Structural and functional studies of protein kinase C regulation

蛋白激酶C（PKC）是一个脂质激活激酶家族，是磷脂酰肌醇的关键效应子 信号传导-真核细胞的主要胞内信号传导途径。与这一中心活动相一致， PKC信号传导的失调与癌症进展、心脏病、糖尿病和阿尔茨海默病有关 疾病由于PKC在信号转导中的基础作用， PKC活性--无论是用于治疗还是基础研究目的--被广泛认为是主要的 外地的挑战。解决这些挑战需要对PKC控制的原子级理解- 特别是脂质如何调节PKC活性。这个主题定义了一个主要的差距，在理解蛋白激酶C 监管机制。这项研究计划的中心目标是破译 触发PKC激活的关键事件-其与二酰基甘油（DAG）的相互作用。目前未知 DAG如何被PKC的保守同源1（C1）结构域识别和捕获在膜中。到 解决这一关键的知识差距，先进的溶液NMR方法的协同组合， 原子分子动力学模拟和生物化学策略将用于解决以下问题 具体目的：（1）了解C1结构域识别甘油二酯的分子基础，（2） 精确地破译C1结构域最初是如何被募集到膜上的，以及（3）确定串联C1结构域是如何被募集到膜上的。 结构域执行DAG和阴离子磷脂的重合检测。对DAG传感的见解 从拟议的研究中获得的机制将产生超越已经很大的影响， PKC调节的问题。关于C1-DAG相互作用的原子级信息将直接 适用于依赖于C1结构域调节其DAG的其他五个信号蛋白大家族， 相关活动。这一信息也将有助于设计合理的药理学试剂， 通过直接靶向其C1结构域调节PKC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.