayama M., Zachariae W., Ciosk R., Nasmyth K. The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae. EMBO J. 1998 Mar 2;17(5):1336–1349. doi: 10.1093/emboj/17.5.1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sigrist S. J., Lehner C. F. Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. Cell. 1997 Aug 22;90(4):671–681. doi: 10.1016/s0092-8674(00)80528-0. [DOI] [PubMed] [Google Scholar]
  46. Sigrist S., Jacobs H., Stratmann R., Lehner C. F. Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3. EMBO J. 1995 Oct 2;14(19):4827–4838. doi: 10.1002/j.1460-2075.1995.tb00164.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Stewart E., Kobayashi H., Harrison D., Hunt T. Destruction of Xenopus cyclins A and B2, but not B1, requires binding to p34cdc2. EMBO J. 1994 Feb 1;13(3):584–594. doi: 10.1002/j.1460-2075.1994.tb06296.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sudakin V., Ganoth D., Dahan A., Heller H., Hershko J., Luca F. C., Ruderman J. V., Hershko A. The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. Mol Biol Cell. 1995 Feb;6(2):185–197. doi: 10.1091/mbc.6.2.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sunkel C. E., Glover D. M. polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. J Cell Sci. 1988 Jan;89(Pt 1):25–38. doi: 10.1242/jcs.89.1.25. [DOI] [PubMed] [Google Scholar]
  50. Surana U., Amon A., Dowzer C., McGrew J., Byers B., Nasmyth K. Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. EMBO J. 1993 May;12(5):1969–1978. doi: 10.1002/j.1460-2075.1993.tb05846.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Takenaka K., Gotoh Y., Nishida E. MAP kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit in Xenopus egg cell cycle extracts. J Cell Biol. 1997 Mar 10;136(5):1091–1097. doi: 10.1083/jcb.136.5.1091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tavares A. A., Glover D. M., Sunkel C. E. The conserved mitotic kinase polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts. EMBO J. 1996 Sep 16;15(18):4873–4883. [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Varshavsky A. The ubiquitin system. Trends Biochem Sci. 1997 Oct;22(10):383–387. doi: 10.1016/s0968-0004(97)01122-5. [DOI] [PubMed] [Google Scholar]
  55. Visintin R., Prinz S., Amon A. CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. Science. 1997 Oct 17;278(5337):460–463. doi: 10.1126/science.278.5337.460. [DOI] [PubMed] [Google Scholar]
  56. Watanabe N., Hunt T., Ikawa Y., Sagata N. Independent inactivation of MPF and cytostatic factor (Mos) upon fertilization of Xenopus eggs. Nature. 1991 Jul 18;352(6332):247–248. doi: 10.1038/352247a0. [DOI] [PubMed] [Google Scholar]
  57. Yamada H., Kumada K., Yanagida M. Distinct subunit functions and cell cycle regulated phosphorylation of 20S APC/cyclosome required for anaphase in fission yeast. J Cell Sci. 1997 Aug;110(Pt 15):1793–1804. doi: 10.1242/jcs.110.15.1793. [DOI] [PubMed] [Google Scholar]
  58. Zachariae W., Shin T. H., Galova M., Obermaier B., Nasmyth K. Identification of subunits of the anaphase-promoting complex of Saccharomyces cerevisiae. Science. 1996 Nov 15;274(5290):1201–1204. doi: 10.1126/science.274.5290.1201. [DOI] [PubMed] [Google Scholar]

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