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

本申请的主要目的是1-了解调节AGC蛋白激酶ζ和ι/λ的分子机制；2-表征介导AGC蛋白激酶激活的构象转变。蛋白激酶在信号转导中起着核心作用。蛋白激酶的失控是癌症和糖尿病等疾病的罪魁祸首。在过去的几年里，我们已经发现并鉴定了一个存在于蛋白激酶(AGC蛋白激酶)大家族上的调控位点(PIF-Pocket)。特别是，这个口袋被证明是调节胰岛素/生长因子信号下游的蛋白激酶的重要组成部分，如PDK1，PKB/Akt，S6K等。本申请涉及另一种AGC蛋白激酶，非典型蛋白激酶C(APKCs，PKCζ/λ)，它与刺激胰岛素信号下游的葡萄糖摄取有关。作为本应用的一部分，我们将生产一组不同的人PKCζ突变体，在没有或存在各种假定激活剂的情况下进行表达、纯化和活性测试，包括脂质、神经酰胺、洗涤剂，并使用不同的体外底物。这一分析将有助于我们建立一个全面的分子模型来调控aPKC的活性，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AGC激酶PIF-Pocket新化合物的激活和抑制的分子机制。我们将使用荧光分析和氚交换技术在体外表征激活AGC激酶的构象变化。最后，我们将评估在体外影响aPKC活性的小化合物对aPKC在胰岛素刺激的葡萄糖摄取中的可能作用的影响。

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.