Regulation and Function of PP2A-B56delta

PP2A-B56delta 的调节和功能

• 批准号: 10619441
• 负责人: Kali Smolen
• 金额: $ 5.27万
• 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/10619441
• 关键词: 3-Dimensional; Binding; Biochemical; Biochemistry; Biological; Biological Process; Cell Line; Cell Survival; Cells; Cellular biology; Communication; DNA Damage; Data; Data Analyses; Development; Disease; Environment; Epilepsy; Epitopes; Experimental Designs; FRAP1 gene; Family; Family member; Genes; Genome Stability; Glutamic Acid; Holoenzymes; Immunoprecipitation; Impaired cognition; In Vitro; Intervention; Knock-out; Knowledge; Maintenance; Mass Spectrum Analysis; Mediating; Mentors; Metabolism; 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; Proliferating; Protein Dephosphorylation; Protein Deregulation; Protein Isoforms; Protein phosphatase; Proteins; Proteomics; Proto-Oncogene Proteins c-akt; RNA Splicing; Regulation; Reporting; Research; Resistance; Role; Scientist; Serine; Signal Pathway; Signal Transduction; Site; 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.

项目摘要 蛋白质磷酸化参与了许多生物学过程的调节，包括对 DNA损伤。虽然激酶信号传导已得到广泛研究，但磷酸酶的作用仍然 研究了。磷酸蛋白磷酸酶2a（PP2A）负责大多数磷酸氨氨酸和 细胞内的磷酸硫氨酸去磷酸化，并通过其调节性B赋予其底物特异性 亚基。 B56家族是PP2A监管亚基四个家族中最大的。识别的所有B56同工型 其底物和相互作用器上的LXXIXE短线性基序（Slim）。我们已经开发了一个细长的介导的 竞争性抑制剂方法来询问pp2a-b56依赖性磷蛋白体，从而扩展 了解B56特异性底物。但是，我们对管理活动的机制的了解 PP2A-B56的含量仍然不完整。在B56抑制后，我们发现B56Δ的磷s列88-90为 差异调节。我们的初步数据表明，这些站点本质上正在灭活。 B56Δ编码 通过基因PPP2R5D，该基因在以认知为特征的PPP2R5D与发育障碍中突变 障碍和癫痫。此外，我们确定了氧化抗性1（OXR1），含Lxxixe基序 参与细胞对氧化应激的反应和基因组稳定性维持的蛋白质，为B56 基材。然而，对OXR1的调节知之甚少。在这里，我们建议确定如何磷酸化 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的关键调节位点，在B56Δ上起着至关重要的作用 通过OXR1信号传导调节DNA损伤响应。特定目标1研究B56δ的调节 S88-90磷脂，磷酸化的激酶，并使用创新的生物学功能 质谱，体外和细胞研究的方法。特定目标2试图确定 pp2a-b56在OXR1的去磷酸化中 DNA损伤信号传导中B56-OXR1轴的生物学性。这些成功完成 目的将阐明B56δ的磷酸调节以及PP2A-B56在OXR1的去磷酸化中的作用。 通过了解与各种人类疾病有关的这些蛋白质的调节，我们将 更好地了解其与疾病相关的生理学，潜在的治疗靶点和点 治疗干预。本应用程序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，科学交流， 指导和翻译研究。达特茅斯学院和诺里斯棉花的协作环境 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 完成本应用程序中提出的研究，并支持我作为物理科学家的发展。