Phosphorylation Regulates the p31Comet-Mitotic Arrest-deficient 2 (Mad2) Interaction to Promote Spindle Assembly Checkpoint (SAC) Activity

Dipali A Date,Amy C Burrows,Matthew K Summers

doi:10.1074/jbc.m113.520841

Abstract

The spindle assembly checkpoint (SAC) ensures the faithful segregation of the genome during mitosis by ensuring that sister chromosomes form bipolar attachments with microtubules of the mitotic spindle. p31(Comet) is an antagonist of the SAC effector Mad2 and promotes silencing of the SAC and mitotic progression. However, p31(Comet) interacts with Mad2 throughout the cell cycle. We show that p31(Comet) binds Mad2 solely in an inhibitory manner. We demonstrate that attenuating the affinity of p31(Comet) for Mad2 by phosphorylation promotes SAC activity in mitosis. Specifically, phosphorylation of Ser-102 weakens p31(Comet)-Mad2 binding and enhances p31(Comet)-mediated bypass of the SAC. Our results provide the first evidence for regulation of p31(Comet) and demonstrate a previously unknown event controlling SAC activity.

Highlights

The spindle assembly checkpoint (SAC)2 is an evolutionarily conserved and essential surveillance mechanism that ensures that chromosome segregation during mitosis proceeds with high fidelity
Recovery from SAC activity occurs in two steps: termination of mitotic checkpoint complex (MCC) formation and disassembly of preexisting MCCs. p31Comet is a key factor in the recovery of cells from SAC activity [12,13,14,15,16]. p31Comet binds C-Mad2 via the same binding interface as O-Mad2 [9, 12, 17, 18]. p31Comet silences SAC activity in vitro and in vivo in a manner that requires this interaction with Mad2 [18]. p31Comet interacts with C-Mad2 in both the MCC and the C-Mad21⁄7Mad1 complex and has been demonstrated to antagonize both [9, 12, 17,18,19]
We confirmed that immunoprecipitation of Mad2 using an antibody that binds C-Mad2 via the O-Mad2/p31Comet binding surface does not co-precipitate p31Comet as would be expected if an additional binding interface exists (Fig. 3D). These data indicate that p31Comet interacts with C-Mad2 and the C-Mad2:Mad1 heterodimer solely via the O-Mad2 binding interface

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—HeLa and HCT116 cells were obtained from ATCC and maintained in DMEM supplemented with 10% FBS. Isogenic p31Comet WT and S102A cell lines were generated with the use of pGLAP2 as described [24]. Equal amounts of cell lysates were incubated with the indicated antibodies for 2–12 h and washed in EBC buffer, including inhibitors. Lysates and p31Comet or Mad immunoprecipitates (IP) were resolved by SDS-PAGE and blotted with the indicated antibodies. Cyclins A and B were probed as markers for the indicated cell cycle phases. C, asynchronous lysates were immunoprecipitated with control IgGs or the indicated antibodies and analyzed as described in A. Statistical Analysis—One-way analysis of variance with Bonferroni post tests were performed using GraphPad Prism

RESULTS

DISCUSSION