DETERMINING THE ROLE OF PP5 IN THE HSP90 CHAPERONE COMPLEX

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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的主要结合蛋白，是一种分子伴侣，负责多种细胞蛋白的折叠和成熟。PP5在Hsp90分子伴侣复合体中的作用尚不清楚。在这个项目中，我们将确定PP5在Hsp90伴侣复合体中的作用和靶点。 我们将从表达野生型PP5、催化失活的PP5或不能结合Hsp90的突变型PP5的细胞中分离出PP5复合体。利用质谱学，我们将鉴定与PP5共免疫沉淀的蛋白质伙伴。不能结合Hsp90的PP5突变体的合作伙伴预计代表与Hsp90复合体无关的PP5合作伙伴。我们将确定含有野生型或非活性PP5的复合体中Hsp90和其他蛋白质的磷酸化状态，以确定Hsp90复合体中哪些成员的磷酸化随着PP5的活性而发生变化，并确定PP5去磷酸化的位置。由于PP5与糖皮质激素受体(GRs)和Hsp90结合，GRs可被磷酸化事件激活，并被PP5负性调节。我们还将具体评估GRs的磷酸化状态。最后，我们将分析大鼠脑中PP5：HSP90复合体的组成，以验证我们细胞研究中看到的蛋白质伙伴是否也存在于含有正常水平PP5的自然组织中。具体目标包括： 1.鉴定存在于PP5-Hsp90复合体中的PP5结合伙伴。 2.确定PP5是否使哺乳动物Hsp90、辅助伴侣和/或客户去磷酸化，并确定随着PP5活性的变化而变化的磷酸化位点。 3.确定PP5-Hsp90复合体中GRs是否被PP5去磷酸化。 4.鉴定大鼠脑内天然PP5-Hsp90复合体中存在的蛋白质。

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.