Polo-like Kinase-1 Is Required for Bipolar Spindle Formation but Is Dispensable for Anaphase Promoting Complex/Cdc20 Activation and Initiation of Cytokinesis

Abstract

Polo-like kinase-1 (Plk1) performs multiple essential functions during the cell cycle. Here we show that human Plk1-deficient cells are unable to separate their centrosomes, fail to form a bipolar spindle, and undergo a Mad2/BubR1-dependent prometaphase arrest. However, electron microscopy demonstrates that kinetochore-microtubule interactions can be established in cells lacking Plk1. In addition, co-depletion of Plk1 and survivin allows mitotic exit. This indicates that Plk1 depletion does not prevent microtubule attachment, but specifically interferes with the generation of tension, as a consequence of a failure to form a bipolar spindle. Moreover, we find that after silencing of the spindle assembly checkpoint, degradation of cyclin B1 is unaffected in cells lacking Plk1. These data indicate that activation of the anaphase promoting complex or cyclosome (APC/C)-Cdc20 complex that is under control of the spindle assembly checkpoint does not require Plk1 activity. Finally, we find that translocation of chromosome passengers and initiation of cleavage furrow ingression is unaffected in cells depleted of Plk1. Thus, our data confirm an important role of Plk1 in bipolar spindle formation, and also demonstrate that Plk1 is dispensable for APC/C-Cdc20 activation and the initiation of cytokinesis. Polo-like kinase-1 (Plk1) performs multiple essential functions during the cell cycle. Here we show that human Plk1-deficient cells are unable to separate their centrosomes, fail to form a bipolar spindle, and undergo a Mad2/BubR1-dependent prometaphase arrest. However, electron microscopy demonstrates that kinetochore-microtubule interactions can be established in cells lacking Plk1. In addition, co-depletion of Plk1 and survivin allows mitotic exit. This indicates that Plk1 depletion does not prevent microtubule attachment, but specifically interferes with the generation of tension, as a consequence of a failure to form a bipolar spindle. Moreover, we find that after silencing of the spindle assembly checkpoint, degradation of cyclin B1 is unaffected in cells lacking Plk1. These data indicate that activation of the anaphase promoting complex or cyclosome (APC/C)-Cdc20 complex that is under control of the spindle assembly checkpoint does not require Plk1 activity. Finally, we find that translocation of chromosome passengers and initiation of cleavage furrow ingression is unaffected in cells depleted of Plk1. Thus, our data confirm an important role of Plk1 in bipolar spindle formation, and also demonstrate that Plk1 is dispensable for APC/C-Cdc20 activation and the initiation of cytokinesis. Polo-like kinases have been associated with many aspects of mitosis (reviewed in Refs. 1Donaldson M.M. Tavares A.A. Hagan I.M. Nigg E.A. Glover D.M. J. Cell Sci. 2001; 114: 2357-2358Crossref PubMed Google Scholar, 2Glover D.M. Ohkura H. Tavares A. J. Cell Biol. 1996; 135: 1681-1684Crossref PubMed Scopus (93) Google Scholar, 3Glover D.M. Hagan I.M. Tavares A.A. Genes Dev. 1998; 12: 3777-3787Crossref PubMed Scopus (393) Google Scholar, 4Nigg E.A. Curr. Opin. Cell Biol. 1998; 10: 776-783Crossref PubMed Scopus (308) Google Scholar). Originally identified as the polo gene in Drosophila, Polo-like kinases were shown to be required for proper mitotic progression in various species. Whereas a single Polo-like kinase has been identified in Saccharomyces cerevisiae (Cdc5), Schizosaccharomyces pombe (Plo1), and Drosophila melanogaster (Polo), mammalian cells appear to express up to four distinct Polo-like kinases (Plk1, Plk2 or SNK, Plk3 or Prk/Fnk, and SAK) (4Nigg E.A. Curr. Opin. Cell Biol. 1998; 10: 776-783Crossref PubMed Scopus (308) Google Scholar). Of these different kinases, the function of Plk1 is most extensively studied, and Plk1 appears to play multiple important roles during mitosis (1Donaldson M.M. Tavares A.A. Hagan I.M. Nigg E.A. Glover D.M. J. Cell Sci. 2001; 114: 2357-2358Crossref PubMed Google Scholar). At the G2-M transition, a role for Polo-like kinases was suggested in activation of the cyclin B1-Cdk1 complex. Experiments in Xenopus extracts showed that the Polo orthologue Plx1 is able to phosphorylate and activate the Cdc25C phosphatase (5Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (468) Google Scholar, 6Qian Y.W. Erikson E. Taieb F.E. Maller J.L. Mol. Biol. Cell. 2001; 12: 1791-1799Crossref PubMed Scopus (141) Google Scholar). Cdc25C in turn activates the Cdk1 subunit of the cyclin B1-Cdk1 complex by dephosphorylation (reviewed in Ref. 7Smits V.A. Medema R.H. Biochim. Biophys. Acta. 2001; 1519: 1-12Crossref PubMed Scopus (302) Google Scholar). Cyclin B1-Cdk1 complexes can phosphorylate and further activate Cdc25C (8Hoffmann I. Clarke P.R. Marcote M.J. Karsenti E. Draetta G. EMBO J. 1993; 12: 53-63Crossref PubMed Scopus (560) Google Scholar, 9Izumi T. Maller J.L. Mol. Biol. Cell. 1993; 4: 1337-1350Crossref PubMed Scopus (251) Google Scholar), resulting in a positive feedback loop that facilitates rapid entry into mitosis. In addition, active Plk1 was shown to induce translocation of both Cdc25C and cyclin B1 to the nucleus (10Toyoshima-Morimoto F. Taniguchi E. Shinya N. Iwamatsu A. Nishida E. Nature. 2001; 410: 215-220Crossref PubMed Scopus (309) Google Scholar, 11Toyoshima-Morimoto F. Taniguchi E. Nishida E. EMBO Rep. 2002; 3: 341-348Crossref PubMed Scopus (267) Google Scholar). More recent work, however, indicates that Plk1-mediated phosphorylation does not cause cyclin B1 to move into the nucleus, but suggests it rather contributes to activation of cyclin B1-Cdk complexes at the centrosomes (12Jackman M. Lindon C. Nigg E.A. Pines J. Nat. Cell Biol. 2003; 5: 143-148Crossref PubMed Scopus (487) Google Scholar). During the early stages of mitosis, Polo-like kinases were shown to mediate the displacement of the chromosome by the subunit A. M. 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PubMed Scopus Google Scholar). of but also of cyclin and cyclin of with the subunit Cell PubMed Scopus Google Scholar). in mitosis, of the with the is required for degradation of mitotic as and In the of Plk1 was suggested to play a Plk1 has been shown to phosphorylate of the in and M. N. C. Mol. Cell. 1998; PubMed Scopus Google Scholar, A. A. J. Biol. 2002; PubMed Scopus Google Scholar). In addition, depletion of Plx1 Xenopus extracts was shown to degradation of cyclin Nigg E.A. EMBO J. 1998; PubMed Scopus Google Scholar). However, cyclin B1-Cdk1 or Plk1 as the as in at cyclin B1-Cdk1 as the in J. Cell Biol. PubMed Scopus Google Scholar). Polo-like kinases also essential roles during the stages of mitosis. In of the gene to failure in formation, a that is to in mammalian cells H. Hagan I.M. Glover D.M. Genes Dev. PubMed Scopus Google Scholar). of in failure to form an as as of of in the Polo-like kinase has been shown to activation of the mitotic by of the phosphatase the F. J. Cell. 2001; PubMed Scopus Google Scholar, F. A. Cell. 2002; PubMed Scopus Google Scholar). of the activation of the of the and to the of mitotic a for mitotic and A. Nat. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). role for Polo kinases in in mammalian cells was also in of a of Plk1 was to with the function of Plk1 E. T. J. Biol. 2002; PubMed Scopus Google Scholar). of the spindle assembly checkpoint, of not and in of In addition, recent suggested that phosphorylation of and by Plk1 is required for proper N. A. Mol. Cell. PubMed Scopus Google Scholar, C. J. Nigg E.A. J. Cell Biol. 2003; PubMed Scopus Google Scholar). Finally, roles for Plk1 in the and of the mitotic spindle have been in a of (reviewed in Refs. 1Donaldson M.M. Tavares A.A. Hagan I.M. Nigg E.A. Glover D.M. J. Cell Sci. 2001; 114: 2357-2358Crossref PubMed Google Scholar, 2Glover D.M. Ohkura H. Tavares A. J. Cell Biol. 1996; 135: 1681-1684Crossref PubMed Scopus (93) Google Scholar, 3Glover D.M. Hagan I.M. Tavares A.A. Genes Dev. 1998; 12: 3777-3787Crossref PubMed Scopus (393) Google Scholar, 4Nigg E.A. Curr. Opin. Cell Biol. 1998; 10: 776-783Crossref PubMed Scopus (308) Google Scholar). In Drosophila, Polo was shown to phosphorylate a that to the centrosomes and is required to microtubule C. Glover D.M. 1998; PubMed Scopus Google Scholar, M. Tavares A. Glover D.M. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). in Polo in the of mitotic and A. Tavares A. C. C. Glover D.M. Genes Dev. 5: PubMed Scopus Google Scholar, Glover D.M. J. Cell Sci. PubMed Google Scholar). In of and in a mitotic Nigg E.A. J. Cell Biol. 1996; 135: PubMed Scopus Google Scholar). were in Xenopus cells that not require activation of Cdc25C for mitotic entry were shown to in mitosis with Plx1 was depleted Y.W. Erikson E. C. Maller J.L. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). More with Plk1 was shown to the of centrosomes to in a Erikson Sci. A. 2002; PubMed Scopus Google Scholar). At it is the multiple functions of Plk1 are in and to progression the different stages of mitosis. In addition, it is not of the cell are a consequence of with Plk1 or as a of a spindle or entry into mitosis. the functions of Plk1 during of human we the roles of Plk1 by were as Science. 2002; PubMed Scopus Google Scholar). and and were a in the of human Plk1 or was by G. of Cyclin was by J. Pines and N. Pines J. J. Cell Biol. 2001; PubMed Scopus Google Scholar). was by J. in the of Plk1 were by the indicate J. T. G. Medema R.H. EMBO J. 2003; PubMed Scopus Google Scholar), V.A. G. Nigg E.A. Medema R.H. Nat. Cell Biol. PubMed Scopus Google Scholar), T. Curr. Biol. 1998; PubMed Scopus Google Scholar), T. PubMed Scopus Google Scholar), and E. Science. 1993; PubMed Scopus Google have been is J. H. and M. A. in and and were was was was and were was was was a of of and has been J. Cell Sci. 2001; 114: PubMed Google Scholar). was and were was was were of in with of or At after cells were was with and kinase were as V.A. G. Nigg E.A. Medema R.H. Nat. Cell Biol. PubMed Scopus Google Scholar). with showed that of Plk1. cyclin kinase cells were with of or a of as of was in cells were in for in the of after cells were cyclin B1 was and kinase were as V.A. G. Nigg E.A. Medema R.H. Nat. Cell Biol. PubMed Scopus Google Scholar). Cell and were in and cells in with and cell at the transition, cells were with for was after cells at the spindle assembly checkpoint, was after the cells were by at after cells were at after were and were by the cells were by a after the were in for after were the and as and At different after cells were and by or cells were by or of of was with of or after cells were by and of cells were and with for At cells were in and were with were and the with or in with cells were by microscopy at after More of the cells were in at as in were to a of a with a and a to and to were a at and a or to were of cells was as V.A. G. Nigg E.A. Medema R.H. Nat. Cell Biol. PubMed Scopus Google Scholar). of cells were at the after a and in were with and with were with a and with Cell of the cells was as V.A. G. Nigg E.A. Medema R.H. Nat. Cell Biol. PubMed Scopus Google Scholar). were with of or a cells were with after mitotic cells were by in in and for and for with were for with with and in were with a electron were by in for were the and At the after the were the and with were for in at and with for and were in with for in were in for by in of of or was to associated with centrosomes by were and with to at a cells were and showed as and cells were in to were a with a Plk1 for to specifically Plk1 human we allows of in cells Science. 2002; PubMed Scopus Google Scholar). of our Plk1 with an Plk1 in human cells in a of This was further by in kinase in the Plk1 was specifically to the of we the in Plk1 by of to be of Plk1 was to of we cells after of a This that the of Plk1 the for Plk1 for human cell we cell specifically the that cells with a that these cells in or in mitosis is shown in at after of cells in mitosis, as by a in This is of the of mitotic cells we after a of cells with that a of the cells in mitosis. were we the of established mitotic Sci. A. PubMed Scopus Google Scholar), or mitotic by microscopy of cells not that the is we Plk1 into we in the of the of Plk1 that is by the Plk1 of a of Plk1 is unaffected by that of Plk1 can be to of Plk1 in cells depleted of Plk1. of the mitotic that was with In of a Plk1 not the mitotic that the kinase of Plk1 is for mitotic of Plk1 in cells to a of formation, that Plk1 has an essential role in cell that the of cell that Plk1 These are in with the of cell after Plk1 depletion G. C. M. G. M. 2002; PubMed Scopus Google Scholar). Cell after Plk1 has been suggested to play an important role in the activation of Cdc25C and in the translocation of both cyclin B1 as as This that Plk1 is essential for mitotic cell progression of the cells in a cell were in for to at the transition, after were and as the cell cycle. Whereas control to the at after cells to the cell and with the of Plk1 cells in mitosis of the of mitotic entry showed that the of cells depleted of Plk1 mitosis with as control that Plk1 does not play an essential role in mitotic M. A. T. and H. Mol. in the most cell after Plk1 depletion during mitosis. in of microscopy to at cells in mitosis depletion of Plk1. cells were with or in with a to chromosome for Plk1 to their and in a prometaphase of cells were in cells failure to the in cells be the of a in mitotic spindle formation, as has been in Polo in Drosophila and in cells with of in cells showed that a of the cells were unable to form a bipolar spindle, in to cells Moreover, form in the but showed as we microtubule the in cells In addition, for showed that cells centrosomes that are as separate However, the centrosomes move to of the cell in control we find a of cells with centrosomes that have associated entry into mitosis and in mitotic cells lacking centrosomes to with the microtubule Thus, centrosomes function as the in cells lacking Plk1. of was the centrosomes in mitotic with centrosomes in control that of centrosomes and is these show Plk1 is essential for the of a bipolar spindle, most of in and of the in Plk1-deficient of Plk1 interferes with of a bipolar spindle, the prometaphase be the of activation of the spindle assembly confirm we the of spindle assembly checkpoint and in Plk1-deficient in and were in but both were as as cells and into anaphase depletion of however, to and and were of a mitotic that is by the spindle assembly an microtubule appears to form the in Plk1-deficient cells is a of cells for This suggests that these to form to of and were in cells with cells for the of the resulting in the of the cells to in prometaphase Science. PubMed Scopus Google Scholar). Thus, it is that the mitotic in cells is of a failure to a bipolar spindle, rather a consequence of an essential function of Plk1 during mitosis. that the spindle assembly checkpoint the mitotic after Plk1 and to Plk1 depletion stages of mitosis, we checkpoint function by of or we have shown that of our and a of spindle assembly checkpoint function J. T. G. Medema R.H. 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EMBO J. 2003; PubMed Scopus Google Scholar), cyclin was in mitotic cells depleted of that the activation of the APC/C-Cdc20 in prometaphase is not by depletion of Plk1 In addition, degradation of showed that both initiation and of are In these the of in cells showed that Plk1 depletion was in the cells for cyclin by was in mitotic cell a of the that in Plk1 play an role in we the degradation of the APC/C-Cdc20 in cells depleted of Plk1 are in cells depleted of the active spindle assembly checkpoint However, in cells in we the spindle assembly checkpoint by co-depletion of was to with control cells also cyclin kinase in cells depleted of Plk1 but was in cells after spindle assembly checkpoint silencing by co-depletion of or of these cells with that spindle assembly checkpoint silencing does not with of cyclin B1-Cdk1 and not These show in the of APC/C-Cdc20 is the spindle assembly checkpoint is and that to proper degradation of and cyclin in silencing not cell progression in Plk1-deficient as these cells with a This suggests mitotic is in Plk1-deficient cells lacking a spindle assembly checkpoint, these cells fail to undergo cell with suggested that mammalian Plk1 an important role during E. T. J. Biol. 2002; PubMed Scopus Google Scholar), to the role for in H. Hagan I.M. Glover D.M. Genes Dev. PubMed Scopus Google Scholar, J. F. Glover D.M. Hagan I.M. EMBO J. 2001; PubMed Scopus Google Scholar). However, it is also that the in as a consequence of a failure to form a bipolar spindle. a we with with in an in to the we after Plk1 depletion we the spindle assembly checkpoint in cells by of or mitotic were and prometaphase was cells as as the to into cells the spindle assembly checkpoint was as by these not with Plk1 not the that the with are the of with the function of Plk1. Thus, spindle assembly checkpoint silencing in both and cells does mitotic but to a of cells at in a we Whereas suggested in or cells after spindle assembly checkpoint and we cleavage furrow ingression in both but of the with of the up at of the furrow of cells of chromosome was after Plk1 and anaphase of was that were to a spindle and in the of furrow as as furrow ingression were both in the and of Plk1 and the cells to as by chromosome and ingression were in of cells that the of Plk1 depletion mitotic progression are in the of a failure to form a bipolar spindle. Thus, our data indicate that Plk1 is not required for the initiation of in human In the of however, in a as a of spindle further the of cells to undergo the of chromosome was are at the At to the spindle play a role in cell cleavage M. Cell Biol. 2001; PubMed Scopus Google Scholar). of showed in mitotic as in control mitotic cells and data not In control to the during assembly checkpoint silencing in as as cells also in to to the nucleus we in further by to prometaphase of and as as cells and During anaphase to the spindle in control cells and after spindle assembly checkpoint also to the of cleavage furrow and to the to the of and cells of and of cells and and These and with not show that translocation of to is of Plk1. In addition, these show that formation, a that cleavage furrow of Plk1. Here we to show the of Plk1 in different in mitosis. of we showed that the depletion was In addition, cells showed of Plk1 of Plk1 that were not by showed of the mitotic that the are for Plk1 Plk1-deficient cells in a mitotic was This essential role for Polo-like kinases in mitotic progression in Drosophila A. Tavares A. C. C. Glover D.M. Genes Dev. 5: PubMed Scopus Google Scholar, Glover D.M. J. Cell Sci. PubMed Google Scholar), H. Hagan I.M. Glover D.M. Genes Dev. PubMed Scopus Google Scholar), and Xenopus Y.W. Erikson E. C. Maller J.L. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). Moreover, these confirm the mitotic that was after of in cells Nigg E.A. J. Cell Biol. 1996; 135: PubMed Scopus Google or a Plk1 in cells E. T. J. Biol. 2002; PubMed Scopus Google Scholar). showed that kinase of Plk1 is required to role in mitosis, of the mitotic was not a was different have shown a role for Polo-like kinases in mitotic entry (5Kumagai A. Dunphy W.G. Science. 1996; 273: 1377-1380Crossref PubMed Scopus (468) Google Scholar, Y.W. Erikson E. C. Maller J.L. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). data indicate that Plk1 is dispensable for mitotic entry in human as we find that the of cells depleted of Plk1 mitosis with cells to their and to a bipolar spindle entry into mitosis. showed that is most by a in with that Polo-like kinases play an important role in the E. T. J. 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Thus, our data that depletion of Plk1 spindle assembly checkpoint by the generation of tension, rather with microtubule electron microscopy showed the of interactions in assembly checkpoint silencing by of or the mitotic after Plk1 cell however, was not and most cells This suggested that in spindle assembly checkpoint function is an cell were after of Plk1 and E. T. J. Biol. 2002; PubMed Scopus Google Scholar), to the that Plk1 a role during cytokinesis. However, we that the in cell also as an consequence of a failure to form a bipolar spindle. we show that cleavage furrow as as furrow ingression in that Plk1 is not required for these aspects of cytokinesis. This is with recent that bipolar spindle is not essential for furrow and ingression A. G. Nature. 2003; PubMed Scopus Google Scholar). Moreover, we were able to show that to be required for to the during furrow ingression in cells depleted of Plk1 and these that Plk1 is not required for translocation of to the and after spindle assembly checkpoint these show that the furrow ingression in cells depleted of Plk1 with the of at the of cell as in cells and shown to be required for M. Cell Biol. 2001; PubMed Scopus Google Scholar). Thus, the cell of cells appears to be an of the of a bipolar spindle, and these data be as for a role for Plk1 in cytokinesis. is important to however, that depletion of Plk1 of Plk1 that are to cytokinesis. However, our that a role for Plk1 in were to cells Plk1 depletion of a spindle. Thus, we a role for of Plk1 in the initiation of our data demonstrate that Plk1 is depleted a required for proper spindle formation, initiation of is not Experiments with show that of Plk1 in mitotic cells with cyclin B1-Cdk1 Erikson Sci. A. 2002; PubMed Scopus Google Scholar). was suggested that was a of of However, our data show that the in mitosis and the cyclin kinase are of a spindle that to a spindle assembly arrest. of a role for Polo-like kinases in M. N. C. Mol. Cell. 1998; PubMed Scopus Google Scholar, A. A. J. Biol. 2002; PubMed Scopus Google Scholar, Nigg E.A. EMBO J. 1998; PubMed Scopus Google Scholar). of Plk1 in in human we a of the is not by the spindle assembly we of cyclin to of APC/C-Cdc20 activity. Whereas cells cells More initiation and of APC/C-Cdc20 activation were in both that APC/C-Cdc20 activation is in with recent of C. F. C. A. Pines J. EMBO J. 2003; PubMed Scopus Google Scholar). these to a separate of under control of the spindle assembly checkpoint, we checkpoint function by for or These showed that cyclin B1 and can be in the of that Plk1 is not required for activation of under these This that the Xenopus Plk1 was depleted Xenopus This in a in cyclin degradation mitotic was by the of Nigg E.A. EMBO J. 1998; PubMed Scopus Google Scholar). for for in of is not a role for Polo-like kinases in activation of the during Maller J.L. Genes Dev. 2003; PubMed Scopus Google Scholar). Moreover, the be in a different the in Xenopus extracts not a subunit of the and in these extracts of mitotic APC/C-Cdc20 In we data that show that Plk1 is required for proper of a bipolar spindle but not for or the initiation of in human data also show that of the of cells for Plk1 as a consequence of a failure to form a bipolar spindle. of cyclin B1-Cdk1 is a of we show that degradation of cyclin B1 does not require Plk1 in cells with an spindle assembly Thus, our data show that Plk1 does multiple roles mitosis, but not as many as can the T. G. E. J. and J. for and and for with also the of the of Medema for with