Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint

• DOI: 10.1371/journal.pgen.1006310
• Publication Date: 2016-09-15T17:55:02Z
• Authors: M. Kasim Diril; Xavier Bisteau; Mayumi Kitagawa; Matias J. Caldez; Sheena Wee; Jayantha Gunaratne; Sang Hyun Lee; Philipp Kaldis
• Keywords: 0301 basic medicine; M phase cell cycle checkpoint; Protein Phosphatase 2 -- genetics -- metabolism; animal cell; QH426-470; Microtubules; fibroblast; MPS1 protein; Mice; M Phase Cell Cycle Checkpoints -- genetics; Chromosome Segregation; CDC2 Protein Kinase -- genetics; phosphoprotein phosphatase 2A; enzyme phosphorylation; Protein Phosphatase 2; Phosphorylation; Kinetochores; cellular distribution; Mastl gene; Mice, Knockout; phosphoprotein phosphatase 2; Kinetochores -- metabolism; adult; Protein-Serine-Threonine Kinases -- genetics -- metabolism; Sciences bio-médicales et agricoles; Mitosis -- genetics; unclassified drug; enzyme activity; mitosis inhibition; cell enzyme; centromere; gene inactivation; Biologie; Microtubule-Associated Proteins; microtubule; Research Article; 570; in vitro study; enzyme localization; Knockout; Biochimie; animal experiment; chromosome segregation; embryo; Mitosis; cytokinesis; Spindle Apparatus; Protein Serine-Threonine Kinases; Article; animal tissue; Microtubules -- genetics -- metabolism; knockout gene; Microtubule-Associated Proteins -- genetics -- metabolism; 03 medical and health sciences; okadaic acid; protein serine threonine kinase; CDC2 Protein Kinase; greatwall protein; Genetics; Ttk protein; Animals; protein Mad1; controlled study; phosphotransferase; cyclin dependent kinase 1; mouse; Ppp2r2a protein; Cytokinesis; nonhuman; Biologie moléculaire; embryo development; gene loss; essential gene; cell proliferation; gene function; Spindle Apparatus -- genetics; Chromosome Segregation -- genetics; M Phase Cell Cycle Checkpoints; Biologie cellulaire; microtubule associated protein; Cytokinesis -- genetics; knockout mouse; protein dephosphorylation; protein determination

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.