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Posttranslational modification of p53 plays important roles in regulating its stability and activity. In the first part of my PhD, my main goal was to study the target sites for Mdm2-mediated ubiquitination within the p53 protein molecule and to examine the regulation of p53 levels and activity by different types of Mdm2-mediated ubiquitination. For this purpose I established in vitro and in vivo systems for Mdm2- mediated p53 ubiquitination. Based on these two systems I was able to show that in contrast to the previously published data, which reported that the six lysines at the extreme carboxyl-terminal domain of p53 are the main target for Mdm2-mediated p53 ubiquitination and degradation, a mutant p53 protein with substitution of the six Cterminal lysines to arginines (p53-6KR) can still undergo ubiquitination very efficiently by Mdm2. These results imply the presence of additional acceptor lysines within p53. In order to identify the lysine residues that serve as sites for Mdm2- mediated polyubiquitination, a series of p53 molecules were generated. By using in vitro ubiquitination assays, I showed that neither of the tested lysines are truly acceptors for ubiquitination by Mdm2. This result, together with the accumulated data showing that Mdm2 is able to promote either mono, oligo or polyubiquitination of p53 and additionally to use different types of ubiquitin chains, may imply that under different conditions Mdm2 is able to target different lysines of p53 with different ubiquitin chains, and this may have different biological consequences. In order to understand under which conditions Mdm2 catalyzes mono versus poly ubiquitination of p53, I tested whether different amounts of Mdm2 catalyze different patterns of p53 ubiquitination, in a dose-dependent manner, in my cell-free ubiquitination assay. Interestingly, while polyubiquitinated forms of p53 were generated quite efficiently when high amounts of Mdm2 were added, lower amounts stimulated preferentially p53 monoubiquitination. Therefore, I propose that Mdm2 can catalyze both mono and polyubiquitination, in a dosage-dependent manner. Since the levels of endogenous Mdm2 are dynamically regulated through the p53-Mdm2 feedback loop, these findings raise the possibility that different forms of ubiquitinated p53, perhaps with different biochemical and biological roles, can be formed within the same cell at different times and under different conditions. The goal of the second part of my PhD was to determine whether the p53 cofactor ASPP2 might affect Mdm2-mediated neddylation of p53. I found that ASPP2 increases overall protein neddylation in cells and Mdm2 inhibits it. Importantly, the addition of ASPP2 increases the conjugation of Nedd8 to p53, and Mdm2 inhibits this effect of ASPP2 and imposes instead the Mdm2-mediated p53 neddylation pattern. Finally, results that I obtained in the in vivo ubiquitination assay suggest that Mdm2 can inhibit the ubiquitination of ASPP2 by a yet unknown E3. These results suggest that Mdm2 and ASPP2 exert antagonistic functions and that Mdm2 can inhibit the effect of ASPP2 on p53. In the last part of my PhD I focused on the regulation of Mdm2 ubiquitination. It is well known that, like several other RING finger E3 ligases, Mdm2 is capable of catalyzing its own ubiquitination. I found that Mdm2 deletion mutants lacking the RING domain are still capable of being ubiquitinated, suggesting additional mechanisms for Mdm2 regulation. One protein that I considered in that regard is Fbw7γ, an F-box protein component of the SCF ubiquitin ligase complex. In coimmunoprecipitation experiments I found that Flag-Fbw7γ can be efficiently coprecipitated with Mdm2 in cells. Additionally, I found that Mdm2 can bind free ΔFbox-Flag-Fbw7γ, which is not associated with other components of the SCF complex. (Abstract shortened by UMI.)
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