IDENTIFICATION OF PHYSIOLOGICAL SUBSTRATES FOR SER/THR PROTEIN PHOSPHATASE 5

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这个由NINDS资助的项目的目标是确定蛋白磷酸酶5(PP5)的神经元底物。PP5是一种广泛表达的丝氨酸/苏氨酸磷酸酶，在结构上与蛋白磷酸酶1、2A和钙调神经磷酸酶相关。PP5参与了多种信号转导途径，但对其底物及其在这些信号转导途径中的作用知之甚少，对其生理调控也知之甚少。我们将使用蛋白质组学策略来确定PP5是否调节关键信号蛋白，确定PP5的新靶点，并比较从表达野生型PP5或突变形式的PP5的培养神经元细胞系中分离出的磷蛋白对磷酸酶抑制剂冈田酸的敏感性改变。其磷酸化状态随着冈田酸抗性PP5活性的变化而变化的蛋白质将被鉴定并接受进一步的分析，从而为PP5在神经元中的功能和调节产生可检验的假说。在MS分析中，由于磷酸基团的存在抑制了多肽的电离，并且pSer和pThr磷酸基团可能在电离过程中丢失，因此在MS分析过程中，磷酸肽产生低信号。由于这些技术挑战和尽可能广泛地对细胞蛋白质组进行采样的需要，这项研究需要一个具有高灵敏度和高通量能力的样本分析系统，具有分离磷酸肽和解释来自磷酸肽的MS模式的经验。Rossie博士在PNNL花了6个月的时间学习样品准备和处理技术、使用在线毛细管LC的IMac亲和层析，并进行试点研究，以探索从匹配样品中比较细胞磷蛋白的工作策略。在她的实验室里，两名学生现在接受了样本制备方面的培训，并正在生成两种类型的生物样本：如上所述的用于全细胞磷酸蛋白质组学的全细胞蛋白质提取物，以及PP5和结合伙伴的免疫纯化复合体。样品将被送往PNNL，利用高灵敏度和高通量的LC/MS系统进行磷酸肽的纯化和分析。

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. The goal of this NINDS-funded project is to identify neuronal substrates for protein phosphatase 5 (PP5). PP5 is a widely expressed Ser/Thr phosphatase structurally related to protein phosphatases 1, 2A and calcineurin. PP5 has been implicated in numerous signaling pathways, however little is known concerning its substrates and roles in these pathways and nothing is known concerning its physiologic regulation. We will use a proteomics strategy to determine if PP5 regulates key signaling proteins, to identify novel targets for PP5, and to compare phosphoproteins isolated from cultured neuronal cell lines expressing either wild type PP5 or a mutant form of PP5 with altered sensitivity to the phosphatase inhibitor okadaic acid. Proteins whose phosphorylation status changes as a function of okadaic acid-resistant PP5 activity will be identified and subjected to further analysis generating testable hypotheses for the function and regulation of PP5 in neurons. Phosphopeptides yield low signals during MS analysis because peptide ionization is suppressed by the presence of phosphate groups, and pSer and pThr phosphate groups can be lost during the ionization process. Due to these technical challenges and the need to sample the cellular proteome as broadly as possible, this study requires a sample analysis system with high sensitivity and high throughput capability, experience isolating phosphopeptides and interpreting MS patterns arising from phosphopeptides. Dr. Rossie spent 6 months at PNNL learning sample preparation and processing techniques, IMAC affinity chromatography using on-line capillary LC, and performing pilot studies to explore working strategies for comparing cellular phosphoproteins from matched samples. In her lab, two students are now trained in sample preparation and are generating two types of biological samples; whole cell protein extracts for whole cell phosphoproteomics as described above, and immunopurified complexes of PP5 and binding partners. Samples are being sent to PNNL on an ongoi ng basis for phosphopeptide purification and analysis utilizing the high sensitivity and high throughput LC/MS systems.