IDENTIFICATION OF PHYSIOLOGICAL SUBSTRATES FOR SER/THR PROTEIN PHOSPHATASE 5

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。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 这个NINDS资助的项目的目标是确定蛋白磷酸酶5（PP 5）的神经元底物。 PP 5是一种广泛表达的Ser/Thr磷酸酶，结构上与蛋白磷酸酶1、2A和钙调神经磷酸酶相关。 PP 5与许多信号通路有关，然而关于其底物和在这些通路中的作用知之甚少，关于其生理调节也一无所知。 我们将使用蛋白质组学策略，以确定是否PP 5调节关键信号蛋白，以确定新的目标PP 5，并比较磷蛋白分离培养的神经元细胞系表达野生型PP 5或突变形式的PP 5与改变敏感性的磷酸酶抑制剂冈田酸。 蛋白质的磷酸化状态的变化作为冈田酸抗性PP 5活性的函数将被确定，并进行进一步的分析，产生可测试的假设PP 5在神经元中的功能和调节。 磷酸肽在MS分析期间产生低信号，因为磷酸基团的存在抑制了肽电离，并且pSer和pThr磷酸基团在电离过程中可能丢失。 由于这些技术挑战和需要尽可能广泛地对细胞蛋白质组进行采样，本研究需要具有高灵敏度和高通量能力的样品分析系统，分离磷酸肽和解释磷酸肽产生的MS模式的经验。 Rossie博士在PNNL学习了6个月的样品制备和处理技术，使用在线毛细管LC的IMAC亲和色谱，并进行了试点研究，以探索比较来自匹配样品的细胞磷蛋白的工作策略。 在她的实验室中，两名学生现在接受样品制备培训，并正在生成两种类型的生物样品;如上所述用于全细胞磷酸蛋白质组学的全细胞蛋白质提取物，以及PP 5和结合伴侣的免疫纯化复合物。 正在将样品送往PNNL，利用高灵敏度和高通量LC/MS系统进行磷酸肽纯化和分析。

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 ongoing basis for phosphopeptide purification and analysis utilizing the high sensitivity and high throughput LC/MS systems.