Structural and functional studies of protein kinase C regulation

蛋白激酶C(PKC)是磷脂酰肌醇的关键效应物，它是一类脂类激活的蛋白激酶 信号转导--真核细胞内的主要信号转导途径。与这一中心活动相一致， PKC信号的失调与癌症进展、心脏疾病、糖尿病和阿尔茨海默氏症有关 疾病。由于PKC在信号转导中的基础作用，近年来对PKC调节剂的研究进展 PKC活动--用于治疗和基础研究目的--被广泛认为是主要的 这一领域的挑战。应对这些挑战需要对PKC控制的原子级别的了解- 特别是，脂类如何调节PKC的活性。本主题定义了对PKC的理解上的主要差距 监管机制。这项研究建议的中心目标是破译 触发PKC激活的关键事件--它与二酰基甘油(DAG)的相互作用。目前还不清楚 DAG是如何被PKC的保守的同源1(C1)结构域识别和捕获的。至 解决知识中的这一关键差距，高级解决方案核磁共振方法的协同组合， 原子分子动力学模拟和生化策略将应用于解决以下问题 具体目标：(1)了解C1域识别二酰甘油的分子基础，(2) 准确地破译C1域最初是如何招募到膜上的，以及(3)确定如何串联C1域 结构域执行DAG和阴离子磷脂的符合检测。对DAG传感的洞察 从拟议的研究中获得的机制将产生超出已经很大的影响 PKC监管问题。关于C1-DAG相互作用的原子级信息将直接 适用于其他五大类信号蛋白，它们依赖于C1域来调节其DAG- 从属活动。这些信息也将有助于Rational的药理药物的设计 通过直接靶向PKC的C1域来调节其功能。

Protein Kinases C (PKC) define a family of lipid-activated kinases that are key effectors of phosphoinositide signaling – a major intracellular signaling pathway of eukaryotic cells. Consistent with this central activity, dysregulation of PKC signaling is implicated in cancer progression, cardiac disease, diabetes, and Alzheimer's disease. Because of the fundamental role of PKC in signal transduction, the development of modulators of PKC activity – both for therapeutic and basic research purposes – is widely recognized as one of the major challenges in the field. Addressing these challenges requires an atomic-level understanding of PKC control – in particular, how lipids regulate PKC activity. This subject defines a major gap in understanding of PKC regulatory mechanisms. The central objective of this research proposal is to decipher the mechanism of the key event that triggers activation of PKC – its interaction with diacylglycerol (DAG). It is currently unknown how DAG is recognized and captured in membranes by the conserved homology 1 (C1) domains of PKC. To address this critical gap in knowledge, a synergistic combination of advanced solution NMR approaches, atomistic molecular dynamics simulations, and biochemical strategies will be applied to address the following Specific Aims: (1) to understand the molecular basis of diacylglycerol recognition by C1 domains, (2) to decipher precisely how C1 domains are initially recruited to membranes, and (3) to determine how tandem C1 domains execute a coincidence detection of DAG and anionic phospholipids. Insights into the DAG-sensing mechanism obtained from the proposed studies will have impact that extends beyond the already large problem of PKC regulation. The atomic-level information regarding C1-DAG interactions will be directly applicable to five other large families of signaling proteins that rely on C1 domains for regulation of their DAG- dependent activities. This information will also facilitate the design of pharmacological agents for rational modulation of PKC function by direct targeting of its C1 domains.