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，调节p53的因子引起了极大的兴趣。PML通过将p53募集到称为PML-核小体（PML-NB）的多蛋白复合物中来激活p53，而MDM 2通过促进其降解来灭活p53。在我的实验室进行的实验表明，在体外和体内的PML和MDM 2之间的相互作用是p53独立的。MDM 2与PML在瞬时表达两种蛋白质的细胞中共免疫沉淀，并且重组纯化的MDM 2与GST-PML融合蛋白形成强复合物。此外，共聚焦显微镜揭示了内源性PML和MDM 2在p53缺失细胞中的共定位。我们预计PML和MDM 2之间的相互作用可能间接影响p53水平或活性，也可能影响p53独立的功能。该基金将在体外和体内表征PML和MDM 2之间的相互作用，并确定这种相互作用对PML和MDM 2的p53依赖和独立功能的影响。初步结果表明，PML：MDM 2相互作用是由PML类小泛素化和响应DNA损伤应力。我们将详细阐述sumoylation的PML和MDM 2之间的相互作用在体外和体内的影响，并评估PML和MDM 2之间的相互作用正常和应激反应。初步结果表明PML可以抑制MDM 2介导的自身和p53的泛素化。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MDM 2泛素化活性的能力。MDM 2破坏PML-NB并促进转染细胞中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，MDM 2抑制PML刺激核受体信号传导的能力。我们将确定MDM 2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.