Abstract
SummaryDefects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6–8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest.
Highlights
Results and Discussion mph1-D459A Is a Kinase-Dead Allele S. pombe Mph1 is the homolog of S. cerevisiae Mps1, but it is neither required for spindle pole duplication nor essential for cell viability [9]
To test whether Mph1 kinase activity was required for distinct functions in fission yeast, we made two ‘‘kinase-dead’’ alleles
Mph1 Mutants Display Reduced Mad2 and Mad3-APC/C Binding Fission yeast Mad2p and Mad3p bind to the APC/C in mitosis [15], and we employed cdc25 block and release to determine whether this interaction was perturbed in the absence of Mph1 kinase activity. cdc25 cells containing Lid1(Apc4)-tandem affinity purification (TAP), Mad2-GFP, and Mad3-GFP were arrested in G2 at 36C and released, to pass through a synchronous mitosis at 25C
Summary
Mph1 Mutants Display Reduced Mad2 and Mad3-APC/C Binding Fission yeast Mad2p and Mad3p bind to the APC/C in mitosis [15], and we employed cdc25 block and release to determine whether this interaction was perturbed in the absence of Mph1 kinase activity. Mph1 kinase is required for stable association of fission yeast spindle checkpoint proteins with mitotic APC/C.
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