טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentYudkovsky Yana
SubjectThe Mechanisms of the Regulation of Cyclosome Activity
in Somatic Human Cells
DepartmentDepartment of Medicine
Supervisor ? 18? Avram Hershko


Abstract

 The cyclosome/Anaphase Promoting Complex (APC) is a multi-subunit ubiquitin ligase, which targets for degradation specific cell cycle regulatory proteins, such as mitotic cyclins and securin, an inhibitor of anaphase initiation.  It is  activated at the end of mitosis  by  phosphorylation  and  association with the WD-40 protein  Cdc20/Fizzy and is  kept  active in the G1 phase by  association with  Cdh1/Fizzy-related.  The mitotic  checkpoint  system that  keeps  cells with defective spindles from leaving mitosis interacts with Cdc20 and prevents its stimulatory  action on the cyclosome/APC.  The aim of this study was to examine the mode of the regulation of the cyclosome/APC in somatic mammalian cells.

          It was found that, in contrast to Cdh1, Cdc20 specifically activated cyclosome from M-phase cells and that the phosphorylation of mammalian cyclosome was required for this activation.  It was furthermore observed that activity of Cdc20 is negatively regulated by Cdk-mediated phosphorylation, and   this may provide a safeguard, in addition to the mitotic checkpoint system to prevent the premature or inappropriate activation of the cyclosome/APC in early mitosis. This notion was supported by the observation that a non-phosphorylatable derivative of Cdc20  stimulated cyclin-ubiquitin ligation in extracts from nocodazole-arrested cells.

            Next, an attempt was made to gain some insight into the molecular mechanisms of the cyclosome/APC regulation by the checkpoint proteins Mad2 and BubR1. It was found that combination of low concentrations of Mad2 and BubR1 specifically inhibited the activity of the mitotic (phosphorylated) form of the cyclosome-Cdc20 in an additive mode.