Structural and functional studies of protein kinase C regulation

项目摘要 控制细胞生长、分化、凋亡和运动的信号转导途径失调， 与许多人类疾病有关参与调节这些途径的关键激酶之一是 蛋白激酶C（PKC）家族同工酶。由于这些酶在信号传导中的核心作用， 转导和人类疾病，需要PKC活性的亚型特异性调节剂-无论是用于 治疗和研究目的-被广泛认为是该领域的主要挑战之一。的 由于对蛋白激酶C的分子基础缺乏了解，这一领域的进展受到严重阻碍 激活和调节。事实上，PKC对传统的结构生物学提出了重大挑战 由于其多模块化结构，相关的域间灵活性和两亲性， 在酶活化时经历膜插入的N-末端调节结构域的性质。的 我实验室的长期目标是通过以下途径了解PKC亚型激活的分子基础： 生物物理和生物化学研究其最可变的域。本提案的具体目标是 是表征关键的域间和域-辅因子相互作用，并测试几种新的 关于它们在PKC亚型激活过程中的作用的假说。我们的实验 这种方法广泛利用了PKC结构域的结构和功能自主性， 集成了先进的溶液NMR技术，荧光光谱，X射线晶体学，诱变， 和体外膜结合测定。本建议的具体目标是：（1）确定 PKC膜插入步骤所必需的脂质结合结构域的结构和功能相互作用， 肿瘤促进反应，和（2）测试的假设，即PKC的C-末端结构域作为一个 膜锚和分子内蛋白质相互作用模块。我们预计，我们的研究结果将： 通过识别参与相互作用的关键残基，深入了解PKC的亚型特异性调节 具有膜/膜嵌入配体和结构域间相互作用;提供分子平台， 设计亚型特异性试剂，通过干扰其活性来调节PKC的活性， 膜结合和/或结构域间相互作用;并建立一个结构框架，用于解释 常规PKC亚型的体外和体内功能数据。

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.