Regulation and Function of PP2A-B56delta

项目摘要 蛋白质磷酸化参与了许多生物过程的调节，包括对 DNA损伤。虽然已经广泛研究了激酶信号，但磷酸酶的作用仍然存在。 未得到充分研究。磷酸蛋白磷酸酶2A(PP2A)负责大部分的磷酸丝氨酸和 细胞内的磷酸苏氨酸去磷酸化，并通过其调节B赋予底物专一性 亚单位。B56家族是PP2A调节亚基四个家族中最大的一个。所有B56亚型都能识别 在它们的底物和相互作用体上有一个LxxIxE短线状基序(SLIM)。我们已经开发出一种由SLIM中介的 竞争性抑制物方法询问依赖PP2A-B56的磷酸蛋白质组，从而扩展 了解B56特定的底物。然而，我们对管理这一活动的机制的了解 PP2A-B56的同源性仍未完成。通过对B56的抑制，我们发现B56δ的磷酸丝氨酸88-90是 差异化监管。我们的初步数据表明，这些站点在自然界中处于失活状态。B56δ编码 由PPP2R5D基因突变，在PPP2R5D相关的发育障碍中表现为认知功能障碍 损伤和癫痫。此外，我们还鉴定了氧化抗性1(OXR1)，这是一个包含LxxIxE基序的基因 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，将成为B56 底物。然而，人们对OXR1的调控知之甚少。在这里，我们建议确定磷酸化如何 S88-90调节B56的δ，并在我们理解的基础上扩展对B56依赖的OXR1.我们的 中心假说是B56δ的S88-90是B56δ上的关键调控位点，它在 通过OXR1信号调节DNA损伤反应。特异性目标1研究B56δ的调控 S88-90亚磷酸盐，使其磷酸化的激酶(S)，以及它们的生物功能 在质谱学、体外和细胞研究中的方法。具体目标2试图确定 PP2A-B56在OXR1的去磷酸化中，OXR1 LxxIxE基序对这种相互作用的贡献，以及 B56-OXR1轴在DNA损伤信号中的生物学意义。这些项目的顺利完成 AIMS将阐明B56OXR1PP2A-B56的磷酸化调节和δ去磷酸化的作用。 通过了解这些与多种人类疾病有关的蛋白质的调节，我们将 更好地了解他们与疾病相关的生理、潜在的治疗目标和治疗要点 治疗性干预。此申请表中建议的项目和培训计划将提高我在 生化和细胞生物学技术、实验设计、数据分析、科学交流、 指导和翻译研究。达特茅斯学院和诺里斯·科顿的合作环境 癌症中心和强大的导师和合作者网络为我提供了一个理想的环境 完成申请中提出的研究，并支持我作为内科科学家的发展。

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.