טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMoshe Yakir
SubjectRegulation of the APC/C Ubiquitin Ligase by the Protein Emi1
DepartmentDepartment of Medicine
Supervisor ? 18? Avram Hershko
Full Thesis textFull thesis text - English Version


Abstract

The cell division cycle is a highly ordered sequence of events which is strictly regulated. Much of the regulation of the cell cycle is carried out by protein phosphorylation and by ubiquitin mediated degradation. Two of the primary regulators of the cell cycle are the cyclin dependent kinase family, CDKs, and the anaphase promoting complex/cyclosome, APC/C. CDKs are protein kinases which are dependent on the binding to a cyclin subunit for activation. For cells to exit from mitosis and prepare for the next cell division cycle, CDK activity must be extinguished. APC/C, acting with its' co-activators Cdc20 or Cdh1, targets cyclins for destruction by polyubiquitylating them.

Premature activation of APC/C can cause chromosome re-replication and mitotic catastrophes. An inhibitor of APC/C, early mitotic inhibitor 1, or Emi1, prevents premature degradation of cyclins and other APC/C substrates throughout S-phase. The mechanism of inhibition of APC/C by Emi1, and the factors regulating Emi1 function throughout the cell division cycle are unclear.

In this study we have examined the interactions between Emi1, APC/C and the co-activators of APC/C. We report that Emi1, APC/C and co-activators form a trimeric complex. The formation of this trimeric complex is correlated with inhibition of APC/C by Emi1. Inhibition of APC/C is dependent upon two conserved elements in the C-terminal portion of Emi1, but binding of Emi1 to APC/C is unaffected by mutation of these elements, suggesting that additional elements in Emi1 may be necessary to enable functional binding and inhibition of APC/C.

We also examined the inactivation of Emi1 upon entry into mitosis. This inactivation is achieved by two mechanisms. We have discovered that the mitotic kinase polo-like kinase 1 (Plk1) phosphorylates a conserved sequence in Emi1 which allows Emi1 to be ubiquitylated by a Skp1-Cullin1-F-box protein (SCF) complex containing the F-box protein β-TrCP. Phosphorylation of Emi1 by Plk1 is stimulated by the activity of cyclin B/CDK1, thus linking Emi1 degradation to the timing of the activation of these two mitotic kinases at entry into mitosis.

Phosphorylation of Emi1 by CDKs provides an additional mechanism for the inactivation of Emi1 at mitosis. In addition to promoting Emi1 degradation, we show that phosphorylation of Emi1 by CDKs prevents Emi1 from efficiently binding and inhibiting APC/C. CDK mediated phosphorylation of Emi1 in mitotic, but not S-phase extracts inactivates Emi1. Finally we suggest that a certain putative factor protects Emi1 throughout S-phase from CDK mediated inactivation.