title: Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint.
abstract: The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing.
authors.affiliation: Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Republic of Singapore.
authors.forename: M Kasim
authors.identifier: 0000-0002-3644-4178
authors.initials: MK
authors.lastname: Diril
authors.affiliation: Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Republic of Singapore.
authors.forename: Xavier
authors.identifier: 0000-0002-8896-5098
authors.initials: X
authors.lastname: Bisteau
authors.affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Republic of Singapore.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Kitagawa
authors.affiliation: Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Republic of Singapore.|Department of Biochemistry, National University of Singapore (NUS), Singapore, Republic of Singapore.
authors.forename: Matias J
authors.initials: MJ
authors.lastname: Caldez
authors.affiliation: Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Republic of Singapore.
authors.forename: Sheena
authors.identifier: 0000-0003-2536-3433
authors.initials: S
authors.lastname: Wee
authors.affiliation: Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Republic of Singapore.|Department of Anatomy, National University of Singapore (NUS), Singapore, Republic of Singapore.
authors.forename: Jayantha
authors.initials: J
authors.lastname: Gunaratne
authors.affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Republic of Singapore.
authors.forename: Sang Hyun
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Republic of Singapore.|Department of Biochemistry, National University of Singapore (NUS), Singapore, Republic of Singapore.
authors.forename: Philipp
authors.identifier: 0000-0002-7247-7591
authors.initials: P
authors.lastname: Kaldis
mesh_terms: D000818:Animals; D016203:CDC2 Protein Kinase / Q000235:genetics*; D020090:Chromosome Segregation / Q000235:genetics; D048749:Cytokinesis / Q000235:genetics; D018386:Kinetochores / Q000378:metabolism; D059566:M Phase Cell Cycle Checkpoints / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D008870:Microtubules / Q000235:genetics / Q000378:metabolism; D008938:Mitosis / Q000235:genetics; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D008941:Spindle Apparatus / Q000235:genetics
chemical_list: D008869:Microtubule-Associated Proteins; C441620:Ttk protein, mouse; D017346:Protein Serine-Threonine Kinases; C585164:greatwall protein, mouse; D016203:CDC2 Protein Kinase; C581986:Ppp2r2a protein, mouse; D054648:Protein Phosphatase 2