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• W2777334852 abstract "•Eg5 localization and centrosome separation in prophase depend on TPX2•Eg5[Ser1033] phosphorylation downstream of Nek9 controls Eg5 binding to TPX2•TPX2 localizes to the centrosomes before NEBD in a RHAMM- and Nek9-dependent manner•Nek9 phosphorylation of TPX2 NLS prevents importin interaction and nuclear import Centrosomes [1Conduit P.T. Wainman A. Raff J.W. Centrosome function and assembly in animal cells.Nat. Rev. Mol. Cell Biol. 2015; 16: 611-624Crossref PubMed Scopus (318) Google Scholar, 2Fu J. Hagan I.M. Glover D.M. The centrosome and its duplication cycle.Cold Spring Harb. Perspect. Biol. 2015; 7: a015800Crossref PubMed Scopus (143) Google Scholar] play a central role during spindle assembly in most animal cells [3Meraldi P. Centrosomes in spindle organization and chromosome segregation: A mechanistic view.Chromosome Res. 2016; 24: 19-34Crossref PubMed Scopus (35) Google Scholar]. In early mitosis, they organize two symmetrical microtubule arrays that upon separation define the two poles of the forming spindle. Centrosome separation is tightly regulated [4Agircan F.G. Schiebel E. Mardin B.R. Separate to operate: Control of centrosome positioning and separation.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2014; 369: 20130461Crossref PubMed Scopus (70) Google Scholar, 5van Ree J.H. Nam H.-J. van Deursen J.M. Mitotic kinase cascades orchestrating timely disjunction and movement of centrosomes maintain chromosomal stability and prevent cancer.Chromosome Res. 2016; 24: 67-76Crossref PubMed Scopus (14) Google Scholar], occurring through partially redundant mechanisms that rely on the action of microtubule-based dynein and kinesin motors and the actomyosin system [6Tanenbaum M.E. Medema R.H. Mechanisms of centrosome separation and bipolar spindle assembly.Dev. Cell. 2010; 19: 797-806Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar]. While centrosomes can separate in prophase or in prometaphase after nuclear envelope breakdown (NEBD), prophase centrosome separation optimizes spindle assembly and minimizes the occurrence of abnormal chromosome attachments that could end in aneuploidy [7Kaseda K. McAinsh A.D. Cross R.A. Dual pathway spindle assembly increases both the speed and the fidelity of mitosis.Biol. Open. 2012; 1: 12-18Crossref PubMed Scopus (61) Google Scholar, 8Silkworth W.T. Nardi I.K. Paul R. Mogilner A. Cimini D. Timing of centrosome separation is important for accurate chromosome segregation.Mol. Biol. Cell. 2012; 23: 401-411Crossref PubMed Scopus (116) Google Scholar]. Prophase centrosome separation relies on the activity of Eg5/KIF11, a mitotic kinesin [9Ferenz N.P. Gable A. Wadsworth P. Mitotic functions of kinesin-5.Semin. Cell Dev. Biol. 2010; 21: 255-259Crossref PubMed Scopus (119) Google Scholar] that accumulates around centrosomes in early mitosis under the control of CDK1 and the Nek9/Nek6/7 kinase module [10Blangy A. Lane H.A. d’Hérin P. Harper M. Kress M. Nigg E.A. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo.Cell. 1995; 83: 1159-1169Abstract Full Text PDF PubMed Scopus (783) Google Scholar, 11Sawin K.E. Mitchison T.J. Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.Proc. Natl. Acad. Sci. USA. 1995; 92: 4289-4293Crossref PubMed Scopus (227) Google Scholar, 12Whitehead C.M. Rattner J.B. Expanding the role of HsEg5 within the mitotic and post-mitotic phases of the cell cycle.J. Cell Sci. 1998; 111: 2551-2561PubMed Google Scholar, 13Tanenbaum M.E. Macůrek L. Galjart N. Medema R.H. Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly.EMBO J. 2008; 27: 3235-3245Crossref PubMed Scopus (120) Google Scholar, 14Woodcock S.A. Rushton H.J. Castañeda-Saucedo E. Myant K. White G.R.M. Blyth K. Sansom O.J. Malliri A. Tiam1-Rac signaling counteracts Eg5 during bipolar spindle assembly to facilitate chromosome congression.Curr. Biol. 2010; 20: 669-675Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 15Smith E. Hégarat N. Vesely C. Roseboom I. Larch C. Streicher H. Straatman K. Flynn H. Skehel M. Hirota T. et al.Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1.EMBO J. 2011; 30: 2233-2245Crossref PubMed Scopus (76) Google Scholar, 16Bertran M.T. Sdelci S. Regué L. Avruch J. Caelles C. Roig J. Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.EMBO J. 2011; 30: 2634-2647Crossref PubMed Scopus (116) Google Scholar, 17Mardin B.R. Agircan F.G. Lange C. Schiebel E. Plk1 controls the Nek2A-PP1γ antagonism in centrosome disjunction.Curr. Biol. 2011; 21: 1145-1151Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar]. Here, we show that Eg5 localization and centrosome separation in prophase depend on the nuclear microtubule-associated protein TPX2 [18Neumayer G. Belzil C. Gruss O.J. Nguyen M.D. TPX2: Of spindle assembly, DNA damage response, and cancer.Cell. Mol. Life Sci. 2014; 71: 3027-3047Crossref PubMed Scopus (111) Google Scholar], a pool of which localizes to the centrosomes before NEBD. This localization involves RHAMM/HMMR [19Groen A.C. Cameron L.A. Coughlin M. Miyamoto D.T. Mitchison T.J. Ohi R. XRHAMM functions in ran-dependent microtubule nucleation and pole formation during anastral spindle assembly.Curr. Biol. 2004; 14: 1801-1811Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar] and the kinase Nek9 [20Roig J. Mikhailov A. Belham C. Avruch J. Nercc1, a mammalian NIMA-family kinase, binds the Ran GTPase and regulates mitotic progression.Genes Dev. 2002; 16: 1640-1658Crossref PubMed Scopus (114) Google Scholar], which phosphorylates TPX2 nuclear localization signal (NLS) preventing its interaction with importin and nuclear import. The pool of centrosomal TPX2 in prophase has a critical role for both microtubule aster organization and Eg5 localization, and thereby for centrosome separation. Our results uncover an unsuspected role for TPX2 before NEBD and define a novel regulatory mechanism for centrosome separation in prophase. They furthermore suggest NLS phosphorylation as a novel regulatory mechanism for spindle assembly factors controlled by the importin/Ran system. Centrosomes [1Conduit P.T. Wainman A. Raff J.W. Centrosome function and assembly in animal cells.Nat. Rev. Mol. Cell Biol. 2015; 16: 611-624Crossref PubMed Scopus (318) Google Scholar, 2Fu J. Hagan I.M. Glover D.M. The centrosome and its duplication cycle.Cold Spring Harb. Perspect. Biol. 2015; 7: a015800Crossref PubMed Scopus (143) Google Scholar] play a central role during spindle assembly in most animal cells [3Meraldi P. Centrosomes in spindle organization and chromosome segregation: A mechanistic view.Chromosome Res. 2016; 24: 19-34Crossref PubMed Scopus (35) Google Scholar]. In early mitosis, they organize two symmetrical microtubule arrays that upon separation define the two poles of the forming spindle. Centrosome separation is tightly regulated [4Agircan F.G. Schiebel E. Mardin B.R. Separate to operate: Control of centrosome positioning and separation.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2014; 369: 20130461Crossref PubMed Scopus (70) Google Scholar, 5van Ree J.H. Nam H.-J. van Deursen J.M. Mitotic kinase cascades orchestrating timely disjunction and movement of centrosomes maintain chromosomal stability and prevent cancer.Chromosome Res. 2016; 24: 67-76Crossref PubMed Scopus (14) Google Scholar], occurring through partially redundant mechanisms that rely on the action of microtubule-based dynein and kinesin motors and the actomyosin system [6Tanenbaum M.E. Medema R.H. Mechanisms of centrosome separation and bipolar spindle assembly.Dev. Cell. 2010; 19: 797-806Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar]. While centrosomes can separate in prophase or in prometaphase after nuclear envelope breakdown (NEBD), prophase centrosome separation optimizes spindle assembly and minimizes the occurrence of abnormal chromosome attachments that could end in aneuploidy [7Kaseda K. McAinsh A.D. Cross R.A. Dual pathway spindle assembly increases both the speed and the fidelity of mitosis.Biol. Open. 2012; 1: 12-18Crossref PubMed Scopus (61) Google Scholar, 8Silkworth W.T. Nardi I.K. Paul R. Mogilner A. Cimini D. Timing of centrosome separation is important for accurate chromosome segregation.Mol. Biol. Cell. 2012; 23: 401-411Crossref PubMed Scopus (116) Google Scholar]. Prophase centrosome separation relies on the activity of Eg5/KIF11, a mitotic kinesin [9Ferenz N.P. Gable A. Wadsworth P. Mitotic functions of kinesin-5.Semin. Cell Dev. Biol. 2010; 21: 255-259Crossref PubMed Scopus (119) Google Scholar] that accumulates around centrosomes in early mitosis under the control of CDK1 and the Nek9/Nek6/7 kinase module [10Blangy A. Lane H.A. d’Hérin P. Harper M. Kress M. Nigg E.A. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo.Cell. 1995; 83: 1159-1169Abstract Full Text PDF PubMed Scopus (783) Google Scholar, 11Sawin K.E. Mitchison T.J. Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.Proc. Natl. Acad. Sci. USA. 1995; 92: 4289-4293Crossref PubMed Scopus (227) Google Scholar, 12Whitehead C.M. Rattner J.B. Expanding the role of HsEg5 within the mitotic and post-mitotic phases of the cell cycle.J. Cell Sci. 1998; 111: 2551-2561PubMed Google Scholar, 13Tanenbaum M.E. Macůrek L. Galjart N. Medema R.H. Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly.EMBO J. 2008; 27: 3235-3245Crossref PubMed Scopus (120) Google Scholar, 14Woodcock S.A. Rushton H.J. Castañeda-Saucedo E. Myant K. White G.R.M. Blyth K. Sansom O.J. Malliri A. Tiam1-Rac signaling counteracts Eg5 during bipolar spindle assembly to facilitate chromosome congression.Curr. Biol. 2010; 20: 669-675Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 15Smith E. Hégarat N. Vesely C. Roseboom I. Larch C. Streicher H. Straatman K. Flynn H. Skehel M. Hirota T. et al.Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1.EMBO J. 2011; 30: 2233-2245Crossref PubMed Scopus (76) Google Scholar, 16Bertran M.T. Sdelci S. Regué L. Avruch J. Caelles C. Roig J. Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.EMBO J. 2011; 30: 2634-2647Crossref PubMed Scopus (116) Google Scholar, 17Mardin B.R. Agircan F.G. Lange C. Schiebel E. Plk1 controls the Nek2A-PP1γ antagonism in centrosome disjunction.Curr. Biol. 2011; 21: 1145-1151Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar]. Here, we show that Eg5 localization and centrosome separation in prophase depend on the nuclear microtubule-associated protein TPX2 [18Neumayer G. Belzil C. Gruss O.J. Nguyen M.D. TPX2: Of spindle assembly, DNA damage response, and cancer.Cell. Mol. Life Sci. 2014; 71: 3027-3047Crossref PubMed Scopus (111) Google Scholar], a pool of which localizes to the centrosomes before NEBD. This localization involves RHAMM/HMMR [19Groen A.C. Cameron L.A. Coughlin M. Miyamoto D.T. Mitchison T.J. Ohi R. XRHAMM functions in ran-dependent microtubule nucleation and pole formation during anastral spindle assembly.Curr. Biol. 2004; 14: 1801-1811Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar] and the kinase Nek9 [20Roig J. Mikhailov A. Belham C. Avruch J. Nercc1, a mammalian NIMA-family kinase, binds the Ran GTPase and regulates mitotic progression.Genes Dev. 2002; 16: 1640-1658Crossref PubMed Scopus (114) Google Scholar], which phosphorylates TPX2 nuclear localization signal (NLS) preventing its interaction with importin and nuclear import. The pool of centrosomal TPX2 in prophase has a critical role for both microtubule aster organization and Eg5 localization, and thereby for centrosome separation. Our results uncover an unsuspected role for TPX2 before NEBD and define a novel regulatory mechanism for centrosome separation in prophase. They furthermore suggest NLS phosphorylation as a novel regulatory mechanism for spindle assembly factors controlled by the importin/Ran system. We initially investigated the molecular requirements for Eg5 localization in prophase using different drugs (Figure S1). As previously described, in control prophase cells Eg5 localized around the centrosomes. This localization was completely disturbed by treatment with nocodazole at concentrations that depolymerize microtubules. Monastrol and S-trityl-L-cysteine (STLC), two compounds that inhibit Eg5 motor activity and decrease microtubule binding [21Maliga Z. Kapoor T.M. Mitchison T.J. Evidence that monastrol is an allosteric inhibitor of the mitotic kinesin Eg5.Chem. Biol. 2002; 9: 989-996Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 22Skoufias D.A. DeBonis S. Saoudi Y. Lebeau L. Crevel I. Cross R. Wade R.H. Hackney D. Kozielski F. S-trityl-L-cysteine is a reversible, tight binding inhibitor of the human kinesin Eg5 that specifically blocks mitotic progression.J. Biol. Chem. 2006; 281: 17559-17569Crossref PubMed Scopus (189) Google Scholar], similarly resulted in a strong disruption of Eg5 localization. We thus concluded that Eg5 depends on its ability to efficiently bind microtubules and/or its motor activity to correctly localize in prophase. Eg5 inhibition with monastrol or STLC resulted in prophase cells with unseparated centrosomes, similarly to what is observed upon Eg5 downregulation [16Bertran M.T. Sdelci S. Regué L. Avruch J. Caelles C. Roig J. Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.EMBO J. 2011; 30: 2634-2647Crossref PubMed Scopus (116) Google Scholar]. Nocodazole in contrast did not seem to interfere with centrosome separation. Accordingly, nocodazole has been shown to result in centrosome separation even in interphase cells [23Jean C. Tollon Y. Raynaud-Messina B. Wright M. The mammalian interphase centrosome: Two independent units maintained together by the dynamics of the microtubule cytoskeleton.Eur. J. Cell Biol. 1999; 78: 549-560Crossref PubMed Scopus (36) Google Scholar, 24Meraldi P. Nigg E.A. Centrosome cohesion is regulated by a balance of kinase and phosphatase activities.J. Cell Sci. 2001; 114: 3749-3757Crossref PubMed Google Scholar], something that we indeed observed with this drug, but not with STLC or monastrol (data not shown). Eg5 pericentrosomal localization is controlled by Ser1033 phosphorylation by Nek6/7 downstream of Nek9 [16Bertran M.T. Sdelci S. Regué L. Avruch J. Caelles C. Roig J. Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.EMBO J. 2011; 30: 2634-2647Crossref PubMed Scopus (116) Google Scholar]. We have previously shown that mutation of Ser1033 does not affect significantly Eg5 oligomerization or binding to microtubules [25Rapley J. Nicolàs M. Groen A. Regué L. Bertran M.T. Caelles C. Avruch J. Roig J. The NIMA-family kinase Nek6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation.J. Cell Sci. 2008; 121: 3912-3921Crossref PubMed Scopus (98) Google Scholar], thus suggesting that it regulates an interaction with an unidentified partner. Efforts to directly identify proteins that would preferentially bind to phosphomimetic forms of Eg5 were not successful. We therefore took a candidate-based approach and focused on TPX2, previously shown to regulate the localization of Eg5 on the (pro)metaphase spindle [26Ma N. Titus J. Gable A. Ross J.L. Wadsworth P. TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle.J. Cell Biol. 2011; 195: 87-98Crossref PubMed Scopus (68) Google Scholar, 27Gable A. Qiu M. Titus J. Balchand S. Ferenz N.P. Ma N. Collins E.S. Fagerstrom C. Ross J.L. Yang G. Wadsworth P. Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2.Mol. Biol. Cell. 2012; 23: 1254-1266Crossref PubMed Scopus (41) Google Scholar]. In prophase cells, downregulation of TPX2 resulted in a reduction of centrosomal Eg5 and interfered with centrosome separation (Figure 1A). TPX2 downregulation decreased as well the amount of microtubules around centrosomes. However, the ratio of Eg5 to microtubules was also reduced when compared to control conditions, and therefore this alone could not explain the observed reduction of pericentrosomal Eg5, suggesting that in prophase TPX2 is necessary for both microtubule organization and Eg5 localization around the centrosomes. A similar dependence of Eg5 prophase localization on TPX2 was observed in transgenic mouse embryonic fibroblast (MEF) cells (Figure S2). Surprisingly, both transformed and untransformed MEFs show separated centrosomes in G2, when no Eg5 can be detected at centrosomes, suggesting a defective intercentrosomal cohesion in these cells and therefore precluding their use to study centrosome separation. TPX2 has been shown to directly interact with Eg5 in vitro and to slow down its movement toward microtubule plus ends [26Ma N. Titus J. Gable A. Ross J.L. Wadsworth P. TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle.J. Cell Biol. 2011; 195: 87-98Crossref PubMed Scopus (68) Google Scholar, 28Eckerdt F. Eyers P.A. Lewellyn A.L. Prigent C. Maller J.L. Spindle pole regulation by a discrete Eg5-interacting domain in TPX2.Curr. Biol. 2008; 18: 519-525Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 29Balchand S.K. Mann B.J. Titus J. Ross J.L. Wadsworth P. TPX2 inhibits Eg5 by interactions with both motor and microtubule.J. Biol. Chem. 2015; 290: 17367-17379Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]. We confirmed their interaction in vivo as we could consistently coimmunoprecipitate GFP-TPX2 with a small pool of endogenous Eg5 (Figure 1B). This interaction was abolished when the extreme C terminus of TPX2 (residues 711–747) was eliminated, as expected from in vitro results. Yeast two hybrid assays indicated that the interaction was direct and mapped it to Eg5 C-terminal domain (Eg5[762–1057], data not shown). Once we confirmed that TPX2 and Eg5 interact, we sought to determine whether phosphorylation of Eg5[Ser1033] could modulate this interaction. For this, we studied the ability of different Eg5 mutants to coimmunoprecipitate with TPX2 in mitotic cells (Figure 1C). We found that the mutation of Ser1033 to a non-phosphorylatable residue (Eg5[S1033A]) robustly interfered with Eg5 ability to interact with TPX2. Moreover, mutation to an acidic residue (Eg5[S1033D]) rescued the interaction. Eg5[S1033D] interacted with TPX2 only slightly more than wild-type Eg5, suggesting that the mutation only partially behaves as a bona fide phosphomimetic substitution, as has been suggested by previous functional studies where this mutant could not totally rescue endogenous Eg5 downregulation [25Rapley J. Nicolàs M. Groen A. Regué L. Bertran M.T. Caelles C. Avruch J. Roig J. The NIMA-family kinase Nek6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation.J. Cell Sci. 2008; 121: 3912-3921Crossref PubMed Scopus (98) Google Scholar]. Additional acidic mutants similarly showed higher TPX2 binding levels to these of the non-phosphorylatable Ser1033Ala mutant. Importantly, incubation with phosphatase (Figure 1D) or Nek9 downregulation (Figure 1E) resulted both in a strong reduction of the amount of Eg5 coimmunoprecipitating with TPX2, demonstrating the importance of phosphorylation for the interaction between the two proteins. TPX2 is a RanGTP regulated spindle assembly factor (SAF) that interacts with importins through its NLS. In interphase, it accumulates in the nucleus, and after nuclear envelope breakdown it performs essential functions for spindle assembly when dissociated from importins by RanGTP in the vicinity of the chromosomes [30Gruss O.J. Carazo-Salas R.E. Schatz C.A. Guarguaglini G. Kast J. Wilm M. Le Bot N. Vernos I. Karsenti E. Mattaj I.W. Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity.Cell. 2001; 104: 83-93Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 31Cavazza T. Vernos I. The RanGTP pathway: From nucleo-cytoplasmic transport to spindle assembly and beyond.Front. Cell Dev. Biol. 2016; 3: 82Crossref PubMed Scopus (77) Google Scholar]. Our results, however, indicate that TPX2 is also necessary in prophase for the formation of centrosomal microtubule asters and to maintain the pericentrosomal pool of Eg5, thus performing essential roles in the cytoplasm before NEBD. To solve this discrepancy, we first sought to establish whether TPX2 is present in the cytoplasm of prophase cells. Using a monoclonal anti-TPX2 antibody that recognizes poorly nuclear TPX2, we could indeed detect a cytoplasmic pool of TPX2 that colocalized with centrosomes in prophase cells (Figure 2A, lamin staining was used to assess nuclear envelope integrity). The specificity of the signal was confirmed by TPX2 RNAi in human cells (Figure S3A) and abrogation of expression in MEF cells (Figure S2). Moreover, using synchronized cells to minimize the toxic effects of TPX2 overexpression in mitosis, we also detected recombinant GFP- and FLAG-tagged TPX2 at centrosomes in prophase cells (Figure S3B). Our results agree with what was previously hinted in previous reports in mammalian cells [32Gruss O.J. Wittmann M. Yokoyama H. Pepperkok R. Kufer T. Silljé H. Karsenti E. Mattaj I.W. Vernos I. Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells.Nat. Cell Biol. 2002; 4: 871-879Crossref PubMed Scopus (257) Google Scholar] and in plants [33Vos J.W. Pieuchot L. Evrard J.-L.L. Janski N. Bergdoll M. de Ronde D. Perez L.H. Sardon T. Vernos I. Schmit A.-C.C. The plant TPX2 protein regulates prospindle assembly before nuclear envelope breakdown.Plant Cell. 2008; 20: 2783-2797Crossref PubMed Scopus (88) Google Scholar], where the TPX2 ortholog has been shown to be functionally active in the cytoplasm. TPX2 centrosomal localization depended on RHAMM (Figure 2B) similarly to what has been described in later phases of mitosis [34Chen H. Mohan P. Jiang J. Nemirovsky O. He D. Fleisch M.C. Niederacher D. Pilarski L.M. Lim C.J. Maxwell C.A. Spatial regulation of Aurora A activity during mitotic spindle assembly requires RHAMM to correctly localize TPX2.Cell Cycle. 2014; 13: 2248-2261Crossref PubMed Scopus (26) Google Scholar]. We could therefore specifically interfere with the localization of TPX2 in early mitosis without altering TPX2 total levels. Consistently, RHAMM downregulation resulted in the disruption of both Eg5 centrosomal localization and centrosome separation in prophase cells (Figure 2C). Reasoning that TPX2 prophase localization could be regulated by the same kinases that regulate Eg5, we investigated whether it depended on Nek9. Upon Nek9 RNAi, TPX2 was greatly diminished from centrosomes in prophase cells (Figure 3A). To further understand how Nek9 controls TPX2 localization, we next sought to determine whether TPX2 is a direct Nek9 substrate. TPX2 was readily phosphorylated by Nek9 as detected both by 32P incorporation and electrophoretic mobility shift (Figure S4). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified 19 high confidence phosphosites (Figures 3B and S4). 10 of the sites conform to the motif [FLV]XX[ST] similar to that of sites modified by Nek2 and Nek6 [35Lizcano J.M. Deak M. Morrice N. Kieloch A. Hastie C.J. Dong L. Schutkowski M. Reimer U. Alessi D.R. Molecular basis for the substrate specificity of NIMA-related kinase-6 (NEK6). Evidence that NEK6 does not phosphorylate the hydrophobic motif of ribosomal S6 protein kinase and serum- and glucocorticoid-induced protein kinase in vivo.J. Biol. Chem. 2002; 277: 27839-27849Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 36Alexander J. Lim D. Joughin B.A. Hegemann B. Hutchins J.R.A. Ehrenberger T. Ivins F. Sessa F. Hudecz O. Nigg E.A. et al.Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling.Sci. Signal. 2011; 4: ra42Crossref PubMed Scopus (0) Google Scholar], thus indicating that the three kinases have a similar consensus sequence. Most of the sites identified correspond to phosphorylated residues detected in vivo (13 out of 19), several of them specific for mitosis or dependent on the Nek9 upstream activator Plk1 [37Santamaria A. Wang B. Elowe S. Malik R. Zhang F. Bauer M. Schmidt A. Silljé H.H.W. Körner R. Nigg E.A. The Plk1-dependent phosphoproteome of the early mitotic spindle.Mol. Cell. Proteomics. 2011; 10 (M110.004457)Crossref Scopus (176) Google Scholar, 38Kettenbach A.N. Schweppe D.K. Faherty B.K. Pechenick D. Pletnev A.A. Gerber S.A. Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells.Sci. Signal. 2011; 4: rs5Crossref PubMed Scopus (364) Google Scholar]. A number of Nek9 sites, including some of the most heavily modified ones, clustered around the main NLS and importin binding site of Xenopus TPX2 (residues 313–315 in human TPX2, called herein NLS1, see Figure 3B) [39Schatz C.A. Santarella R. Hoenger A. Karsenti E. Mattaj I.W. Gruss O.J. Carazo-Salas R.E. Importin alpha-regulated nucleation of microtubules by TPX2.EMBO J. 2003; 22: 2060-2070Crossref PubMed Scopus (143) Google Scholar, 40Giesecke A. Stewart M. Novel binding of the mitotic regulator TPX2 (target protein for Xenopus kinesin-like protein 2) to importin-alpha.J. Biol. Chem. 2010; 285: 17628-17635Crossref PubMed Scopus (68) Google Scholar]. Another site was adjacent to a region that was shown in human TPX2 to also be important for nuclear localization (NLS2, residues 158–159 in human TPX2 [41Kahn O.I. Ha N. Baird M.A. Davidson M.W. Baas P.W. TPX2 regulates neuronal morphology through kinesin-5 interaction.Cytoskeleton. 2015; 72: 340-348Crossref Scopus (15) Google Scholar]). We thus sought to explore whether the modification of these sites could modulate the function of TPX2 NLSs and thus control its nucleocytoplasmic distribution. First, we confirmed that human TPX2 is maintained in the nucleus by the combined action of NLS1 and NLS2 by mutating two key basic residues of NLS2 (TPX2[K158M;K159M]) generating TPX2 NLS2-MM and three of NLS1 (TPX2[K313M;K314M;R315Q]) generating TPX2 NLS1-MMQ. Although mutating NLS2 or NLS2 is effective in changing TPX2 nucleocytoplasmic distribution (Figure 3C), combining the mutations of both NLSs has a synergistic effect, resulting in a very significant increase of the amount of cytoplasmic TPX2 (mean cytoplasm/nucleus ratio is 0.6 for GFP-TPX2 wild-type while being 2.3 for GFP-TPX2 NLS2-MM;NLS1-MMQ). Thus, we confirmed that human TPX2 localization is controlled jointly by two import signals, agreeing with work in neurons [41Kahn O.I. Ha N. Baird M.A. Davidson M.W. Baas P.W. TPX2 regulates neuronal morphology through kinesin-5 interaction.Cytoskeleton. 2015; 72: 340-348Crossref Scopus (15) Google Scholar]. To determine the effects of phosphorylation around TPX2 NLSs, we next produced different phosphomimetic mutants corresponding to the sites found phosphorylated by Nek9 around NLS2 (TPX2[S157D], TPX2 NLS2-D) as well as around NLS1 (TPX2[S310D;T320E;S322D;T323E], TPX2 NLS1-4DE, the latter two being detected in our MS analysis as low-probability sites). We then observed the effect of the substitutions on the nucleocytoplasmic distribution of TPX2. Both GFP-TPX2 NLS2-D and GFP-TPX2 NLS1-4DE mutants have a cytoplasmic to nuclear ration that is similar to the wild-type form of GFP-TPX2 indicating that the independent modification of the sequences surrounding each NLS has no effect on the nucleocytoplasmic distribution of TPX2. In contrast, introduction of phosphomimetic mutants in forms of TPX2 with a single functional NLS resulted in a significant shift of the distribution of TPX2 toward the cytoplasm that resembled that of the mutant with no functional NLS (Figure 3C, TPX2 NLS2-MM;NLS1-4DE and NLS2-D;NLS1-MMQ). Remarkably, the combination of phosphomimetic mutations around both NLS2 and NLS1 (TPX2 NLS2-D;NLS1-4DE) resulted in a form of TPX2 with a localization significantly shifted toward the cytoplasm, with a cytoplasmic/nuclear ration of 1 (cell fractionation experiments failed to detect an increase of TPX2 NLS2-D;NLS1-4DE in the soluble fraction when compared to wild-type TPX2, thus suggesting that cytoplasmic TPX2 NLS2-D;NLS1-4D may be bound to insoluble cellular structures or that its amount is below the detection limits of this biochemical assay). The results suggest that phosphorylation at Nek9 sites around NLS2 and NLS1 promotes the accumulation of TPX2 at the prophase centrosome by interfering with importin binding. To confirm this, we assessed the ability of different TPX2 mutants to directly bind importin in Xenopus egg extracts, where the interaction with the import factor can be more easily studied. Xenopus TPX2 (XTPX2) has two putative NLS sequences at similar regions to that of human NLS1 and NLS2, both presenting conserved putative phosphosites close by (Figure 3B). Two phosphomimetic mutations of the serines in XTPX2 NLS1 corresponding to the Nek9 sites Ser310 and Ser320/2 in human TPX2 (XTPX2[S281D;S291D], XTPX2 NLS1-2D) strongly decreased the binding of XTPX2 to importin (Figure 3D). A single mutation in XTPX2 Ser281 was produced (XTPX2[S281D], XTPX2 NLS1-D) and found to slightly decrease the importin binding to XTPX2 (data not shown). Additional mutations in the second putative import signal, in the homologous region to human TPX2 NLS2 (XTPX2[S128D;S129D;S130D;S281D], XTPX2 NLS2-3D NLS1-D; and XTPX2[S128D;S129D;S130D;S281D; S291D], XTPX2 NLS2-3D NLS1-2D) did not further interfere with importin binding thus confirming that NLS1 is the main sequence for Xenopus TPX2 binding to importin, as previously reported [39Schatz C.A. Santarella R. Hoenger A. Karsenti E. Mattaj I.W. Gruss O.J. Carazo-Salas R.E. Importin alpha-regulated nucleation of microtubules by TPX2.EMBO J. 2003; 22: 2060-2070Crossref PubMed Scopus (143) Google Scholar]. In agreement with these results, the XTPX2 phosphomutants that showed impaired binding to importin showed a reduction in nuclear accumulation compared to wild-type XTPX2 in interphase egg extracts (Figure 3E). Our results indicate that phosphorylation at Nek9 sites surrounding NLS shifts TPX2 distribution toward the cytoplasm by interfering with importin binding. In prophase cells, this could in turn result in a controlled accumulation of TPX2 around the centrosomes, favored by a higher microtubule density and the presence of RHAMM, that is able to interact with TPX2 and may thus favor its concentration around the organelles (how RHAMM own localization is regulated is unknown, although its ability to bind dynein suggest that it may depend on the action of this minus-end-directed motor). Indeed, in prophase, the levels of GFP-TPX2 NLS1-4A (TPX2[S310A;T320A;S322A;T323A]) at centrosomes were significantly lower than those of GFP-TPX2 wild-type, while the phosphomimetic GFP-TPX2 NLS1-4DE mutant accumulated at centrosomes in a similar amount to that of its wild-type counterpart (Figure 4A). Surprisingly, mutation of Ser157 in TPX2 NLS2 to either aspartic acid or alanine strongly interfered with TPX2 centrosomal localization in prophase, suggesting that this residue may be involved in specific interactions with centrosomal proteins that are disrupted by these mutations, and that in this context an acidic residue does not act as a bona fide phosphomimetic mutant. The existence of phosphorylation sites outside of TPX2 NLS1 (or in associated proteins such as RHAMM) controlling TPX2 localization was further supported by the inability of GFP-TPX2 NLS1-4DE mutants to localize to prophase centrosomes in the absence of Nek9 (data not shown). Finally, we sought to determine whether mutation of TPX2 NLS sites interfered with its ability to recruit Eg5 to centrosomes and thus support centrosome separation (Figure 4A). We downregulated endogenous TPX2 and expressed GFP, GFP-TPX2 wild-type, GFP-TPX2 NLS1-4A (that shows a strong impairment for centrosomal localization in prophase, Figure 4A) or GFP-NLS1-4DE (with centrosomal levels similar to these of wild-type TPX2). Cells were synchronized with the aim of studying the first mitotic cycle after transfection and thus to minimize the deleterious effects of overexpressing exogenous TPX2, that like TPX2 downregulation can result in prometaphase arrest [32Gruss O.J. Wittmann M. Yokoyama H. Pepperkok R. Kufer T. Silljé H. Karsenti E. Mattaj I.W. Vernos I. Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells.Nat. Cell Biol. 2002; 4: 871-879Crossref PubMed Scopus (257) Google Scholar]. None of the recombinant TPX2 forms used completely rescued the effects of endogenous TPX2 downregulation on mitotic progression, although wild-type TPX2 and TPX2 NLS1-4DE were better in reducing the number of cells in prometaphase than TPX2 NLS1-4A. Remarkably, wild-type TPX2 clearly rescued Eg5 accumulation at centrosomes and centrosome separation in prophase cells. In contrast, the expression of TPX2 NLS1-4A at similar levels failed to rescue Eg5 accumulation at centrosomes and to support centrosome separation in prophase cells. Finally, expression of TPX2 NLS1-4DE resulted in levels of Eg5 at centrosomes, that, although smaller than these resulting from TPX2 wild-type expression, were greater than levels observed with TPX2 NLS1-4A and rescued centrosome separation. This suggested that the mutations did not completely behave as phosphomimetic substitutions but were able to rescue Eg5 centrosomal levels sufficiently to support centrosome separation. Our results show that Eg5 acts in concert with TPX2 to separate centrosomes in prophase, thus defining a novel function for TPX2 at a time when it is mostly found in the nucleus. We also show how Nek9, previously shown to be a key controller of Eg5-driven centrosome separation [16Bertran M.T. Sdelci S. Regué L. Avruch J. Caelles C. Roig J. Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5.EMBO J. 2011; 30: 2634-2647Crossref PubMed Scopus (116) Google Scholar], can control this process by both retaining a pool of TPX2 at centrosomes and simultaneously inducing Eg5 phosphorylation and its binding to TPX2. Our results are consistent with a scenario in which TPX2, through its ability to interact with both microtubules and Eg5, acts as an Eg5 “receptor” retaining the kinesin around the centrosomes, allowing it to exert the necessary forces needed to separate centrosomes. TPX2 could do this through its ability to slow down Eg5 plus-end-directed movement without inducing detachment from the microtubules [29Balchand S.K. Mann B.J. Titus J. Ross J.L. Wadsworth P. TPX2 inhibits Eg5 by interactions with both motor and microtubule.J. Biol. Chem. 2015; 290: 17367-17379Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]. We are currently studying whether a minus-end-directed motor such as dynein [27Gable A. Qiu M. Titus J. Balchand S. Ferenz N.P. Ma N. Collins E.S. Fagerstrom C. Ross J.L. Yang G. Wadsworth P. Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2.Mol. Biol. Cell. 2012; 23: 1254-1266Crossref PubMed Scopus (41) Google Scholar, 42Uteng M. Hentrich C. Miura K. Bieling P. Surrey T. Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles.J. Cell Biol. 2008; 182: 715-726Crossref PubMed Scopus (72) Google Scholar] may collaborate in the accumulation of Eg5 at the vicinity of centrosomes in prophase." @default.
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• W2777334852 title "Nek9 Phosphorylation Defines a New Role for TPX2 in Eg5-Dependent Centrosome Separation before Nuclear Envelope Breakdown" @default.
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