Polo on the Rise—from Mitotic Entry to Cytokinesis with Plk1
2008; Elsevier BV; Volume: 14; Issue: 5 Linguagem: Inglês
10.1016/j.devcel.2008.04.014
ISSN1878-1551
AutoresMark Petronczki, Péter Lénárt, Jan‐Michael Peters,
Tópico(s)Cellular transport and secretion
ResumoPolo-like kinase 1 (Plk1) is a key regulator of cell division in eukaryotic cells. New techniques, including the application of small-molecule inhibitors, have greatly expanded our knowledge of the functions, targets, and regulation of this key mitotic enzyme. In this review, we focus on how Plk1 is recruited to centrosomes, kinetochores, and the spindle midzone and what the specific tasks of Plk1 at these distinct subcellular structures might be. In particular, we highlight new work on the role of Plk1 in cytokinesis in human cells. Finally, we describe how better understanding of Plk1 functions allows critical evaluation of Plk1 as a potential drug target for cancer therapy. Polo-like kinase 1 (Plk1) is a key regulator of cell division in eukaryotic cells. New techniques, including the application of small-molecule inhibitors, have greatly expanded our knowledge of the functions, targets, and regulation of this key mitotic enzyme. In this review, we focus on how Plk1 is recruited to centrosomes, kinetochores, and the spindle midzone and what the specific tasks of Plk1 at these distinct subcellular structures might be. In particular, we highlight new work on the role of Plk1 in cytokinesis in human cells. Finally, we describe how better understanding of Plk1 functions allows critical evaluation of Plk1 as a potential drug target for cancer therapy. The gene for the serine/threonine kinase known as Polo in Drosophila, Plk1 (Polo-like kinase 1) in vertebrates (often also referred to as Plx1 in Xenopus), and Cdc5 in budding yeast was first identified in genetic yeast and Drosophila screens for mutants defective in cell division (Hartwell et al., 1973Hartwell 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; 74: 267-286Crossref PubMed Google Scholar, Sunkel and Glover, 1988Sunkel C.E. Glover D.M. polo, a mitotic mutant of Drosophila displaying abnormal spindle poles.J. Cell Sci. 1988; 89: 25-38Crossref PubMed Google Scholar). The cloning of the Drosophila polo gene subsequently identified its product as a protein kinase (Llamazares et al., 1991Llamazares S. Moreira A. Tavares A. Girdham C. Spruce B.A. Gonzalez C. Karess R.E. Glover D.M. Sunkel C.E. polo encodes a protein kinase homolog required for mitosis in Drosophila.Genes Dev. 1991; 5: 2153-2165Crossref PubMed Google Scholar). Since this early pioneering work, Polo and its orthologs have been recognized not only as key regulators of mitosis but also cytokinesis (Barr et al., 2004Barr F.A. Sillje H.H. Nigg E.A. Polo-like kinases and the orchestration of cell division.Nat. Rev. Mol. Cell Biol. 2004; 5: 429-440Crossref PubMed Scopus (503) Google Scholar, Carmena et al., 1998Carmena M. Riparbelli M.G. Minestrini G. Tavares A.M. Adams R. Callaini G. Glover D.M. Drosophila polo kinase is required for cytokinesis.J. Cell Biol. 1998; 143: 659-671Crossref PubMed Scopus (145) Google Scholar). Human Plk1 belongs to a family of four related kinases with largely nonoverlapping functions (van de Weerdt and Medema, 2006van de Weerdt B.C. Medema R.H. Polo-like kinases: a team in control of the division.Cell Cycle. 2006; 5: 853-864Crossref PubMed Google Scholar). This review focuses on Plk1 (Figure 1A), the most prominent regulator of cell division within the Plk family. In yeasts and animal cells, Polo kinase is no less important for the cell division cycle than cyclin-dependent kinases (Cdks). Without Polo, animal cells fail to assemble a proper bipolar spindle and instead arrange their mitotic chromosomes in a circular fashion around a monopolar spindle (thus the name Polo; Sunkel and Glover, 1988Sunkel C.E. Glover D.M. polo, a mitotic mutant of Drosophila displaying abnormal spindle poles.J. Cell Sci. 1988; 89: 25-38Crossref PubMed Google Scholar) (Figure 2A). In Xenopus and humans, Plk1 is also required for proper timing of mitotic entry (Qian et al., 1998Qian Y.W. Erikson E. Li C. Maller J.L. Activated polo-like kinase Plx1 is required at multiple points during mitosis in Xenopus laevis.Mol. Cell. Biol. 1998; 18: 4262-4271Crossref PubMed Google Scholar, Sumara et al., 2004Sumara I. Gimenez-Abian J.F. Gerlich D. Hirota T. Kraft C. de la Torre C. Ellenberg J. Peters J.M. Roles of polo-like kinase 1 in the assembly of functional mitotic spindles.Curr. Biol. 2004; 14: 1712-1722Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), and in yeast, Cdc5 is essential for exit from mitosis and cytokinesis (Stegmeier et al., 2002Stegmeier F. Visintin R. Amon A. Separase, polo kinase, the kinetochore protein Slk19, and Spo12 function in a network that controls Cdc14 localization during early anaphase.Cell. 2002; 108: 207-220Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, Yoshida et al., 2006Yoshida S. Kono K. Lowery D.M. Bartolini S. Yaffe M.B. Ohya Y. Pellman D. Polo-like kinase Cdc5 controls the local activation of Rho1 to promote cytokinesis.Science. 2006; 313: 108-111Crossref PubMed Scopus (82) Google Scholar). In addition, Polo orthologs have been implicated in several other mitotic processes, such as dissociation of cohesin complexes from chromosome arms (Losada et al., 2002Losada A. Hirano M. Hirano T. Cohesin release is required for sister chromatid resolution, but not for condensin-mediated compaction, at the onset of mitosis.Genes Dev. 2002; 16: 3004-3016Crossref PubMed Scopus (213) Google Scholar, Sumara et al., 2002Sumara I. Vorlaufer E. Stukenberg P.T. Kelm O. Redemann N. Nigg E.A. Peters J.M. The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase.Mol. Cell. 2002; 9: 515-525Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar), phosphorylation of the anaphase-promoting complex/cyclosome (APC/C) (Golan et al., 2002Golan A. Yudkovsky Y. Hershko A. The cyclin-ubiquitin ligase activity of cyclosome/APC is jointly activated by protein kinases Cdk1-cyclin B and Plk.J. Biol. Chem. 2002; 277: 15552-15557Crossref PubMed Scopus (115) Google Scholar, Kraft et al., 2003Kraft C. Herzog F. Gieffers C. Mechtler K. Hagting A. Pines J. Peters J.M. Mitotic regulation of the human anaphase-promoting complex by phosphorylation.EMBO J. 2003; 22: 6598-6609Crossref PubMed Scopus (237) Google Scholar), and degradation of the APC/C inhibitor Emi1 (Hansen et al., 2004Hansen D.V. Loktev A.V. Ban K.H. Jackson P.K. Plk1 regulates activation of the anaphase promoting complex by phosphorylating and triggering SCFbetaTrCP-dependent destruction of the APC Inhibitor Emi1.Mol. Biol. Cell. 2004; 15: 5623-5634Crossref PubMed Scopus (125) Google Scholar, Lenart et al., 2007Lenart P. Petronczki M. Steegmaier M. Di Fiore B. Lipp J.J. Hoffmann M. Rettig W.J. Kraut N. Peters J.M. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.Curr. Biol. 2007; 17: 304-315Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, Moshe et al., 2004Moshe Y. Boulaire J. Pagano M. Hershko A. Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase promoting complex/cyclosome.Proc. Natl. Acad. Sci. USA. 2004; 101: 7937-7942Crossref PubMed Scopus (125) Google Scholar). In mammalian cells, a more detailed and comprehensive investigation of the many important functions of Plk1 has only become possible recently through the development and application of new research tools and technologies. These include methods for inactivation of Plk1 by RNA interference (RNAi), specific small-molecule inhibitors (reviewed in Taylor and Peters, 2008Taylor S. Peters J.M. Polo and Aurora kinases-lessons derived from chemical biology.Curr. Opin. Cell Biol. 2008; 20: 77-84Crossref PubMed Scopus (87) Google Scholar), and the generation of genetically modified alleles (Burkard et al., 2007Burkard M.E. Randall C.L. Larochelle S. Zhang C. Shokat K.M. Fisher R.P. Jallepalli P.V. Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 4383-4388Crossref PubMed Scopus (114) Google Scholar). Furthermore, the identification of Plk1 substrates and interaction partners has been facilitated by peptide and protein binding studies and mass spectrometry (e.g., Baumann et al., 2007Baumann C. Korner R. Hofmann K. Nigg E.A. PICH, a centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for the spindle checkpoint.Cell. 2007; 128: 101-114Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, Lowery et al., 2007Lowery D.M. Clauser K.R. Hjerrild M. Lim D. Alexander J. Kishi K. Ong S.E. Gammeltoft S. Carr S.A. Yaffe M.B. Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate.EMBO J. 2007; 26: 2262-2273Crossref PubMed Scopus (124) Google Scholar) and the solution of Plk1 structures at atomic level by X-ray crystallography (Kothe et al., 2007aKothe M. Kohls D. Low S. Coli R. Cheng A.C. Jacques S.L. Johnson T.L. Lewis C. Loh C. Nonomiya J. et al.Structure of the catalytic domain of human polo-like kinase 1.Biochemistry. 2007; 46: 5960-5971Crossref PubMed Scopus (61) Google Scholar, Kothe et al., 2007bKothe M. Kohls D. Low S. Coli R. Rennie G.R. Feru F. Kuhn C. Ding Y.H. Selectivity-determining residues in Plk1.Chem. Biol. Drug Des. 2007; 70: 540-546Crossref PubMed Scopus (35) Google Scholar). The development of potent and specific small-molecule Plk1 inhibitors has for the first time allowed acute inactivation of Plk1 after the metaphase to anaphase transition to probe Plk1's late mitotic functions, which have hitherto been eclipsed by its essential earlier functions. These experiments have uncovered novel roles for Plk1 in initiating cleavage furrow ingression and spindle elongation in human anaphase cells (Brennan et al., 2007Brennan I.M. Peters U. Kapoor T.M. Straight A.F. Polo-like kinase controls vertebrate spindle elongation and cytokinesis.PLoS ONE. 2007; 2: e409Crossref PubMed Scopus (56) Google Scholar, Burkard et al., 2007Burkard M.E. Randall C.L. Larochelle S. Zhang C. Shokat K.M. Fisher R.P. Jallepalli P.V. Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells.Proc. Natl. Acad. Sci. USA. 2007; 104: 4383-4388Crossref PubMed Scopus (114) Google Scholar, Petronczki et al., 2007Petronczki M. Glotzer M. Kraut N. Peters J.M. Polo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF Ect2 to the central spindle.Dev. Cell. 2007; 12: 713-725Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, Santamaria et al., 2007Santamaria A. Neef R. Eberspacher U. Eis K. Husemann M. Mumberg D. Prechtl S. Schulze V. Siemeister G. Wortmann L. et al.Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis.Mol. Biol. Cell. 2007; 18: 4024-4036Crossref PubMed Scopus (111) Google Scholar). Furthermore, these novel chemical tools have also enabled studies in which the potential suitability of Plk1 as a target for antiproliferative cancer therapy can be evaluated (Steegmaier et al., 2007Steegmaier M. Hoffmann M. Baum A. Lenart P. Petronczki M. Krssak M. Gurtler U. Garin-Chesa P. Lieb S. Quant J. et al.BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo.Curr. Biol. 2007; 17: 316-322Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, Strebhardt and Ullrich, 2006Strebhardt K. Ullrich A. Targeting polo-like kinase 1 for cancer therapy.Nat. Rev. Cancer. 2006; 6: 321-330Crossref PubMed Scopus (348) Google Scholar). In this review, we discuss these advances in basic and applied Plk1 biology and point to the many important open questions that remain to be addressed in the future. Although Plk1 can also be regulated by phosphorylation (Barr et al., 2004Barr F.A. Sillje H.H. Nigg E.A. Polo-like kinases and the orchestration of cell division.Nat. Rev. Mol. Cell Biol. 2004; 5: 429-440Crossref PubMed Scopus (503) Google Scholar, Yamashiro et al., 2008Yamashiro S. Yamakita Y. Totsukawa G. Goto H. Kaibuchi K. Ito M. Hartshorne D.J. Matsumura F. Myosin phosphatase-targeting subunit 1 regulates mitosis by antagonizing Polo-like kinase 1.Dev. Cell. 2008; 14 (this issue): 787-797Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, this issue) and protein degradation (Lindon and Pines, 2004Lindon C. Pines J. Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells.J. Cell Biol. 2004; 164: 233-241Crossref PubMed Scopus (150) Google Scholar), the most striking feature of Plk1 is its changing localization to various subcellular structures during mitotic progression. Plk1 first associates with centrosomes in prophase, then also becomes enriched at kinetochores in prometaphase and metaphase, is afterwards recruited to the central spindle in anaphase, and finally accumulates in the midbody during telophase (Figure 1B). How is Plk1 recruited to these subcellular structures and what is the functional relevance of these localizations? A first answer to these questions emerged from studies of Elia and colleagues (Elia et al., 2003aElia A.E. Cantley L.C. Yaffe M.B. Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates.Science. 2003; 299: 1228-1231Crossref PubMed Scopus (356) Google Scholar, Elia et al., 2003bElia A.E. Rellos P. Haire L.F. Chao J.W. Ivins F.J. Hoepker K. Mohammad D. Cantley L.C. Smerdon S.J. Yaffe M.B. The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain.Cell. 2003; 115: 83-95Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar) who discovered that the previously recognized conserved sequence elements in the C-terminal portion of Plk1 function as a phosphopeptide binding domain, referred to as the Polo-box domain (PBD) (Elia et al., 2003aElia A.E. Cantley L.C. Yaffe M.B. Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates.Science. 2003; 299: 1228-1231Crossref PubMed Scopus (356) Google Scholar) (Figure 1A). This discovery suggested that the PBD might have an important role in directing Plk1 to substrates. Thereby, the PBD might contribute to substrate specificity and possibly subcellular localization of Plk1. In addition, the observation that the PBD preferentially binds to phosphorylated peptides implied that another kinase first has to "prime" the site before PBD binding. This suggested a mechanism that can efficiently target Plk1 spatially and temporally—timed relative to previous phosphorylation events (Lowery et al., 2004Lowery D.M. Mohammad D.H. Elia A.E. Yaffe M.B. The Polo-box domain: a molecular integrator of mitotic kinase cascades and Polo-like kinase function.Cell Cycle. 2004; 3: 128-131Crossref PubMed Google Scholar, Lowery et al., 2005Lowery D.M. Lim D. Yaffe M.B. Structure and function of Polo-like kinases.Oncogene. 2005; 24: 248-259Crossref PubMed Scopus (140) Google Scholar). These findings also provided an explanation for earlier studies indicating that the PBD is necessary and sufficient for proper localization of Plk1 to centrosomes, kinetochores, and the midbody, implying that Plk1 is recruited to different subcellular sites via direct physical interaction with phosphorylated proteins (Lee et al., 1998Lee K.S. Grenfell T.Z. Yarm F.R. Erikson R.L. Mutation of the polo-box disrupts localization and mitotic functions of the mammalian polo kinase Plk.Proc. Natl. Acad. Sci. USA. 1998; 95: 9301-9306Crossref PubMed Scopus (147) Google Scholar, Seong et al., 2002Seong Y.S. Kamijo K. Lee J.S. Fernandez E. Kuriyama R. Miki T. Lee K.S. A spindle checkpoint arrest and a cytokinesis failure by the dominant-negative polo-box domain of Plk1 in U-2 OS cells.J. Biol. Chem. 2002; 277: 32282-32293Crossref PubMed Scopus (136) Google Scholar). Many phosphoproteins with which Plk1 can interact may themselves be substrates of Plk1, but it is also conceivable that phosphorylated docking proteins exist which help to increase local Plk1 concentrations without being direct targets of Plk1 phosphorylation. Using the PBD as bait, several hundred candidate interaction partners of Plk1 have been identified that bind to the PBD in a phosphorylation-dependent manner (Lowery et al., 2007Lowery D.M. Clauser K.R. Hjerrild M. Lim D. Alexander J. Kishi K. Ong S.E. Gammeltoft S. Carr S.A. Yaffe M.B. Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate.EMBO J. 2007; 26: 2262-2273Crossref PubMed Scopus (124) Google Scholar). Here we discuss those interactors that have been characterized in more detail and highlight the new principles of regulation that emerge from these studies. The original identification of Drosophila Polo as a gene whose mutation causes defective centrosomes and monopolar spindles (Sunkel and Glover, 1988Sunkel C.E. Glover D.M. polo, a mitotic mutant of Drosophila displaying abnormal spindle poles.J. Cell Sci. 1988; 89: 25-38Crossref PubMed Google Scholar) and the localization of Plk1 at centrosomes (Golsteyn et al., 1995Golsteyn 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; 129: 1617-1628Crossref PubMed Scopus (285) Google Scholar) suggested that this kinase has important functions at centrosomes and thus in the assembly of bipolar spindles. One of these functions is the recruitment of γ-tubulin complexes to centrosomes during prophase (Lane and Nigg, 1996Lane 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; 135: 1701-1713Crossref PubMed Scopus (435) Google Scholar). This process, referred to as centrosome maturation, is essential for microtubule nucleation from mitotic centrosomes. The mechanism whereby Plk1 facilitates centrosome maturation is not fully understood, but it has been postulated that Plk1 can contribute to centrosome maturation by phosphorylating the centrosomal protein Nlp. This modification leads to Nlp dissociation from centrosomes during mitosis and may facilitate recruitment of γ-tubulin complexes (Casenghi et al., 2003Casenghi M. Meraldi P. Weinhart U. Duncan P.I. Korner R. Nigg E.A. Polo-like kinase 1 regulates Nlp, a centrosome protein involved in microtubule nucleation.Dev. Cell. 2003; 5: 113-125Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, Casenghi et al., 2005Casenghi M. Barr F.A. Nigg E.A. Phosphorylation of Nlp by Plk1 negatively regulates its dynein-dynactin-dependent targeting to the centrosome.J. Cell Sci. 2005; 118: 5101-5108Crossref PubMed Scopus (43) Google Scholar). In addition to Nlp, a recently identified Plk1 substrate named Kizuna has also been shown to be required for formation of functional mitotic centrosomes (Oshimori et al., 2006Oshimori N. Ohsugi M. Yamamoto T. The Plk1 target Kizuna stabilizes mitotic centrosomes to ensure spindle bipolarity.Nat. Cell Biol. 2006; 8: 1095-1101Crossref PubMed Scopus (62) Google Scholar). If Kizuna is depleted or cannot be phosphorylated by Plk1, centrosomes loose their mechanical stability and therefore are fragmented by the forces generated by spindle microtubules (Oshimori et al., 2006Oshimori N. Ohsugi M. Yamamoto T. The Plk1 target Kizuna stabilizes mitotic centrosomes to ensure spindle bipolarity.Nat. Cell Biol. 2006; 8: 1095-1101Crossref PubMed Scopus (62) Google Scholar). Drosophila Polo has also been shown to phosphorylate the conserved microtubule minus end-binding protein Asp to facilitate nucleation of microtubules by mitotic centrosomes (do Carmo Avides et al., 2001do Carmo Avides M. Tavares A. Glover D.M. Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes.Nat. Cell Biol. 2001; 3: 421-424Crossref PubMed Scopus (97) Google Scholar). Inactivation of Plk1 by addition of small-molecule inhibitors before and after the assembly bipolar spindles has revealed that Plk1 is not only required for the establishment of a bipolar spindle and functional centrosomes, but also for their maintenance (D'Avino et al., 2006D'Avino P.P. Savoian M.S. Capalbo L. Glover D.M. RacGAP50C is sufficient to signal cleavage furrow formation during cytokinesis.J. Cell Sci. 2006; 119: 4402-4408Crossref PubMed Scopus (37) Google Scholar, Lenart et al., 2007Lenart P. Petronczki M. Steegmaier M. Di Fiore B. Lipp J.J. Hoffmann M. Rettig W.J. Kraut N. Peters J.M. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.Curr. Biol. 2007; 17: 304-315Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, McInnes et al., 2006McInnes C. Mazumdar A. Mezna M. Meades C. Midgley C. Scaerou F. Carpenter L. Mackenzie M. Taylor P. Walkinshaw M. et al.Inhibitors of Polo-like kinase reveal roles in spindle-pole maintenance.Nat. Chem. Biol. 2006; 2: 608-617Crossref PubMed Scopus (61) Google Scholar, Peters et al., 2006Peters U. Cherian J. Kim J.H. Kwok B.H. Kapoor T.M. Probing cell-division phenotype space and Polo-like kinase function using small molecules.Nat. Chem. Biol. 2006; 2: 618-626Crossref PubMed Scopus (66) Google Scholar, Santamaria et al., 2007Santamaria A. Neef R. Eberspacher U. Eis K. Husemann M. Mumberg D. Prechtl S. Schulze V. Siemeister G. Wortmann L. et al.Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis.Mol. Biol. Cell. 2007; 18: 4024-4036Crossref PubMed Scopus (111) Google Scholar). However, centrosomes function normally even if the centrosomal localization of Plk1 is disrupted by overexpression of the PBD domain or by expressing a version of Plk1 without PBD (Hanisch et al., 2006Hanisch A. Wehner A. Nigg E.A. Sillje H.H. Different Plk1 functions show distinct dependencies on Polo-Box domain-mediated targeting.Mol. Biol. Cell. 2006; 17: 448-459Crossref PubMed Scopus (72) Google Scholar). One explanation for this observation is that Plk1's centrosomal substrates may not be stably bound and therefore constantly exchange with the soluble cytoplasmic pool that could be sufficient to mediate the phosphorylation reaction. Alternatively, cytoplasmic Plk1 might be capable to modify stably bound centrosomal factors. Although Plk1's PBD docking interactions appear to be dispensable for centrosome maturation, centrosomal recruitment of Plk1 might play an important role in controlling the timing of mitotic entry. Elia and colleagues chose the protein phosphatase Cdc25C for the initial testing of their hypothesis that Plk1 preferentially recognizes "primed" substrates because Cdc25C had been shown to be activated following its phosphorylation by Plk1 in Xenopus oocytes (Kumagai and Dunphy, 1996Kumagai A. Dunphy W.G. Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts.Science. 1996; 273: 1377-1380Crossref PubMed Google Scholar, Qian et al., 1998Qian Y.W. Erikson E. Li C. Maller J.L. Activated polo-like kinase Plx1 is required at multiple points during mitosis in Xenopus laevis.Mol. Cell. Biol. 1998; 18: 4262-4271Crossref PubMed Google Scholar). Elia et al. showed that the PBD is required for efficient binding of Plk1 to Cdc25C and that this interaction is specific to the mitotic form of Cdc25C that has been prephosphorylated by Cdk1 (Elia et al., 2003aElia A.E. Cantley L.C. Yaffe M.B. Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates.Science. 2003; 299: 1228-1231Crossref PubMed Scopus (356) Google Scholar). Because Cdc25C is required for Cdk1-cyclin B1 activation, which is thought to occur during prophase at centrosomes (Jackman et al., 2003Jackman M. Lindon C. Nigg E.A. Pines J. Active cyclin B1-Cdk1 first appears on centrosomes in prophase.Nat. Cell Biol. 2003; 5: 143-148Crossref PubMed Scopus (309) Google Scholar), Cdc25C may be an important centrosomal substrate of Plk1. Cyclin B1 itself is also phosphorylated by Plk1 in prophase at centrosomes, and this modification is thought to contribute to Cdk1-cyclin B1 activation as well (Jackman et al., 2003Jackman M. Lindon C. Nigg E.A. Pines J. Active cyclin B1-Cdk1 first appears on centrosomes in prophase.Nat. Cell Biol. 2003; 5: 143-148Crossref PubMed Scopus (309) Google Scholar, Toyoshima-Morimoto et al., 2001Toyoshima-Morimoto F. Taniguchi E. Shinya N. Iwamatsu A. Nishida E. Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase.Nature. 2001; 410: 215-220Crossref PubMed Scopus (230) Google Scholar). The recruitment of Plk1 to centrosomes may thus promote mitotic entry, a function likely conserved from yeast (Mulvihill et al., 1999Mulvihill D.P. Petersen J. Ohkura H. Glover D.M. Hagan I.M. Plo1 kinase recruitment to the spindle pole body and its role in cell division in Schizosaccharomyces pombe.Mol. Biol. Cell. 1999; 10: 2771-2785Crossref PubMed Google Scholar) to vertebrates. Several observations indicate, however, that neither Plk1 nor its accumulation at centrosomes is essential for entry into mitosis. If Plk1 has been mislocalized (Hanisch et al., 2006Hanisch A. Wehner A. Nigg E.A. Sillje H.H. Different Plk1 functions show distinct dependencies on Polo-Box domain-mediated targeting.Mol. Biol. Cell. 2006; 17: 448-459Crossref PubMed Scopus (72) Google Scholar), and even if Plk1 has been pharmacologically inhibited (Lenart et al., 2007Lenart P. Petronczki M. Steegmaier M. Di Fiore B. Lipp J.J. Hoffmann M. Rettig W.J. Kraut N. Peters J.M. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.Curr. Biol. 2007; 17: 304-315Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar), mammalian cultured cells can still enter mitosis. These cells are delayed for hours in late prophase (Lenart et al., 2007Lenart P. Petronczki M. Steegmaier M. Di Fiore B. Lipp J.J. Hoffmann M. Rettig W.J. Kraut N. Peters J.M. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.Curr. Biol. 2007; 17: 304-315Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar), at the time when Cdk1 activation is believed to occur at centrosomes, but eventually nuclear envelope breakdown occurs in these cells. Activation of Cdk1 may therefore also be possible in the absence of Plk1, although after a long delay. Whereas Plk1 is not essential for mitotic entry in unperturbed divisions, recovery from a DNA damage checkpoint-induced arrest at the G2/M boundary and restart of the cell cycle requires Plk1 activity in both yeast and human cells (Toczyski et al., 1997Toczyski D.P. Galgoczy D.J. Hartwell L.H. CDC5 and CKII control adaptation to the yeast DNA damage checkpoint.Cell. 1997; 90: 1097-1106Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, van Vugt et al., 2004avan Vugt M.A. Bras A. Medema R.H. Polo-like kinase-1 controls recovery from a G2 DNA damage-induced arrest in mammalian cells.Mol. Cell. 2004; 15: 799-811Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). A third group of proteins that are enriched at centrosomes and known to be substrates of Plk1 are the APC/C and its inhibitor Emi1. During S and G2 phase, Emi1 helps to inhibit the ubiquitin ligase activity of the APC/C, which would otherwise precociously target mitotic cyclins and other proteins for destruction by the 26S proteasome. In late prophase, Plk1 phosphorylates Emi1 and thereby allows its recognition by the ubquitin ligase SCFβTrcp1, which mediates degradation of Emi1 (Hansen et al., 2004Hansen D.V. Loktev A.V. Ban K.H. Jackson P.K. Plk1 regulates activation of the anaphase promoting complex by phosphorylating and triggering SCFbetaTrCP-dependent destruction of the APC Inhibitor Emi1.Mol. Biol. Cell. 2004; 15: 5623-5634Crossref PubMed Scopus (125) Google Scholar, Moshe et al., 2004Moshe Y. Boulaire J. Pagano M. Hershko A. Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase promoting complex/cyclosome.Proc. Natl. Acad. Sci. USA. 2004; 101: 7937-7942Crossref PubMed Scopus (125) Google Scholar). Presumably around the same time, Plk1 contributes to phosphorylation of the APC/C (Golan et al., 2002Golan A. Yudkovsky Y. Hershko A. The cyclin-ubiquitin ligase activity of cyclosome/APC is jointly activated by protein kinases Cdk1-cyclin B and Plk.J. Biol. Chem. 2002; 277: 15552-15557Crossref PubMed Scopus (115) Google Scholar, Kraft et al., 2003Kraft C. Herzog F. Gieffers C. Mechtler K. Hagting A. Pines J. Peters J.M. Mitotic regulation of the human anaphase-promoting complex by phosphorylation.EMBO J. 2003; 22: 6598-6609Crossref PubMed Scopus (237) Google Scholar), which is one of several prerequisites for APC/C's activation in mitosis. 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