Molecular Mechanism of Regulation of the atypical Kinase C (PKC)

目前应用的主要对象是1- AGC蛋白激酶PKCζ和PKCι/λ调控的分子机制的理解和2-介导AGC激酶激活的构象转变的表征。蛋白激酶在信号转导中起着核心作用。蛋白激酶的失调是疾病状态的原因，比如癌症和糖尿病。在过去的几年中，我们已经发现并表征了一个存在于蛋白激酶（AGC蛋白激酶）大家族中的调控位点（“PIF- pocket”）。特别是，这个口袋被证明是胰岛素/生长因子信号传导下游蛋白激酶调控的重要组成部分，如PDK1、PKB/Akt、S6K等。目前的应用涉及另一种AGC蛋白激酶，非典型蛋白激酶C (aPKCs, PKCζ/λ)，它涉及刺激胰岛素信号传导下游的葡萄糖摄取。作为本应用程序的一部分，我们将生产一套不同的人类PKCζ突变体，表达，纯化和测试在缺乏或存在各种假定的激活剂，包括脂质，神经酰胺，洗涤剂，并使用不同的体外底物的活性。该分析将帮助我们建立aPKC活性调控的综合分子模型，并确定PIF-pocket在aPKCs中的具体作用。我们将进一步表征我们开发的AGC激酶PIF-口袋的新化合物的激活和抑制的分子机制。我们将在体外利用荧光分析和氘交换技术表征激活AGC激酶的构象变化。最后，我们将评估影响aPKC活性的小化合物在体外对aPKC在胰岛素刺激的葡萄糖摄取中的假定作用的影响。

The main objects of the present application are 1- the understanding of the molecular mechanism of regulation of the AGC protein kinase PKCζ and PKCι/λ and 2- the characterization of the conformational transition that mediates AGC kinase activation. Protein kinases play central roles in signal transduction. De-regulation of protein kinases is responsible for disease states, such as cancer and diabetes. Over the last years, we have discovered and characterized a regulatory site (“PIF- pocket”) present on a large family of protein kinases (AGC protein kinases). In particular, this pocket was proven to be an essential component in the regulation of protein kinases downstream of insulin/growth factor signalling, such as PDK1, PKB/Akt, S6K, etc. The present application deals with another AGC protein kinase, atypical protein kinase C (aPKCs, PKCζ/λ), which has been implicated in stimulating glucose uptake downstream of insulin signalling. As part of the present application we will produce a diverse set of human PKCζ mutants, express, purify and test for activity in the absence or presence of various presumed activators including lipids, ceramide, detergents, and using different in vitro substrates. The analysis will help us build up a comprehensive molecular model for the regulation of aPKC activity and to determine the specific role of the PIF-pocket in aPKCs. We will further characterize the molecular mechanism of activation and inhibition of novel compounds that we developed to the AGC kinase PIF- pocket. We will characterize in vitro the conformational change which activates AGC kinases using a fluorescence assay and deuterium exchange technology. Finally, we will evaluate the effects of small compounds that affect aPKC activity in vitro on the presumed role of aPKCs on insulin stimulated glucose uptake.