טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMiniowitz Shem-Tov Shirly
SubjectMolecular Mechanisms of Release from the Mitotic Checkpoint
DepartmentDepartment of Medicine
Supervisor ? 18? Avram Hershko
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

The mitotic checkpoint system ensures accurate segregation of chromosomes by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle.  When the checkpoint system is turned on, it promotes the formation of the Mitotic Checkpoint Complex (MCC), which inhibits the ubiquitin ligase Anaphase-Promoting Complex/ Cyclosome (APC/C) to target securin for degradation.  APC/C is inhibited by the MCC composed of BubR1, Bub3, Mad2, and Cdc20 . When the checkpoint is satisfied, MCC is disassembled and APC/C becomes active. The molecular mechanisms involved in the disassembly of APC/C inhibitors when the checkpoint is satisfied, and the events leading to the inactivation of the mitotic checkpoint, remain obscure .

In this research we have been studying the mechanisms of the release of APC/C from mitotic checkpoint inhibition. For this purpose, we used extracts from nocodazole-arrested cells that faithfully reproduced downstream events of the mitotic checkpoint system. When such extracts were incubated in the presence of ATP, APC/C was converted to an active form following a lag period. This was accompanied by the release of inhibitory factors from APC/C and the disassembly of MCC.

The first part of this research work focused on the requirement for ATP for the release of APC/C from inhibition by the mitotic checkpoint. Previously, it has been reported that polyubiquitylation by the APC/C is required for the inactivation of the mitotic checkpoint . We confirmed the involvement of polyubiquitylation, but found that another process, which requires ATP cleavage at the β-γ position is essential for the release of APC/C from checkpoint inhibition.

We also found that polyubiquitylation cannot replace ATP (β-γ) cleavage-requiring process to overcome arrest caused by the mitotic checkpoint .

In the second part of this research work, we focused on the possibility that the ATP requirement for MCC dissociation reflects the involvement of the phosphorylation of some protein(s) in this process.

We found that inhibition of cyclin dependent protein kinase blocks a part of the dissociation of MCC that takes place in MCC-containing immunoprecipitates in the presence of ATP and p31comet.

We found that the phosphorylation of Cdc20 in MCC by Cdk was stimulated by p31comet. Mutation of all 8 potential Cdk phosphorylation sites of Cdc20 prevented a part of its release from BubR1. These findings indicated that a part of the disassembly of MCC required Cdk-catalyzed phosphorylation of Cdc20 .

 Further processes in the disassembly to MCC remain to be elucidated .