DETERMINING THE ROLE OF PP5 IN THE HSP90 CHAPERONE COMPLEX

确定 PP5 在 HSP90 伴侣复合物中的作用

• 批准号: 7957013
• 负责人: SANDRA ROSSIE
• 金额: $ 6.5万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957013
• 关键词: Binding; Biology; Brain; Cells; Client; Complex; Computer Retrieval of Information on Scientific Projects Database; Event; Funding; Glucocorticoid Receptor; Goals; Grant; Heat-Shock Proteins 90; Institution; Mass Spectrum Analysis; Molecular Chaperones; PP5 protein-serine-threonine phosphatase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Protein Dephosphorylation; Proteins; Proteomics; Rattus; Research; Research Personnel; Resources; Role; Serine; Site; Source; Threonine; Tissues; United States National Institutes of Health; member; mutant

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Rossie实验室的主要目标是确定丝氨酸/苏氨酸磷酸酶蛋白磷酸酶5（PP 5）的底物。 热休克蛋白90（Heat shock protein 90，Hsp 90）是PP 5的主要细胞结合伴侣，是一种分子伴侣，负责许多细胞蛋白的折叠和成熟。 PP 5在Hsp 90分子伴侣复合物中的作用尚不清楚。 在这个项目中，我们将定义PP 5在Hsp 90伴侣复合物中的作用和靶点。 我们将从表达野生型PP 5、无催化活性的PP 5或不能结合Hsp 90的PP 5突变体的细胞中分离PP 5复合物。 使用质谱，我们将确定与PP 5共免疫沉淀的蛋白质伴侣。 不能结合Hsp 90的PP 5突变体的配偶体预期代表与Hsp 90复合物无关的PP 5配偶体。 我们将确定的磷酸化状态的热休克蛋白90和其他蛋白质的复合物含有野生型或无活性的PP 5，以确定哪些成员的热休克蛋白90复合物进行磷酸化的PP 5活性的函数的变化，并确定PP 5去磷酸化的网站。 由于PP 5与糖皮质激素受体（GR）以及Hsp 90复合，GR可以通过磷酸化事件激活并且由PP 5负调节。 我们还将专门评估GR的磷酸化状态。 最后，我们将分析PP 5的组成：热休克蛋白90复合物从大鼠脑，以验证我们的细胞研究中看到的蛋白质合作伙伴也存在于天然组织中含有正常水平的PP 5。 具体目标包括： 1. 鉴定PP 5-Hsp 90复合物中存在的PP 5结合配偶体。 2. 确定PP 5是否使哺乳动物Hsp 90、辅助分子伴侣和/或客户去磷酸化，并鉴定磷酸化位点随PP 5活性的变化而变化。 3. 为了确定GR是否被PP 5-Hsp 90复合物中的PP 5去磷酸化。 4. 鉴定从大鼠脑中分离的天然PP 5-Hsp 90复合物中存在的蛋白质。