טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBornstein Gil
SubjectConjugation of Cul1 by the Ubiquitin-like Protein NEDD8
and the Mode of its Action on SCFskp2 Ubiquitin
Ligase
DepartmentDepartment of Medicine
Supervisor ? 18? Avram Hershko
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Cullin-containing multiprotein complexes comprise the largest family of ubiquitin-protein ligases. The best studied of these is the SCF (Skp1-Cullin-F-box protein) class of ubiquitin ligases. Our laboratory has been studying an SCF ubiquitin ligase that contains the F-box protein Skp2 (SCFSkp2) that targets the Cdk inhibitor p27 for degradation in the transition of cells from G0/G1 to S phase of the cell cycle. As is the case with other cullin-based ubiquitin ligases, the assembly and activity of SCFSkp2 is regulated by the ligation of the small ubiquitin-like protein Nedd8 to a specific lysine residue at the C-terminal domain of Cul1. This process increases the ubiquitin ligase activity of SCF, in part by increasing its affinity for E2. Neddylation also controls the assembly of cullin-based ubiquitin ligases, since non-neddylated cullins bind tightly to a protein called CAND1. Binding of CAND1 to Cul1 prevents its neddylation, and also the binding of Skp1 to Cul1 and thus the assembly of the SCF complex. The extent of the neddylation of cullins is also affected by the COP9-signalosome (CSN), a large multi-protein complex that has a specific isopeptidase activity that de-neddylates cullins.


                The molecular mechanisms responsible for the regulation of neddylation and de-neddylation of cullins remained unknown. I examined this problem for the case of SCFSkp2. It was found that the supplementation of Skp2/Skp1, p27, Cdk2/cyclin E and Cks1 to extracts of HeLa cells or to reticulocyte lysates markedly increased levels of neddylated Cul1. Fractionation-reconstitution experiments indicated that Skp2-Skp1 abrogates the inhibitory influence of CAND1 on the neddylation of Cul1, while p27 substrate along with Cdk2/cyclin E and Cks1 prevent the action of the CSN complex to de-neddylate Cul1. Based on these findings, we propose the following sequence of events for the neddylation and assembly of the SCF complex: the process is initiated by Skp2/Skp1, that induces the dissociation of the Cul1-CAND1 complex and thus allows the neddylation of Cul1. The neddylated form of Cul1 is released from inhibition by CAND1, can bind Skp2/Skp1 and thus is assembled to the active SCFSkp2 complex. p27 substrate, along with Cdk2/cyclin E and Cks1, maintain Cul1 in the neddylated state by preventing the action of CSN to de-neddylate Cul1. Thus, the increased availability of Skp2, substrate and further components necessary for substrate presentation in transition of cells to the S phase may promote the neddylation and assembly of the SCFSkp2 ubiquitin ligase complex.