p31comet-mediated extraction of Mad2 from the MCC promotes efficient mitotic exit

114

131

Significance The mitotic checkpoint system has an important role to ensure accurate segregation of chromosomes in mitosis. This system regulates the activity of the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) by the formation of a negatively acting Mitotic Checkpoint Complex (MCC). When the checkpoint is satisfied, MCC is disassembled, but the mechanisms of MCC disassembly are not well understood. We show here that the ATP-hydrolyzing enzyme Thyroid Receptor Interacting Protein 13 (TRIP13), along with the MCC-targeting protein p31 comet , promote the disassembly of the mitotic checkpoint complexes and the inactivation of the mitotic checkpoint. The results reveal an important molecular mechanism in the regulation of APC/C by the mitotic checkpoint.

Section: Resultsmentioning

confidence: 99%

Disassembly of mitotic checkpoint complexes by the joint action of the AAA-ATPase TRIP13 and p31 ^comet

Eytan

Wang

Miniowitz-Shemtov

et al. 2014

114

131

“…Still another, long-standing mystery is that though p31 comet binds to MCC already during active mitotic checkpoint (14,26,27), it acts only later to promote checkpoint silencing and exit from mitosis (14,15,(26)(27)(28). It is possible that p31 comet is converted to an active form when the checkpoint is turned off, or that an inhibitor of p31 comet action is eliminated at that time by some signal linked to the state of the mitotic checkpoint system.…”

Section: Discussionmentioning

confidence: 99%

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

Miniowitz-Shemtov

Eytan

Ganoth

et al. 2012

The mitotic checkpoint system delays anaphase until all chromosomes are correctly attached to the mitotic spindle. When the checkpoint is turned on, it promotes the formation of the mitotic checkpoint complex (MCC), which inhibits the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2 bound to the APC/C activator Cdc20. When the checkpoint is satisfied, MCC is disassembled and APC/C becomes active. Previous studies have shown that the Mad2-binding protein p31 comet promotes the dissociation of Cdc20 from BubR1 in MCC in a process that requires ATP. We now show that a part of MCC dissociation is blocked by inhibitors of cyclin-dependent kinases (Cdks) and that purified Cdk1–cyclin B stimulates this process. The mutation of all eight potential Cdk phosphorylation sites of Cdc20 partially prevented its release from BubR1. Furthermore, p31 comet stimulated Cdk-catalyzed phosphorylation of Cdc20 in MCC. It is suggested that the binding of p31 comet to Mad2 in MCC may trigger a conformational change in Cdc20 that facilitates its phosphorylation by Cdk, and that the latter process may promote its dissociation from BubR1.

“…We thus did not perform additional functional assays with these mutants in human cells because any potential defects associated with these mutants could not be cleanly attributed to I-Mad2 formation. Conformationspecific Mad2 antibodies have been used to probe Mad2 conformational dynamics in human cells (33)(34)(35). In particular, an antibody developed by Taylor and coworkers specifically recognizes the dynamically bound copy of Mad2, but not Mad1-bound C-Mad2, at kinetochores (33).…”

Section: I-mad2 Formation Facilitates the Spontaneous O-mad2 To C-mad2mentioning

confidence: 99%

Structure of an intermediate conformer of the spindle checkpoint protein Mad2

Hara

Özkan

Sun

et al. 2015

The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1–Mad2 core complex. In mitosis, kinetochore-bound Mad1–C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. Here, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2–binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2–C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.