Regulation and Function of PP2A-B56delta

项目摘要 蛋白质磷酸化参与许多生物过程的调节，包括对 DNA损伤。虽然激酶信号已被广泛研究，磷酸酶的作用仍然存在 替补演员磷蛋白磷酸酶2A（PP 2A）负责大部分磷酸丝氨酸和磷酸化丝氨酸。 磷酸苏氨酸在细胞内脱磷酸化，并通过其调节B赋予其底物特异性 亚单位B56家族是PP 2A调节亚基的四个家族中最大的家族。所有B56亚型都识别 LxxIxE短线性基序（SLiM）。我们开发了一种SLiM介导的 竞争性抑制剂的方法来询问PP 2A-B56依赖性磷酸化蛋白质组，从而扩大 了解B56特异性底物。然而，我们对控制这种活动的机制的了解 PP 2A-B56仍然不完整。在B56抑制后，我们发现B56δ的磷酸丝氨酸88-90是 差异调节。我们的初步数据表明，这些网站是失活的性质。B56δ编码 PPP 2 R5 D基因，该基因在PPP 2 R5 D相关的发育障碍中发生突变，其特征在于认知障碍， 损伤和癫痫。此外，我们鉴定了抗氧化性1（OXR 1），一个含有LxxIxE基序的 参与细胞对氧化应激的反应和维持基因组稳定性的蛋白质，是B56 衬底然而，对OXR 1的调控知之甚少。在这里，我们建议确定磷酸化是如何 的S88-90调节B56δ和扩大我们的理解B56依赖性调节OXR 1。我们 中心假设是B56δ的S88-90是B56δ上的关键调节位点，其在以下方面起重要作用： 通过OXR 1信号调节DNA损伤反应。具体目标1研究B56δ的调节 S88-90磷酸化位点，使其磷酸化的激酶，以及它们的生物学功能， 在质谱法和体外和细胞研究中的方法。具体目标2旨在确定 PP 2A-B56在OXR 1去磷酸化中的作用，OXR 1 LxxIxE基序对这种相互作用的贡献，以及 B56-OXR 1轴在DNA损伤信号传导中的生物学意义。成功完成这些 目的是阐明B56δ的磷酸化调节以及PP 2A-B56在OXR 1去磷酸化中的作用。 通过了解这些蛋白质的调节，这些蛋白质与多种人类疾病有关，我们将 更好地了解他们与疾病相关的生理学，潜在的治疗靶点，以及 治疗干预在此申请中提出的项目和培训计划将提高我的技能， 生物化学和细胞生物学技术，实验设计，数据分析，科学交流， 指导和转化研究。达特茅斯学院和诺里斯科顿大学的合作环境 癌症中心和强大的导师和合作者网络为我提供了理想的环境， 完成本申请中提出的研究，并支持我成为一名医生科学家。

Project Summary Protein phosphorylation is involved in the regulation of many biological processes, including the response to DNA damage. While kinase signaling has been extensively investigated, the role of phosphatases remains understudied. Phosphoprotein phosphatase 2A (PP2A) is responsible for the majority of phosphoserine and phosphothreonine dephosphorylation within a cell and confers its substrate specificity through its regulatory B subunit. The B56 family is the largest of the four families of PP2A regulatory subunits. All B56 isoforms recognize an LxxIxE short linear motif (SLiM) on their substrates and interactors. We have developed a SLiM-mediated competitive inhibitor approach to interrogate the PP2A-B56-dependent phosphoproteome, thus expanding the understanding of B56-specific substrates. However, our knowledge of the mechanisms that govern the activity of PP2A-B56 remains incomplete. Upon B56 inhibition, we found phosphoserines 88-90 of B56δ to be differentially regulated. Our preliminary data suggest that these sites are inactivating in nature. B56δ is encoded by the gene PPP2R5D, which is mutated in PPP2R5D-related developmental disorder characterized by cognitive impairment and epilepsy. Furthermore, we identified Oxidative Resistance 1 (OXR1), an LxxIxE motif-containing protein involved in the cellular response to oxidative stress and the maintenance of genome stability, to be a B56 substrate. Yet little is known about the regulation of OXR1. Here, we propose to determine how phosphorylation of S88-90 regulates B56δ and to expand upon our understanding of B56-dependent regulation of OXR1. Our central hypothesis is that S88-90 of B56δ are critical regulatory sites on B56δ, which serves an essential role in modulating DNA damage responses through OXR1 signaling. Specific Aim 1 investigates the regulation of B56δ S88-90 phosphosites, the kinase(s) that phosphorylate them, and their biological function using innovative approaches in mass spectrometry and in vitro and in cell studies. Specific Aim 2 seeks to determine the role of PP2A-B56 in the dephosphorylation of OXR1, the contribution of the OXR1 LxxIxE motif to this interaction, and the biological significance of the B56-OXR1 axis in DNA damage signaling. The successful completion of these aims will elucidate the phosphoregulation of B56δ and the role of PP2A-B56 in the dephosphorylation of OXR1. By understanding the regulation of these proteins, which are involved in a variety of human diseases, we will gain a better understanding of their disease-relevant physiology, potential therapeutic targets, and points for therapeutic intervention. The proposed projects and training plan in this application will enhance my skills in biochemical and cell biological techniques, experimental design, data analysis, scientific communication, mentoring, and translational research. The collaborative environment at Dartmouth College and the Norris Cotton Cancer Center and the strong network of mentors and collaborators provide an ideal environment for me to complete the research proposed in this application and support my development as a physician scientist.