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

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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.