M. Kasim Diril1☯¤a, Xavier Bisteau1☯, Mayumi Kitagawa2, Matias J. Caldez1.3, Sheena Wee1,
Jayantha Gunaratne1.4, Sang Hyun Lee2,*, Philipp Kaldis1,3*
1 Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research),
Singapore, Republic of Singapore.
2 Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore, Republic of Singapore.
3 Department of Biochemistry, National University of Singapore (NUS), Singapore, Republic of Singapore.
4 Department of Anatomy, National University of Singapore (NUS), Singapore, Republic of Singapore.
☯These authors contributed equally to this work.
¤a Current address: Izmir Biomedicine and Genome Institute, Dokuz Eylul University, Izmir, Turkey.
Correspondence: Philipp Kaldis (firstname.lastname@example.org)
Published online in PLOS Genetics on 15 September 2016.
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, MastNULL 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 ofMastNULL 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.
Figure 1. Growth analysis of MastlNULL MEFs. (A) Three MEF lines isolated from different embryos were treated with 4-OHT or DMSO to
induce Mastl knockout (MastlNULL) and their proliferative potential was monitored by Alamar Blue proliferation assays for 8 days. AFU,
arbitrary fluorescence units. (B) MEFs were synchronized and recombination in the Mastl and Cdk1 loci were induced as described in the
Methods section. Cells were arrested in mitosis for 4 hours using 5μM Eg5 inhibitor S-Trityl-L-cysteine (STLC) starting from 20 hours after
release into full growth medium. Still pictures were taken using phase-contrast microscopy. Scale bar 100μm. (C) MEFs were fixed 24
hours after release into full growth medium and stained with anti-phospho-histone H3 Ser10 antibodies (pH3) to quantify mitotic cells using
FACS analysis. (D) MEFs expressing the histone H2B-YFP fusion protein were analyzed by time-lapse microscopy. Still images of a
dividing MastlNULL cell were acquired every 5 minutes. Scale bar 10μm. (E) Quantification of appearance of anaphase bridges in MastlFLOX and MastlNULL MEFs expressing the H2B-YFP. (F) MastlNULL cells were fixed and stained with DAPI 72 hours after synchronization and release into full growth medium. Scale bar 20μm.
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