|Ph.D Student||Sitry-Shevah Danielle|
|Subject||Mechanisms of Degradation of p27 and their Control in the|
|Department||Department of Medicine||Supervisors||Professor Herman Wolosker|
|? 18? Avram Hershko|
|Full Thesis text - in Hebrew|
The cyclin-dependent kinase (Cdk) inhibitor p27 has important roles in the control of the proliferation of mammalian cells. p27 binds to and inhibits the action of protein kinases Cdk2/cyclin E and Cdk2/cyclin A, which are necessary for progress in S-phase. The levels of p27 are high in quiescent cells. Following stimulation by mitogenic agents, p27 is rapidly degraded. The ubiquitylation and degradation of p27 in the S phase require its phosphorylation on T187. Phosphorylated p27 is recognized by an SCF (Skp1-Cullin 1-F-box protein) ubiquitin ligase complex, which contains Skp2 (S-phase kinase-associated protein 2) as the specific substrate-binding F-box protein. SCFSkp2 requires an accessory protein, Cks1 (Cdc kinase subunit 1). Cks1 promoted the binding of Skp2 to T187-phosphorylated p27.
We identified the Skp2-binding site of Cks1 and showed that all three sites of Cks1 are required for its action to promote Skp2-p27 binding. We found that the Skp2-binding site of Cks1 is located on a region including the a2 and a1 helices and their immediate vicinity, well separated from the other two binding sites (anion and Cdk binding sites). Cks1 and Skp2 mutually promote the binding of each other to a peptide similar to the 19 C-terminal amino acids of p27 containing phosphorylated T187. This latter process requires the Skp2- and anion-binding sites of Cks1. The interaction of Skp2 with the substrate is further strengthened by the association of the Cdk-binding site of Cks1 with Cdk2/cyclin E, to which phosphorylated p27 is bound.
We found by using Skp2-/- mice fibroblast cell lines that the levels of p27 in the G1 phase were much higher than those in wild-type fibroblasts. Similarly, levels of non-phosphorylatable p27T187V mutant were very high in cells lacking Skp2, in both G1 and S phases. These data suggested that the degradation of both wild-type and non-phosphorylated p27 in G1 requires the action of SCFSkp2. In S-phase cells, the non-phosphorylatable mutant of p27 is degraded in a Skp2-dependent manner, although at a rate slower than wild-type p27. Ubiquitylation of non-phosphorylatable p27T187A mutant was much slower than that of wild-type p27 in S phase extracts, and was only partially dependent on Skp2.
We found that in cells most of the Cul1 which is in the SCF complex is ligated to Nedd8, as compared to the lower extent of neddylation of total Cul1. We proposed that the non-phosphorylated p27 is ligated to ubiquitin by a mechanism dependent on neddylation of Cul1 in the SCF complex.