Regulation and Function of PP2A-B56delta

PP2A-B56delta 的调节和功能

• 批准号: 10388028
• 负责人: Kali Smolen
• 金额: $ 5.18万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10388028
• 关键词: 3-Dimensional; Binding; Biochemical; Biochemistry; Biological; Biological Process; Cell Survival; Cells; Cellular biology; Communication; DNA Damage; Data; Data Analyses; Development; Disease; Environment; Epilepsy; Epitopes; Experimental Designs; FRAP1 gene; Family; Family member; Genome Stability; Genomics; Glutamic Acid; Holoenzymes; Immunoprecipitation; Impaired cognition; In Vitro; Intervention; Knock-out; Knowledge; Maintenance; Mass Spectrum Analysis; Mediating; Mentors; Muscle hypotonia; Mutate; Mutation; Nature; Norris Cotton Cancer Center; Outcomes Research; Oxidative Phosphorylation; Oxidative Regulation; Oxidative Stress; PPP2R5D gene; Pathology; Peptides; Phenotype; Phosphopeptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphoserine; Phosphothreonine; Phosphotransferases; Physicians; Physiology; Post-Translational Protein Processing; Process; Protein Dephosphorylation; Protein Deregulation; Protein Isoforms; Protein phosphatase; Proteins; Proteomics; Proto-Oncogene Proteins c-akt; Regulation; Reporting; Research; Resistance; Role; Scientist; Serine; Signal Pathway; Signal Transduction; Site; Spliced Genes; Substrate Specificity; Techniques; Testing; Therapeutic Intervention; Training; Translational Research; Up-Regulation; Variant; collaborative environment; college; developmental disease; human disease; inhibitor; innovation; insight; knock-down; loss of function; mutant; oxidation; response; scaffold; skills; therapeutic target

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.

项目概要 蛋白质磷酸化参与许多生物过程的调节，包括对 DNA损伤。虽然激酶信号传导已被广泛研究，但磷酸酶的作用仍然存在 待研究。磷蛋白磷酸酶 2A (PP2A) 负责大部分磷酸丝氨酸和 细胞内磷酸苏氨酸去磷酸化并通过其调节 B 赋予其底物特异性 亚基。 B56 家族是 PP2A 调节亚基四个家族中最大的一个。所有 B56 同工型均识别 其底物和相互作用子上有 LxxIxE 短线性基序 (SLiM)。我们开发了一种 SLiM 介导的 竞争性抑制剂方法来询问 PP2A-B56 依赖性磷酸蛋白质组，从而扩大 了解 B56 特异性底物。然而，我们对控制活动的机制的了解 PP2A-B56 的内容仍然不完整。在 B56 抑制后，我们发现 B56δ 的磷酸丝氨酸 88-90 差别化监管。我们的初步数据表明这些站点本质上处于失活状态。 B56δ编码 由基因 PPP2R5D 引起，该基因在 PPP2R5D 相关发育障碍中发生突变，其特征为认知障碍 损伤和癫痫。此外，我们还鉴定了氧化抗性 1 (OXR1)，一种包含 LxxIxE 基序的 参与细胞对氧化应激反应和维持基因组稳定性的蛋白质，为 B56 基材。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 的 S88-90 调节 B56δ 并扩展我们对 OXR1 的 B56 依赖性调节的理解。我们的 中心假设是 B56δ 的 S88-90 是 B56δ 的关键调控位点，在 通过 OXR1 信号传导调节 DNA 损伤反应。具体目标 1 研究 B56δ 的调节 S88-90 磷酸位点、磷酸化它们的激酶及其使用创新技术的生物学功能 质谱法以及体外和细胞研究中的方法。具体目标 2 旨在确定 PP2A-B56 在 OXR1 去磷酸化中的作用，OXR1 LxxIxE 基序对此相互作用的贡献，以及 B56-OXR1 轴在 DNA 损伤信号传导中的生物学意义。这些工作的顺利完成 目的是阐明 B56δ 的磷酸调节以及 PP2A-B56 在 OXR1 去磷酸化中的作用。 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 更好地了解与疾病相关的生理学、潜在的治疗目标和要点 治疗干预。本申请中提出的项目和培训计划将提高我的技能 生化和细胞生物学技术、实验设计、数据分析、科学传播、 指导和转化研究。达特茅斯学院和 Norris Cotton 的协作环境 癌症中心以及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作为一名医师科学家的发展。