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

本申请的主要目的是1-理解AGC蛋白激酶PKC β和PKC 1/λ的调节的分子机制，和2-表征介导AGC激酶活化的构象转变。蛋白激酶在信号转导中起核心作用。蛋白激酶的失调是疾病状态的原因，例如癌症和糖尿病。在过去的几年中，我们已经发现并表征了存在于蛋白激酶（AGC蛋白激酶）大家族上的调节位点（“PIF-口袋”）。特别地，该口袋被证明是调节胰岛素/生长因子信号传导下游的蛋白激酶如PDK 1、PKB/Akt、S6 K等的必要组分。本申请涉及另一种AGC蛋白激酶，非典型蛋白激酶C（aPKC，PKC β/λ），其涉及刺激胰岛素信号传导下游的葡萄糖摄取。作为本申请的一部分，我们将产生一组不同的人PKC β突变体，在不存在或存在各种假定的激活剂（包括脂质、神经酰胺、去污剂）的情况下并使用不同的体外底物表达、纯化和测试活性。这一分析将有助于我们建立一个全面的aPKC活性调控的分子模型，并确定PIF-pocket在aPKC中的具体作用。我们将进一步表征我们开发的新化合物对AGC激酶PIF口袋的激活和抑制的分子机制。我们将在体外表征的构象变化，激活AGC激酶使用荧光测定和氘交换技术。最后，我们将评估影响体外aPKC活性的小分子化合物对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.