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• W3016277935 abstract "The role of O-linked N-acetylglucosamine (O-GlcNAc) modification in the cell cycle has been enigmatic. Previously, both O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) disruptions have been shown to derail the mitotic centrosome numbers, suggesting that mitotic O-GlcNAc oscillation needs to be in concert with mitotic progression to account for centrosome integrity. Here, using both chemical approaches and biological assays with HeLa cells, we attempted to address the underlying molecular mechanism and observed that incubation of the cells with the OGA inhibitor Thiamet-G strikingly elevates centrosomal distances, suggestive of premature centrosome disjunction. These aberrations could be overcome by inhibiting Polo-like kinase 1 (PLK1), a mitotic master kinase. PLK1 inactivation is modulated by the myosin phosphatase targeting subunit 1 (MYPT1)–protein phosphatase 1cβ (PP1cβ) complex. Interestingly, MYPT1 has been shown to be abundantly O-GlcNAcylated, and the modified residues have been detected in a recent O-GlcNAc–profiling screen utilizing chemoenzymatic labeling and bioorthogonal conjugation. We demonstrate here that MYPT1 is O-GlcNAcylated at Thr-577, Ser-585, Ser-589, and Ser-601, which antagonizes CDK1-dependent phosphorylation at Ser-473 and attenuates the association between MYPT1 and PLK1, thereby promoting PLK1 activity. We conclude that under high O-GlcNAc levels, PLK1 is untimely activated, conducive to inopportune centrosome separation and disruption of the cell cycle. We propose that too much O-GlcNAc is equally deleterious as too little O-GlcNAc, and a fine balance between the OGT/OGA duo is indispensable for successful mitotic divisions. The role of O-linked N-acetylglucosamine (O-GlcNAc) modification in the cell cycle has been enigmatic. Previously, both O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) disruptions have been shown to derail the mitotic centrosome numbers, suggesting that mitotic O-GlcNAc oscillation needs to be in concert with mitotic progression to account for centrosome integrity. Here, using both chemical approaches and biological assays with HeLa cells, we attempted to address the underlying molecular mechanism and observed that incubation of the cells with the OGA inhibitor Thiamet-G strikingly elevates centrosomal distances, suggestive of premature centrosome disjunction. These aberrations could be overcome by inhibiting Polo-like kinase 1 (PLK1), a mitotic master kinase. PLK1 inactivation is modulated by the myosin phosphatase targeting subunit 1 (MYPT1)–protein phosphatase 1cβ (PP1cβ) complex. Interestingly, MYPT1 has been shown to be abundantly O-GlcNAcylated, and the modified residues have been detected in a recent O-GlcNAc–profiling screen utilizing chemoenzymatic labeling and bioorthogonal conjugation. We demonstrate here that MYPT1 is O-GlcNAcylated at Thr-577, Ser-585, Ser-589, and Ser-601, which antagonizes CDK1-dependent phosphorylation at Ser-473 and attenuates the association between MYPT1 and PLK1, thereby promoting PLK1 activity. We conclude that under high O-GlcNAc levels, PLK1 is untimely activated, conducive to inopportune centrosome separation and disruption of the cell cycle. We propose that too much O-GlcNAc is equally deleterious as too little O-GlcNAc, and a fine balance between the OGT/OGA duo is indispensable for successful mitotic divisions. The centrosomes are the primary microtubule-organizing centers that nucleate the mitotic spindle apparatus to ensure subsequent faithful sister chromatid segregation during mitosis. The centrosome cycle is tightly coordinated with other cell cycle events (1Vertii A. Hehnly H. Doxsey S. The centrosome, a multitalented renaissance organelle.Cold Spring Harb. Perspect. Biol. 2016; 8 (27908937): a02504910.1101/cshperspect.a025049Crossref PubMed Scopus (58) Google Scholar), and its aberrancy could culminate in chromosome segregation defects and aneuploidy (2Silkworth 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 (22130796): 401-41110.1091/mbc.e11-02-0095Crossref PubMed Scopus (106) Google Scholar). The entire centrosome cycle encompasses centrosome duplication during S phase, disjunction in late G2 phase, and further separation during prophase or prometaphase, and eventual segregation into the two daughter cells. Centrosomes duplicate concomitant with DNA replication, after which the sister centrosomes are glued together by two proteinaceous linkers, c-Nap1 and rootletin (3Bahe S. Stierhof Y.D. Wilkinson C.J. Leiss F. Nigg E.A. Rootletin forms centriole-associated filaments and functions in centrosome cohesion.J. Cell Biol. 2005; 171 (16203858): 27-3310.1083/jcb.200504107Crossref PubMed Scopus (241) Google Scholar, 4Mardin B.R. Agircan F.G. Lange C. Schiebel E. Plk1 controls the Nek2A-PP1γ antagonism in centrosome disjunction.Curr. Biol. 2011; 21 (21723128): 1145-115110.1016/j.cub.2011.05.047Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), as well as other components such as Cep68, Cep215, and LRRC45 (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 (26615533): 67-7610.1007/s10577-015-9501-9Crossref PubMed Scopus (13) Google Scholar). C-Nap1, a large coiled-coiled protein, links rootletin to the centrioles so that the centrosome pair is joined by fibrous polymers (3Bahe S. Stierhof Y.D. Wilkinson C.J. Leiss F. Nigg E.A. Rootletin forms centriole-associated filaments and functions in centrosome cohesion.J. Cell Biol. 2005; 171 (16203858): 27-3310.1083/jcb.200504107Crossref PubMed Scopus (241) Google Scholar). In late G2, the Never In Mitosis (NIMA) 3The abbreviations used are: NIMANever In MitosisPTMpost-translational modificationO-GlcNAcO-linked N-acetylglucosamineOGTO-GlcNAc transferaseTMGThiamet-GPBDpolo-box binding domainMYPT1myosin phosphatase targeting subunit 1Cdk1cyclin-dependent kinase 1PLK1Polo-like kinase 15S-Gacetyl-5S-GlcNAcPP1cβprotein phosphatase 1cβFLfull-lengthNocnocodazoleIPimmunoprecipitationIBimmunoblotOGAO-GlcNAcaseIP-phosphataseimmunoprecipitation-phosphataseMTHFRmethylenetetrahydrofolate reductaseATMataxia telangiectasia–mutatedNuMAnuclear mitotic apparatus proteinDAPI4′,6-diamidino-2-phenylindole. -related serine/threonine kinase Nek2A phosphorylates and displaces c-Nap1 and rootletin, leading to disjointed centrosomes (6Hardy T. Lee M. Hames R.S. Prosser S.L. Cheary D.M. Samant M.D. Schultz F. Baxter J.E. Rhee K. Fry A.M. Multisite phosphorylation of C-Nap1 releases it from Cep135 to trigger centrosome disjunction.J. Cell Sci. 2014; 127 (24695856): 2493-250610.1242/jcs.142331Crossref PubMed Scopus (44) Google Scholar). Centrosomal accumulation of Nek2A is mediated by the Hippo pathway, among which sterile 20-like kinase 2 (Mst2) and Salvador (Sav1) play critical roles. In particular, Mst2 phosphorylates and activates Nek2A (7Mardin B.R. Lange C. Baxter J.E. Hardy T. Scholz S.R. Fry A.M. Schiebel E. Components of the Hippo pathway cooperate with Nek2 kinase to regulate centrosome disjunction.Nat. Cell Biol. 2010; 12 (21076410): 1166-117610.1038/ncb2120Crossref PubMed Scopus (136) Google Scholar). Upstream of Mst2 is the mitotic master kinase, Polo-like kinase 1 (PLK1) (8Barr F.A. Silljé H.H. Nigg E.A. Polo-like kinases and the orchestration of cell division.Nat. Rev. Mol. Cell Biol. 2004; 5 (15173822): 429-44010.1038/nrm1401Crossref PubMed Scopus (877) Google Scholar). Never In Mitosis post-translational modification O-linked N-acetylglucosamine O-GlcNAc transferase Thiamet-G polo-box binding domain myosin phosphatase targeting subunit 1 cyclin-dependent kinase 1 Polo-like kinase 1 acetyl-5S-GlcNAc protein phosphatase 1cβ full-length nocodazole immunoprecipitation immunoblot O-GlcNAcase immunoprecipitation-phosphatase methylenetetrahydrofolate reductase ataxia telangiectasia–mutated nuclear mitotic apparatus protein 4′,6-diamidino-2-phenylindole. Following centrosome disjunction, the kinesin Eg5 accounts for centrosome positioning in the beginning of the M phase. Cyclin-dependent kinase 1 (CDK1) phosphorylates and activates Eg5 at Thr-927 by stimulating the engagement between Eg5 and microtubules (9Blangy 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 (8548803): 1159-116910.1016/0092-8674(95)90142-6Abstract Full Text PDF PubMed Scopus (762) Google Scholar). Independently, centrosomal localization of Eg5 requires PLK1 (10Smith E. Hégarat N. Vesely C. Roseboom I. Larch C. Streicher H. Straatman K. Flynn H. Skehel M. Hirota T. Kuriyama R. Hochegger H. Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1.EMBO J. 2011; 30 (21522128): 2233-224510.1038/emboj.2011.120Crossref PubMed Scopus (75) Google Scholar), which activates the NIMA-family kinase Nek9, leading to Eg5 phosphorylation at Ser-1033 by the Nek9/6/7 complex (11Bertran 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 (21642957): 2634-264710.1038/emboj.2011.179Crossref PubMed Scopus (109) Google Scholar). Phosphorylated Eg5 then binds the centrosomal targeting protein for Xenopus kinesin-like protein 2 (TPX2), which is also mediated by Nek9 (12Eibes S. Gallisà-Suñé N. Rosas-Salvans M. Martínez-Delgado P. Vernos I. Roig J. Nek9 phosphorylation defines a new role for TPX2 in Eg5-dependent centrosome separation before nuclear envelope breakdown.Curr. Biol. 2018; 28 (29276125): 121-129.e410.1016/j.cub.2017.11.046Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Besides mitosis, Eg5 also governs centrosome dynamics during interphase (10Smith E. Hégarat N. Vesely C. Roseboom I. Larch C. Streicher H. Straatman K. Flynn H. Skehel M. Hirota T. Kuriyama R. Hochegger H. Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1.EMBO J. 2011; 30 (21522128): 2233-224510.1038/emboj.2011.120Crossref PubMed Scopus (75) Google Scholar). Hence, the centrosomal role of Plk1 is 2-fold: centrosome disjunction via the PLK1–Mst2–Nek2A signaling cascade and centrosome separation through PLK1–Nek9/6/7–Eg5 (13Wang G. Jiang Q. Zhang C. The role of mitotic kinases in coupling the centrosome cycle with the assembly of the mitotic spindle.J. Cell Sci. 2014; 127 (25128564): 4111-412210.1242/jcs.151753Crossref PubMed Scopus (71) Google Scholar). Besides centrosomes, PLK1 also orchestrates a multitude of cell cycle events, including replication, mitotic entry, chromosome segregation, and cytokinesis (4Mardin B.R. Agircan F.G. Lange C. Schiebel E. Plk1 controls the Nek2A-PP1γ antagonism in centrosome disjunction.Curr. Biol. 2011; 21 (21723128): 1145-115110.1016/j.cub.2011.05.047Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 14Zitouni S. Nabais C. Jana S.C. Guerrero A. Bettencourt-Dias M. Polo-like kinases: structural variations lead to multiple functions.Nat. Rev. Mol. Cell Biol. 2014; 15 (24954208): 433-45210.1038/nrm3819Crossref PubMed Scopus (272) Google Scholar, 15Song B. Liu X.S. Liu X. Polo-like kinase 1 (Plk1): an unexpected player in DNA replication.Cell Div. 2012; 7 (22309699): 310.1186/1747-1028-7-3Crossref PubMed Scopus (40) Google Scholar, 16Li J. Wang J. Jiao H. Liao J. Xu X. Cytokinesis and cancer: polo loves ROCK'n' Rho(A).J. Genet. Genomics. 2010; 37 (20347825): 159-17210.1016/S1673-8527(09)60034-5Crossref PubMed Scopus (39) Google Scholar). It contains an N-terminal kinase domain and a C-terminal Polo-box binding domain (PBD). Phosphorylation of PLK1 at Thr-210 at the T-loop is mediated by the Aurora A–Bora complex (17Seki A. Coppinger J.A. Jang C.Y. Yates J.R. Fang G. Bora and the kinase aurora A cooperatively activate the kinase Plk1 and control mitotic entry.Science. 2008; 320 (18566290): 1655-165810.1126/science.1157425Crossref PubMed Scopus (439) Google Scholar), resulting in dissociation of PBD from the kinase domain and thus activating PLK1. Dephosphorylation of PLK1 is modulated by the protein phosphatase 1cβ (PP1cβ), targeted by the myosin phosphatase targeting subunit 1 (MYPT1) (18Yamashiro 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 (18477460): 787-79710.1016/j.devcel.2008.02.013Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Specifically, CDK1 phosphorylates MYPT1 at Ser-473, creating a binding pocket between MYPT1 and the PBD of PLK1. Subsequently, MYPT1 recruits PP1cβ to dephosphorylate pThr-210 of PLK1 (18Yamashiro 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 (18477460): 787-79710.1016/j.devcel.2008.02.013Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Such interaction at the kinetochore destabilizes kinetochore–microtubule attachments (19Dumitru A.M.G. Rusin S.F. Clark A.E.M. Kettenbach A.N. Compton D.A. Cyclin A/Cdk1 modulates Plk1 activity in prometaphase to regulate kinetochore-microtubule attachment stability.Elife. 2017; 6 (29154753): e2930310.7554/eLife.29303Crossref PubMed Scopus (27) Google Scholar). Besides phosphorylation, PLK1 is also methylated at Lys-209 (20Feldman M. Vershinin Z. Goliand I. Elia N. Levy D. The methyltransferase SETD6 regulates mitotic progression through PLK1 methylation.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (30622182): 1235-124010.1073/pnas.1804407116Crossref PubMed Scopus (14) Google Scholar, 21Li W. Wang H.Y. Zhao X. Duan H. Cheng B. Liu Y. Zhao M. Shu W. Mei Y. Wen Z. Tang M. Guo L. Li G. Chen Q. Liu X. Du H.N. A methylation-phosphorylation switch determines Plk1 kinase activity and function in DNA damage repair.Sci. Adv. 2019; 5 (30854428): eaau756610.1126/sciadv.aau7566Crossref PubMed Scopus (24) Google Scholar), which vies with pThr-210 and hence blocking Plk1 activity. Because of the vital role of PLK1 in mitosis, MYPT1 is subject to multifaceted regulations as follows: the Hippo pathway kinase LATS1/WARTS phosphorylates MYPT1 at Ser-445 to inactivate PLK1 (22Chiyoda T. Sugiyama N. Shimizu T. Naoe H. Kobayashi Y. Ishizawa J. Arima Y. Tsuda H. Ito M. Kaibuchi K. Aoki D. Ishihama Y. Saya H. Kuninaka S. LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression.J. Cell Biol. 2012; 197 (22641346): 625-64110.1083/jcb.201110110Crossref PubMed Scopus (42) Google Scholar); optineurin, another phosphatase, promotes MYPT1 activity (23Kachaner D. Filipe J. Laplantine E. Bauch A. Bennett K.L. Superti-Furga G. Israël A. Weil R. Plk1-dependent phosphorylation of optineurin provides a negative feedback mechanism for mitotic progression.Mol. Cell. 2012; 45 (22365832): 553-56610.1016/j.molcel.2011.12.030Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar); checkpoint kinase 1 (CHK1) phosphorylates MYPT1 at Ser-20 and enhances MYPT1-PP1cβ binding (24Hu X. Li Z. Ding Y. Geng Q. Xiahou Z. Ru H. Dong M.Q. Xu X. Li J. Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1cβ (PP1cβ).Cell Cycle. 2018; 17 (29262732): 421-42710.1080/15384101.2017.1418235Crossref PubMed Scopus (6) Google Scholar); checkpoint kinase 2 (CHK2) phosphorylates MYPT1 at Ser-507 to attenuate pSer-473 (25Nai S. Shi Y. Ru H. Ding Y. Geng Q. Li Z. Dong M.Q. Xu X. Li J. Chk2-dependent phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) regulates centrosome maturation.Cell Cycle. 2019; 18 (31416392): 2651-265910.1080/15384101.2019.1654795Crossref PubMed Scopus (3) Google Scholar). Previous investigations have identified that MYPT1 is also subject to O-linked N-acetylglucosamine (O-GlcNAc) modifications (26Cheung W.D. Sakabe K. Housley M.P. Dias W.B. Hart G.W. O-Linked β-N-acetylglucosaminyltransferase substrate specificity is regulated by myosin phosphatase targeting and other interacting proteins.J. Biol. Chem. 2008; 283 (18840611): 33935-3394110.1074/jbc.M806199200Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). O-GlcNAcylation is an emerging post-translational modification (PTM) that integrates the metabolic signals with transcription, nutrient sensing, stress responses, and cell cycle events (27Hart G.W. Slawson C. Ramirez-Correa G. Lagerlof O. Cross-talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.Annu. Rev. Biochem. 2011; 80 (21391816): 825-85810.1146/annurev-biochem-060608-102511Crossref PubMed Scopus (851) Google Scholar, 28Yang X. Qian K. Protein O-GlcNAcylation: emerging mechanisms and functions.Nat. Rev. Mol. Cell Biol. 2017; 18 (28488703): 452-46510.1038/nrm.2017.22Crossref PubMed Scopus (438) Google Scholar). It is catalyzed by the sole transferase O-GlcNAc transferase (OGT) and reversed by the only O-GlcNAcase (OGA) (27Hart G.W. Slawson C. Ramirez-Correa G. Lagerlof O. Cross-talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.Annu. Rev. Biochem. 2011; 80 (21391816): 825-85810.1146/annurev-biochem-060608-102511Crossref PubMed Scopus (851) Google Scholar). Chemical inhibitors of OGT (acetyl-5S-GlcNAc (5S-G)) and OGA (Thiamet-G (TMG)) have been developed to interrogate various biological processes (29Gloster T.M. Zandberg W.F. Heinonen J.E. Shen D.L. Deng L. Vocadlo D.J. Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells.Nat. Chem. Biol. 2011; 7 (21258330): 174-18110.1038/nchembio.520Crossref PubMed Scopus (215) Google Scholar). During the cell cycle, O-GlcNAcylation levels fluctuate as the cells proceed through different stages (30Liu C. Li J. O-GlcNAc: a sweetheart of the cell cycle and DNA damage response.Front. Endocrinol. 2018; 9 (30105004): 41510.3389/fendo.2018.00415Crossref PubMed Scopus (20) Google Scholar). In particular, overproduction of both OGT and OGA results in multipolar spindles (31Tan E.P. Caro S. Potnis A. Lanza C. Slawson C. O-Linked N-acetylglucosamine cycling regulates mitotic spindle organization.J. Biol. Chem. 2013; 288 (23946484): 27085-2709910.1074/jbc.M113.470187Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). However, myriad targets of O-GlcNAc and its quintessential functions remain largely unexplored. Here, we identify the O-GlcNAc–modified residues of MYPT1. We show that O-GlcNAcylation of MYPT1 antagonizes pSer-473 and results in its dissociation from PLK1. Elevated O-GlcNAc levels thus fuel PLK1 activity towards centrosomes and render ill-timed centrosome separation, disrupting the mitotic cell cycle. Previously, overproduction of both OGT and OGA has been linked with the multipolar spindle (31Tan E.P. Caro S. Potnis A. Lanza C. Slawson C. O-Linked N-acetylglucosamine cycling regulates mitotic spindle organization.J. Biol. Chem. 2013; 288 (23946484): 27085-2709910.1074/jbc.M113.470187Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). We sought to identify whether O-GlcNAc could also be linked with centrosome dynamics. Strikingly, when HeLa cells were treated with TMG (OGAi), the inter-centrosomal distance was significantly augmented 4-fold (Fig. 1A), reminiscent of the phenotype of Nek2A overexpression or overactivation (4Mardin B.R. Agircan F.G. Lange C. Schiebel E. Plk1 controls the Nek2A-PP1γ antagonism in centrosome disjunction.Curr. Biol. 2011; 21 (21723128): 1145-115110.1016/j.cub.2011.05.047Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 32Chen C. Tian F. Lu L. Wang Y. Xiao Z. Yu C. Yu X. Characterization of Cep85-a new antagonist of Nek2A that is involved in the regulation of centrosome disjunction.J. Cell Sci. 2015; 128 (Correction) (26471995) (26220856): 3290-3303J. Cell Sci. 2015; 128 (Correction) (26471995) (26220856): 383710.1242/jcs.18046310.1242/jcs.171637Crossref PubMed Scopus (15) Google Scholar). As the centrosome cycle is tightly governed by PLK1, we attempted to inhibit PLK1. When BI2536 (PLK1i) was adopted in conjuncture with TMG, the centrosomal distances shortened considerably (Fig. 1, A–C). When BI2536 was utilized alone (Fig. 1A), the cells reduced centrosomal distances as reported previously (4Mardin B.R. Agircan F.G. Lange C. Schiebel E. Plk1 controls the Nek2A-PP1γ antagonism in centrosome disjunction.Curr. Biol. 2011; 21 (21723128): 1145-115110.1016/j.cub.2011.05.047Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). These cytological studies suggest that high O-GlcNAc culminates in premature centrosomal separation, probably via PLK1. Previous investigation has identified the inactivating phosphatase of PLK1 as PP1cβ, which is targeted by MYPT1 (18Yamashiro 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 (18477460): 787-79710.1016/j.devcel.2008.02.013Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Intriguingly, MYPT1 is O-GlcNAcylated (26Cheung W.D. Sakabe K. Housley M.P. Dias W.B. Hart G.W. O-Linked β-N-acetylglucosaminyltransferase substrate specificity is regulated by myosin phosphatase targeting and other interacting proteins.J. Biol. Chem. 2008; 283 (18840611): 33935-3394110.1074/jbc.M806199200Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Therefore, we reasoned that O-GlcNAc might exert its effect through MYPT1. We validated the interaction between MYPT1 and OGT through biochemical assays. As shown in Fig. 2A, GST–OGT pulled down HA–MYPT1 from cell extracts. Then both OGT and MYPT1 proteins were purified from Escherichia coli. Upon incubation, His–OGT pulled down GST–MYPT1 (Fig. 2B), suggesting that the interaction is direct. Then we mapped which domain of MYPT1 interacts with OGT. As MYPT1 is a relatively large protein, we constructed several fragments of MYPT1 as described previously: F1(1–306), F2(297–600), F3(586–901), and F4(886–1030) (Fig. 2C) (24Hu X. Li Z. Ding Y. Geng Q. Xiahou Z. Ru H. Dong M.Q. Xu X. Li J. Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1cβ (PP1cβ).Cell Cycle. 2018; 17 (29262732): 421-42710.1080/15384101.2017.1418235Crossref PubMed Scopus (6) Google Scholar). To investigate which fragment interacts with OGT, recombinant full-length (FL) and F1–F4 MYPT1 proteins were utilized in pulldown experiments, and the FL, F2, and F3 MYPT1 pulled-down Myc-OGT (Fig. 2D), suggesting that the potential modification sites could be residing in F2 and F3. A recent quantitative proteomic analysis of protein O-GlcNAc sites using an isotope-tagged cleavable linker (isoTCL) strategy identified the potential O-GlcNAc sites of MYPT1 to be Thr-577, Ser-585, Ser-589, and Ser-601 (Fig. 3, A–D) (33Qin K. Zhu Y. Qin W. Gao J. Shao X. Wang Y.L. Zhou W. Wang C. Chen X. Quantitative profiling of protein O-GlcNAcylation sites by an isotope-tagged cleavable linker.ACS Chem. Biol. 2018; 13 (30059200): 1983-198910.1021/acschembio.8b00414Crossref PubMed Scopus (43) Google Scholar), all of which locate on F2 and F3. We constructed the T577A/S585A/S589A/S601A (4A) mutant accordingly and assessed its effect. When HA–MYPT1–WT and 4A plasmids were transfected into cells, the 4A mutant significantly abrogated O-GlcNAcylation (Fig. 4A), suggesting that these four amino acids are major O-GlcNAc sites. Considering that MYPT1 is abundantly O-GlcNAcylated, and other proteomic screens have also identified extra glycosylation sites (34Woo C.M. Lund P.J. Huang A.C. Davis M.M. Bertozzi C.R. Pitteri S.J. Mapping and quantification of over 2000 O-linked glycopeptides in activated human T cells with isotope-targeted glycoproteomics (Isotag).Mol. Cell. Proteomics. 2018; 17 (29351928): 764-77510.1074/mcp.RA117.000261Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), our results do not exclude the possibility that there could be more O-GlcNAcylated residues on MYPT1.Figure 4O-GlcNAcylation of MYPT1 antagonizes CDK1-dependent phosphorylation at Ser-473. A, MYPT1–WT and 4A plasmids together with Myc–OGT or empty vectors (vec) were transfected into 293T cells and then blotted with the antibodies indicated. B, cells were transfected with HA–MYPT1–WT or 4A plasmids, and then the lysates were immunoblotted (IB) with the antibodies indicated. C, cells were treated with Noc or Noc with Ro-3306 for the time indicated. D, HeLa cells were transfected with HA–MYPT1 and treated or untreated with 5S-G (OGT inhibitor). E, cells were treated with Noc or Noc + 5S-G. F, cells were transfected with MYPT1–WT plasmids and then treated with Noc or Noc plus TMG + Glu as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because CDK1 phosphorylates MYPT1 at Ser-473 during mitosis and creates a binding motif between MYPT1 and the PBD of PLK1 (18Yamashiro 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 (18477460): 787-79710.1016/j.devcel.2008.02.013Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), we surmised that O-GlcNAc of MYPT1 might interplay with pSer-473. To address this possibility, we used a phospho-specific antibody targeting pSer-473 that has been previously described and utilized (25Nai S. Shi Y. Ru H. Ding Y. Geng Q. Li Z. Dong M.Q. Xu X. Li J. Chk2-dependent phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) regulates centrosome maturation.Cell Cycle. 2019; 18 (31416392): 2651-265910.1080/15384101.2019.1654795Crossref PubMed Scopus (3) Google Scholar). Then the WT and 4A plasmids are compared for the pSer-473 levels, and it is significantly bolstered in the 4A mutant (Fig. 4B). When Noc was used to synchronize cells in the M phase, O-GlcNAc levels decreased while pSer-473 levels increased (Fig. 4C). As pSer-473 is mediated by CDK1, we adopted RO-3306 again, and we observed that RL2 levels decreased while pSer-473 levels increased in the RO-3306–treated cells (Fig. 4C). This is consistent with our conjecture that O-GlcNAc antagonizes pSer-473. Finally, we utilized the 5S-G inhibitor for OGT (29Gloster T.M. Zandberg W.F. Heinonen J.E. Shen D.L. Deng L. Vocadlo D.J. Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells.Nat. Chem. Biol. 2011; 7 (21258330): 174-18110.1038/nchembio.520Crossref PubMed Scopus (215) Google Scholar), and 5S-G treatment substantially boosted pSer-473 levels of transfected HA–MYPT1(Fig. 4D). We also examined the effects of 5S-G on endogenous MYPT1. Noc treatment enhanced pSer-473 levels, and Noc plus 5S-G elevated pSer-473 markedly (Fig. 4E). In contrast, glucose plus TMG (TMG + Glu) treatment during Noc would down-regulate pSer-473 compared with Noc alone (Fig. 4F). Taken together, O-GlcNAc of MYPT1 attenuates pSer-473. Because pSer-473 promotes MYPT1–PLK1 association (18Yamashiro 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 (18477460): 787-79710.1016/j.devcel.2008.02.013Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), we then explored the effect of O-GlcNAc on the interaction between MYPT1 and PLK1 by treating the cells with TMG + Glu to enhance O-GlcNAc (35Ramakrishnan P. Clark P.M. Mason D.E. Peters E.C. Hsieh-Wilson L.C. Baltimore D. Activation of the transcriptional function of the NF-κB protein c-Rel by O-GlcNAc glycosylation.Sci. Signal. 2013; 6 (23982206): ra7510.1126/scisignal.2004097Crossref PubMed Scopus (112) Google Scholar, 36Tian J. Geng Q. Ding Y. Liao J. Dong M.Q. Xu X. Li J. O-GlcNAcylation antagonizes phosphorylation of CDH1 (CDC20 homologue 1).J. Biol. Chem. 2016; 291 (27080259): 12136-1214410.1074/jbc.M116.717850Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). As shown in Fig. 5A, Noc increased PLK1–MYPT1 association discernably as reported (18Yamashiro 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 (18477460): 787-79710.1016/j.devcel.2008.02.013Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), but TMG + Glu together with Noc obliterated PLK1–MYPT1 affinity. As phosphorylated MYPT1 binds with PLK1–PBD (18Yamashiro 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 (18477460): 787-79710.1016/j.devcel.2008.02.013Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), we adopted GST pulldown experiments using PLK1–PBD, and GST–PLK1–PBD modestly increased binding with HA–MYPT1–4A (Fig. 5B). Then we employed FL–PLK1. When we directly utilized the 4A mutant to coIP PLK1, the interaction between MYPT1 and PLK1 substantially up-regulated (Fig. 5C). When His–PLK1 was applied in pulldown assays, 4A again manifested more robust association with PLK1 (Fig. 5D). In sum, the binding between PLK1 and MYPT1 was abolished during high O-GlcNAc. As the MYPT1 associates PLK1 to target" @default.
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• W3016277935 title "O-GlcNAcylation of myosin phosphatase targeting subunit 1 (MYPT1) dictates timely disjunction of centrosomes" @default.
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