Physical and Functional Interactions Between PML & MDM2

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DESCRIPTION (provided by applicant): Inactivation of the p53 tumor suppressor is important in the development of most cancers. Factors that regulate p53 are therefore of great interest. PML activates p53 by recruiting it to multiprotein complexes termed PML-nuclear bodies (PML-NBs), whereas MDM2 inactivates p53 by promoting its degradation. Experiments performed in my laboratory demonstrate an in vitro and in vivo interaction between PML and MDM2 that is p53-independent. MDM2 co-immunoprecipitates with PML in cells transiently expressing both proteins, and recombinant, purified MDM2 forms a strong complex with a GST-PML fusion protein. Further, confocal microscopy reveals co-localization of endogenous PML and MDM2 in p53-null cells. We anticipate that interactions between PML and MDM2 may indirectly affect p53 levels or activity, and may also affect p53-independent functions of either protein. This grant will characterize the interaction between PML and MDM2 in vitro and in vivo, and determine the effect of this interaction on p53-dependent and independent functions of both PML and MDM2. Preliminary results suggest PML:MDM2 interaction is regulated by PML sumoylation and in response to DNA damaging stress. We will elaborate the effect of sumoylation on the interaction between PML and MDM2 in vitro and in vivo, and assess the interaction between PML and MDM2 normally and in response to stress. Preliminary results suggest PML can inhibit MDM2-mediated ubiquitination of itself and p53. We will determine the molecular basis for this inhibitory effect of PML. Further, we will assess the ability of the cancer-associated PML-RAR fusion protein to affect MDM2 ubiquitination activity. MDM2 disrupts PML-NBs and promotes nuclear exclusion of PML in transfected cells. We will determine the molecular basis for this effect. Our preliminary results indicate that MDM2 inhibits PML ability to stimulate nuclear receptor signaling. We will determine the mechanism by which MDM2 inhibits this ability of PML.