IDENTIFICATION OF PHYSIOLOGICAL SUBSTRATES FOR SER/THR PROTEIN PHOSPHATASE 5

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。这个ninds资助的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。PP5参与了许多信号通路，但对其底物和在这些通路中的作用知之甚少，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，确定PP5的新靶点，并比较从表达野生型PP5或突变型PP5的培养神经细胞系中分离的磷酸化蛋白，这些磷酸化蛋白对磷酸酶抑制剂冈田酸的敏感性发生了改变。磷酸化状态随着抗冈田酸PP5活性的变化而变化的蛋白质将被识别并进行进一步分析，从而产生关于PP5在神经元中的功能和调节的可测试假设。磷酸肽在质谱分析中产生低信号，因为磷酸基团的存在抑制了肽的电离，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质谱模式的经验。罗西博士在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进行IMAC亲和色谱，并进行试点研究，探索比较匹配样品的细胞磷酸化蛋白的工作策略。在她的实验室里，两名学生正在接受样品制备方面的培训，并正在生成两种类型的生物样品；用于上述全细胞磷酸化蛋白质组学的全细胞蛋白提取物，以及PP5和结合伙伴的免疫纯化复合物。样品将持续送往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 ongoi ng basis for phosphopeptide purification and analysis utilizing the high sensitivity and high throughput LC/MS systems.