טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDiab Sammer
SubjectThe Involvement of the E3 Ubiquitin Ligase Mdm2 in the
Regulation of the Oncoprotein c-Myc
DepartmentDepartment of Medicine
Supervisor ? 18? Aaron Ciechanover
Full Thesis textFull thesis text - English Version


Abstract

The c-myc proto-oncogene encodes a short-lived multifunctional protein that plays important roles in a broad array of cellular processes, including cell cycle  regulation, cell growth, differentiation, apoptosis, and malignant transformation. The many, and at times apparently opposing, roles played by c-Myc imply that the cellular levels of the protein must be tightly regulated.  c-Myc is an extremely short-lived protein with a half life of ~30 min.  Several groups, initially ours, have shown that c-Myc is degraded in vitro and in vivo by the ubiquitin-proteasome pathway. Importantly and not surprisingly, several E3s have been implicated in the targeting of Myc, including the E6/E6-AP complex, Fbw7, Skp2, Hect9, and p300. These findings clearly demonstrate the complexity involved with Myc regulation and suggest that the different E3s do not overlap and are not functionally redundant. Interestingly, the finding that Myc and p53 can, under certain conditions, cooperate in induction of apoptosis, has raised the interesting possibility that under these conditions, they may be targeted by the same ligases, thus ensuring their coordinated regulation.  Hence, it was attractive to test the hypothesis that Mdm2, the main ubiquitin ligase that regulates p53, catalyzes also c-Myc ubiquitination, leading to its degradation.  Indeed, our results provide evidence that Mdm2 interacts with c-Myc in vitro and in vivo. Moreover, we show, in vitro, that Mdm2 can promote ubiquitination of c-Myc, that is dependent on the RING domain, since mutant Mdm2 lacking the RING finger failed to ubiquitinate c-Myc. Also, we demonstrated that the level of c-Myc is decreased upon WT Mdm2 overexpression, and Mdm2-mediated c-Myc degradation is slowed down by a mutation in the RING domain. Decreasing the level of Mdm2 by silencing its gene, results in an increase in the level of endogenous and exogenous c-Myc. Using the temperature-sensitive p53Val135 mutant results in induction of Mdm2 and a decrease in the level of c-Myc in the permissive but not in the non-permissive temperature, and also causes shortening of the half life of c-Myc. In the context of DNA damage induced by Doxorubicin, the level of endogenous c-Myc was inversely correlated to the level of Mdm2, which implies that Mdm2 can degrade c-Myc, and the degradation is elicited following DNA damage. The same result was obtained when we used cells that lack p53. The inverse correlation between the two proteins was prevented by silencing Mdm2, or in the presence of the proteasome inhibitor MG132.