IDENTIFICATION OF PHYSIOLOGICAL SUBSTRATES FOR SER/THR PROTEIN PHOSPHATASE 5

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。子项目和 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。 该 NINDS 资助项目的目标是鉴定蛋白磷酸酶 5 (PP5) 的神经元底物。 PP5 是一种广泛表达的 Ser/Thr 磷酸酶，其结构与蛋白磷酸酶 1、2A 和钙调神经磷酸酶相关。 PP5 与许多信号传导途径有关，然而对其底物和在这些途径中的作用知之甚少，并且对其生理调节也一无所知。 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，识别 PP5 的新靶标，并比较从表达野生型 PP5 或对磷酸酶抑制剂冈田酸敏感性改变的 PP5 突变型的培养神经元细胞系中分离的磷蛋白。 磷酸化状态随冈田酸抗性 PP5 活性而变化的蛋白质将被鉴定并进行进一步分析，从而为神经元中 PP5 的功能和调节产生可测试的假设。 磷酸肽在 MS 分析过程中产生低信号，因为肽电离因磷酸基团的存在而受到抑制，并且 pSer 和 pThr 磷酸基团可能在电离过程中丢失。 由于这些技术挑战以及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，具有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 花了 6 个月的时间学习样品制备和处理技术、使用在线毛细管 LC 的 IMAC 亲和色谱，并进行试点研究以探索比较匹配样品中的细胞磷蛋白的工作策略。 在她的实验室中，两名学生现在正在接受样品制备培训，并正在生成两种类型的生物样品：用于如上所述的全细胞磷酸化蛋白质组学的全细胞蛋白提取物，以及PP5和结合配偶体的免疫纯化复合物。 样品被持续发送至 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.