Structural and functional studies of protein kinase C regulation

蛋白激酶C调节的结构和功能研究

• 批准号: 8614155
• 负责人: Tatyana I. Igumenova
• 金额: $ 26.19万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8614155
• 关键词: 1,2-diacylglycerol; Address; Alzheimer' s Disease; Apoptosis; Area; Avidity; Binding; Biochemical; Biological Assay; C-terminal; C2 Domain; Complex; DAG/PE-Binding Domain; Data; Development; Diabetes Mellitus; Diagnostic; Diglycerides; Disease; Drug or chemical Tissue Distribution; Enzyme Activation; Enzymes; Family; Fluorescence Spectroscopy; Geometry; Heart Diseases; Heart failure; Human Pathology; Immunoglobulin Variable Region; Impairment; In Vitro; Ions; Isoenzymes; Laboratories; Learning; Length; Ligands; Lipid Binding; Lipids; Malignant Neoplasms; Membrane; Memory; Molecular; Molecular Conformation; Mutagenesis; N-terminal; Nature; Parents; Pathway interactions; Peptides; Peripheral; Phorbol Esters; Phosphotransferases; Process; Protein Isoforms; Protein Kinase C; Proteins; Regulation; Relative (related person); Research Proposals; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Solutions; Specificity; Structure; Techniques; Testing; Therapeutic Human Experimentation; X-Ray Crystallography; base; cell growth; cell motility; cofactor; design; flexibility; human disease; in vivo; insight; mimetics; mutant; novel; prevent; protein phosphatase inhibitor-2; public health relevance; response; structural biology; tumor; tumor progression

PROJECT SUMMARY Dysregulation of signal transduction pathways that control cell growth, differentiation, apoptosis, and motility is associated with many human pathologies. One of the key kinases involved in regulation of these pathways is the Protein Kinase C (PKC) family of isoenzymes. Because of the central role of these enzymes in signal transduction and human disease, the need for isoform-specific modulators of PKC activity - both for therapeutic and research purposes - is widely recognized as one of the major challenges in the field. The progress in this area has been significantly impeded by poor understanding of the molecular basis of PKC activation and regulation. Indeed, PKC presents significant challenges for conventional structural biology approaches due to its multi-modular structure, the associated inter-domain flexibility, and the amphiphilic nature of the N-terminal regulatory domain that undergoes membrane insertion upon enzyme activation. The long-term objective of my laboratory is to understand the molecular basis of activation of PKC isoforms through biophysical and biochemical studies of their most variable domains. The specific objective of this proposal is to characterize the key inter-domain and domain-cofactor interactions and test several novel hypotheses about their role in the activation process of the ¿ isoform of PKC. Our experimental approach makes an extensive use of the structural and functional autonomy of the PKC¿ domains and integrates advanced solution NMR techniques, fluorescence spectroscopy, X-ray crystallography, mutagenesis, and in-vitro membrane binding assays. The Specific Aims of this proposal are directed at (1) identifying the structural and functional interplay of lipid-binding domains essential for the PKC¿ membrane-insertion step and tumor-promoting response, and (2) testing the hypotheses that the C-terminal domain of PKC¿ serves as a membrane anchor and an intra-molecular protein interaction module. We anticipate that our findings will: generate insight into isoform-specific regulation of PKC¿ by identifying key residues involved in the interactions with membranes/membrane-embedded ligands and inter-domain interactions; provide a molecular platform for the design of isoform-specific agents that modulate the activity of PKC¿ through interference with its membrane binding and/or inter-domain interactions; and establish a structural framework for interpretation of in vitro and in vivo functional data on conventional PKC isoforms.

项目概要 控制细胞生长、分化、凋亡和运动的信号转导途径失调 与许多人类病理有关。参与调节这些途径的关键激酶之一是 蛋白激酶 C (PKC) 同工酶家族。由于这些酶在信号中的核心作用 转导和人类疾病，需要 PKC 活性的异构体特异性调节剂 - 两者都用于 治疗和研究目的——被广泛认为是该领域的主要挑战之一。这 由于对 PKC 分子基础了解不足，该领域的进展受到严重阻碍 激活和调节。事实上，PKC 对传统结构生物学提出了重大挑战 方法由于其多模块结构、相关的域间灵活性和两亲性 N 末端调节结构域的性质，在酶激活时进行膜插入。这 我实验室的长期目标是通过以下方式了解 PKC 同工型激活的分子基础： 对其变化最大的领域进行生物物理和生物化学研究。本提案的具体目标 是表征关键的域间和域辅因子相互作用并测试几种新颖的 关于它们在 PKC 亚型激活过程中的作用的假设。我们的实验 方法广泛利用了 PKC 域的结构和功能自主性， 集成了先进的溶液核磁共振技术、荧光光谱、X 射线晶体学、诱变、 和体外膜结合测定。该提案的具体目标是 (1) 确定 PKC 膜插入步骤所必需的脂质结合域的结构和功能相互作用 肿瘤促进反应，以及 (2) 检验 PKC 的 C 末端结构域作为 膜锚和分子内蛋白质相互作用模块。我们预计我们的发现将： 通过识别相互作用中涉及的关键残基，深入了解 PKC 的异构体特异性调节 具有膜/膜嵌入配体和域间相互作用；提供分子平台 设计异构体特异性药物，通过干扰 PKC 来调节 PKC 的活性 膜结合和/或域间相互作用；并建立解释的结构框架 常规 PKC 亚型的体外和体内功能数据。