Proteolysis plays an important role in regulating cellular proliferation, differentiation and stress response. Studies supported by the current funding period have established and characterized JNK targeting of the ubiquitination and degradation of c-Jun, AFT2, JunB, and p53. In all cases JNK targeting takes place in non-stressed cells when the targeted protein is non-phosphorylated. Upon stress, the phosphorylation of JNK substrates renders them protected from JNK targeting. Since the duration and magnitude of activities elicited by key regulatory proteins is determined by the delicate balance between protein protection from- or targeting for- degradation, our competing renewal application focuses on mechanisms that underlie the protection of stress regulatory proteins from ubiquitination and degradation. Most recent studies from our laboratory have focused on SUMO-1 - an ubiquitin-like protein that is conjugated to a rapidly growing number of substrates. In these preliminary studies, we have established, using the model of Mdm2 E3 ubiquitin ligase, that protein sumoylation (conjugating a single SUMO-1 molecule via respective E1 and E2 to target protein) is key to its stabilization. Further, our studies establish that Mdm2 sumoylation takes place on the same lysine residue that is otherwise ubiquitinated. Importantly, sumoylation of Mdm2 increases its ubiquitin ligase activity. These findings provide the foundation for our working hypothesis, which proposes that SUMO-1 modification of ubiquitin ligase proteins, using Mdm2 as a model, is a key regulatory event in its stability and ubiquitin ligase activity. Our proposed studies are aimed at understanding the mechanisms that regulate Mdm2 sumoylation as opposed to ubiquitination both in normal and transformed cells, using malignant melanoma as a model. Accordingly, we propose to: (1) Identify the effects of post-translational modifications on Mdm2 sumoylation, Mdm2 self-ubiquitination, and on Mdm2-targeted ubiquitination of p53; (2) Determine the contribution of Mdm2-associated proteins to its sumoylation and ubiquitin ligase activities. (3) Test the hypothesis that Mdm2 sumoylation contributes to its oncogenic potential. (4) Determine binding sites for Ubc9 and UbcH5b - E2 for SUMO-1 and ubiquitin, respectively. Identify RING finger proteins that share the same structural motifs. (5) Identify (via computer modeling and experimental approaches) minimal size fragments from Mdm2 (or its regulatory proteins) that would attenuate its sumoylation or E3 ligase activities. In all, our proposed studies will establish the mechanism underlying Sumo-conjugation to Mdm2, its significance to the biology of this oncogene and develop reagents that alter Mdm2 sumoylation.
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