Correction: Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests.
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ABSTRACT:
SUBMITTER: Zich J
PROVIDER: S-EPMC4839610 | biostudies-literature | 2016 Apr
REPOSITORIES: biostudies-literature
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Correction: Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests.
Zich Judith J May Karen K Paraskevopoulos Konstantinos K Sen Onur O Syred Heather M HM van der Sar Sjaak S Patel Hitesh H Moresco James J JJ Sarkeshik Ali A Yates John R JR Rappsilber Juri J Hardwick Kevin G KG
PLoS genetics 20160421 4
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Project description:The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1(Mph1) kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1(Mph1) kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20(Slp1)-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1(Mph1) kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.
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Project description:The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mads/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We chow biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20Slp1-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1Mph1 kinase-dependant modification of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.
2016-03-31 | PXD003806 | Pride
Project description:Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset.
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