Physical and Functional Interactions Between PML & MDM2

描述（由申请人提供）：p53肿瘤抑制因子的失活在大多数癌症的发展中是重要的。因此，调控p53的因子引起了极大的兴趣。PML通过将p53招募到称为PML-核小体（PML- nbs）的多蛋白复合物中来激活p53，而MDM2通过促进其降解来灭活p53。在我的实验室进行的实验表明，PML和MDM2之间的体外和体内相互作用是p53独立的。MDM2在短暂表达两种蛋白的细胞中与PML共免疫沉淀，重组纯化的MDM2与GST-PML融合蛋白形成强复合物。此外，共聚焦显微镜显示内源性PML和MDM2在p53-null细胞中共定位。我们预计PML和MDM2之间的相互作用可能间接影响p53的水平或活性，也可能影响两种蛋白的p53独立功能。该资助将表征PML和MDM2在体外和体内的相互作用，并确定这种相互作用对PML和MDM2依赖p53和独立功能的影响。初步结果表明，PML与MDM2的相互作用受PML酰化调控，并对DNA损伤应激作出反应。我们将详细阐述sumoylation对PML和MDM2在体内外相互作用的影响，并评估PML和MDM2在正常情况下和应激反应下的相互作用。初步结果表明PML可以抑制mdm2介导的自身泛素化和p53。我们将确定PML这种抑制作用的分子基础。此外，我们将评估癌症相关PML-RAR融合蛋白影响MDM2泛素化活性的能力。MDM2破坏PML- nb，促进转染细胞中PML的核排斥。我们将确定这种效应的分子基础。我们的初步结果表明，MDM2抑制PML刺激核受体信号的能力。我们将确定MDM2抑制PML这种能力的机制。

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.