p31 ^comet promotes disassembly of the mitotic checkpoint complex in an ATP-dependent process

Abstract

Accurate segregation of chromosomes in mitosis is ensured by a surveillance mechanism called the mitotic (or spindle assembly) checkpoint. It prevents sister chromatid separation until all chromosomes are correctly attached to the mitotic spindle through their kinetochores. The checkpoint acts by inhibiting the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. The activity of APC/C is inhibited by a mitotic checkpoint complex (MCC), composed of the APC/C activator Cdc20 bound to the checkpoint proteins MAD2, BubR1, and Bub3. When all kinetochores acquire bipolar attachment the checkpoint is inactivated, but the mechanisms of checkpoint inactivation are not understood. We have previously observed that hydrolyzable ATP is required for exit from checkpoint-arrested state. In this investigation we examined the possibility that ATP hydrolysis in exit from checkpoint is linked to the action of the Mad2-binding protein p31 comet in this process. It is known that p31 comet prevents the formation of a Mad2 dimer that it thought to be important for turning on the mitotic checkpoint. This explains how p31 comet blocks the activation of the checkpoint but not how it promotes its inactivation. Using extracts from checkpoint-arrested cells and MCC isolated from such extracts, we now show that p31 comet causes the disassembly of MCC and that this process requires β,γ-hydrolyzable ATP. Although p31 comet binds to Mad2, it promotes the dissociation of Cdc20 from BubR1 in MCC.

Role of phosphorylation of Cdc20 in p31 comet -stimulated disassembly of the mitotic checkpoint complex

“…We found that the hydrolysis of ATP at the β−γ position was required for both for the activation of APC/C and for the disassembly of MCC (10); this was different from the previously described requirement of checkpoint inactivation for ubiquitylation (11,12), because the latter process involves the cleavage of the α−β bond of ATP (13). We have furthermore observed that β−γ-hydrolyzable ATP is needed for the action of p31 comet (a Mad2-binding protein known to be involved in checkpoint silencing and mitotic exit) (14-16) to promote MCC dissociation (17). Surprisingly, this process caused the dissociation of Cdc20 from BubR1 in MCC, even though p31 comet binds to Mad2 in this complex (17).…”

“…We previously found that ATP was required for exit from mitotic checkpoint (10) and observed that ATP cooperated with the Mad2-binding protein p31 comet to promote the disassembly of MCC (17). However, the mode of the action of ATP in these processes remained unknown.…”

“…1A, the influence of protein kinase inhibitors on the disassembly of MCC was examined. For this purpose, we used a previously established assay that followed the release of MCC components from BubR1 immunoprecipitates derived from checkpoint extracts (17). In this partially purified system, the joint supplementation of ATP and p31 comet caused synergistic release of Cdc20 and Mad2 from BubR1 (17).…”

“…We have furthermore observed that β−γ-hydrolyzable ATP is needed for the action of p31 comet (a Mad2-binding protein known to be involved in checkpoint silencing and mitotic exit) (14-16) to promote MCC dissociation (17). Surprisingly, this process caused the dissociation of Cdc20 from BubR1 in MCC, even though p31 comet binds to Mad2 in this complex (17). The mode of action of ATP in MCC dissociation remained unknown.…”

The Biochemistry of Mitosis

“…But recent attention has focused on mutual inhibition between the MCC and the APC/C (Reddy et al 2007), which requires a particular APC/C subunit, APC15 (Mansfeld et al 2011;Foster and Morgan 2012;Uzunova et al 2012). In animal cells, a second important player for MCC disassembly is the p31 comet protein (Habu et al 2002;Teichner et al 2011;Westhorpe et al 2011), which has recently been implicated in targeting an AAA ATPase to the MCC to initiate its disassembly (Eytan et al 2014;Wang et al 2014).…”