טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGolan Amnon
SubjectMolecular Processes Responsible for Cyclosome Activation
at Mitosis
DepartmentDepartment of Medicine
Supervisor ? 18? Avram Hershko


Abstract

Proteolysis of cell cycle regulators is carried out by the ubiquitin pathway.  Proteins are targeted for degradation by ligation to ubiquitin, a process that requires the action of 3 enzymes: the ubiquitin activating enzyme (E1), a ubiquitin carrier protein (E2) and a ubiquitin protein ligase (E3).  Proteins ligated to polyubiquitin chains are usually degraded by the 26S proteasome. The cyclosome/APC is a multiprotein complex whose E3-ubiquitin ligase activity is required for cell division. Cdk1-cyclin B is active at the beginning of the mitosis and phosphorylates the cyclosome. There is also some evidence that Plk plays a role as a cyclosome kinase. Four cyclosome subunits are known to be phosphorylated at the stage of mitosis: APC1, Cdc23, Cdc16 and Cdc27. The phosphorylation of the cyclosome in mitosis is necessary for its activation by a protein called fizzy.

The first goal in this study was to purify cyclosome to homogeneity and high enzyme activity, by biochemical methods. Next, the activation by phosphorylation of the cyclosome, which was purified from human cells was examined. The main objective was to investigate whether cyclosome subunits directly phosphorylated by the Cdk1-cyclin B and Plk1 or if there is a cascade of regulators that mediate between the kinases and the ligase. In contrast to the results of other groups, it was found that Plk1 is not activated by Cdk1-cyclin B. In addition, it was found that Cdk1-cyclin B and Plk1 phosphorylate different subunits of purified cyclosome. Another important finding of this study was that three of the cyclosome subunits (APC7, APC5, APC2) are also phosphorylated by Cdk1-cyclin B and Plk1.

We found that protein kinases Cdk1-cyclin B and Plk1 synergistically activate the cyclosome and suggest that this is due to the complementary pattern of phosphorylation of different cyclosome subunits by the two protein kinases.