DETERMINING THE ROLE OF PP5 IN THE HSP90 CHAPERONE COMPLEX

确定 PP5 在 HSP90 伴侣复合物中的作用

基本信息

批准号：
8365466
负责人：
SANDRA ROSSIE
金额：
$ 2.87万
依托单位：
BATTELLE PACIFIC NORTHWEST LABORATORIES
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2012-06-30
项目状态：
已结题

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A major goal of the Rossie lab is to determine the substrates for the serine/threonine phosphatase protein phosphatase 5 (PP5). Heat shock protein 90 (Hsp90), the major cellular binding partner for PP5, is a molecular chaperone responsible for the folding and maturation of numerous cellular proteins. The role of PP5 in Hsp90 chaperone complexes is not understood. In this project, we will define the role and targets of PP5 in the Hsp90 chaperone complex. We will isolate PP5 complexes from cells expressing wild-type PP5, catalytically inactive PP5 or a mutant form of PP5 unable to bind Hsp90. Using mass spectrometry centric capabilities in the Center, we will identify protein partners that are co-immunoprecipitated with PP5. Partners of the PP5 mutant that cannot bind Hsp90 are expected to represent PP5 partners unrelated to the Hsp90 complex. We will determine the phosphorylation state of Hsp90 and other proteins in complexes containing either wild-type or inactive PP5 to determine which members of the Hsp90 complex undergo changes in phosphorylation as a function of PP5 activity and identify sites of PP5 dephosphorylation. Since PP5 is complexed with glucocorticoid receptors (GRs) together with Hsp90, GRs can be activated by phosphorylation events and are negatively regulated by PP5. We will also specifically evaluate the phosphorylation status of GRs. Finally we will analyze the composition of PP5:Hsp90 complexes from rat brain in order to verify that the protein partners seen in our cell studies also exist in native tissue containing normal levels of PP5. Specific Aims include: 1. To identify PP5 binding partners present in the PP5-Hsp90 complexes. 2. To determine whether PP5 dephosphorylates mammalian Hsp90, co-chaperones and/or clients, and identify sites of phosphorylation that change as a function of PP5 activity. 3. To determine if GRs are dephosphorylated by PP5 in PP5-Hsp90 complexes. 4. To identify proteins present in native PP5-Hsp90 complexes isolated from rat brain.

该子项目是利用资源的众多研究子项目之一由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持并且子项目的主要研究者可能是由其他来源提供的，包括其他 NIH 来源。子项目可能列出的总成本代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 Rossie 实验室的一个主要目标是确定丝氨酸/苏氨酸磷酸酶蛋白磷酸酶 5 (PP5) 的底物。热休克蛋白 90 (Hsp90) 是 PP5 的主要细胞结合伴侣，是负责众多细胞蛋白折叠和成熟的分子伴侣。 PP5 在 Hsp90 伴侣复合物中的作用尚不清楚。在这个项目中，我们将定义 PP5 在 Hsp90 伴侣复合物中的作用和目标。我们将从表达野生型 PP5、催化失活 PP5 或无法结合 Hsp90 的 PP5 突变形式的细胞中分离 PP5 复合物。利用该中心以质谱为中心的功能，我们将鉴定与 PP5 免疫共沉淀的蛋白质伙伴。不能结合 Hsp90 的 PP5 突变体伴侣预计代表与 Hsp90 复合物无关的 PP5 伴侣。我们将确定含有野生型或失活 PP5 的复合物中 Hsp90 和其他蛋白质的磷酸化状态，以确定 Hsp90 复合物的哪些成员随着 PP5 活性的变化而发生磷酸化变化，并鉴定 PP5 去磷酸化位点。由于 PP5 与糖皮质激素受体 (GR) 以及 Hsp90 复合，因此 GR 可以通过磷酸化事件激活，并受到 PP5 的负向调节。我们还将专门评估GRs的磷酸化状态。最后，我们将分析大鼠大脑中 PP5:Hsp90 复合物的组成，以验证我们的细胞研究中看到的蛋白质伴侣也存在于含有正常水平 PP5 的天然组织中。具体目标包括： 1. 鉴定 PP5-Hsp90 复合物中存在的 PP5 结合伴侣。 2. 确定 PP5 是否使哺乳动物 Hsp90、共伴侣和/或客户去磷酸化，并鉴定随 PP5 活性而变化的磷酸化位点。 3. 确定 PP5-Hsp90 复合物中的 GR 是否被 PP5 去磷酸化。 4. 鉴定从大鼠脑中分离的天然 PP5-Hsp90 复合物中存在的蛋白质。