DETERMINING THE ROLE OF PP5 IN THE HSP90 CHAPERONE COMPLEX

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 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。 使用质谱法，我们将鉴定与 PP5 免疫共沉淀的蛋白质伴侣。 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。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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, 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.