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.

项目摘要 控制细胞生长，分化，凋亡和运动性的信号转移途径的失调是 与许多人类病理有关。这些途径调节涉及的关键激酶之一是 同工酶的蛋白激酶C（PKC）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活性的同工型特异性调节剂 - 均用于 治疗和研究目的被广泛认为是该领域的主要挑战之一。 由于对PKC的分子基础的不良理解，这一领域的进展受到了极大的阻碍 激活和调节。确实，PKC对常规结构生物学提出了重大挑战 由于其多模二型结构，相关的域间柔韧性和两亲性的方法 酶激活后经历膜插入的N末端调节结构域的性质。这 我实验室的长期目标是了解PKC同工型激活的分子基础 其最可变领域的生物物理和生化研究。该提议的具体目标 是要表征关键的域间和域 - 伴侣相互作用并测试几种新颖的 假设它们在PKC的同工型的激活过程中的作用。我们的实验 方法广泛使用PKC域的结构和功能自主权 整合先进的溶液NMR技术，荧光光谱，X射线晶体学，诱变， 和体外膜结合测定法。该提案的具体目的针对（1）确定 脂质结合结构域的结构和功能相互作用，对PKC¿MEMBRANE-insertion步骤和 促进肿瘤的反应，以及（2）检验PKC的C末端结构域作为A的假设 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： 通过识别相互作用涉及的密钥保留，从而对PKC？的同工型特异性调节产生洞察力 带有膜/膜包裹的配体和域间相互作用；提供一个分子平台 同工型特异性药物的设计，通过干扰PKC？ 膜结合和/或域间相互作用；并建立一个结构框架来解释 常规PKC同工型的体外和体内功能数据。