Structural and Functional Studies of Protein Kinase C Regulation

Texas A&M Agrilife Research, College Station TX

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Abstract

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 description of PKC control – in particular, how lipids and exogenous agonists regulate PKC activity. This requirement defines a major gap in understanding of PKC regulatory mechanisms. This research proposal builds upon two key discoveries made by our laboratory. First, an atomistic resolution of how DAG is recognized and captured in membranes by the conserved homology 1 (C1) domains of PKC that eluded the field for 30 years. Second, the discovery that novel PKC isoforms have an unexplored lipid-sensing function that adds a novel facet to control of PKC activity. This renewal application proposes to apply an integrated research strategy consisting of solution NMR spectroscopy, x-ray crystallography, atomistic molecular dynamics simulations, and imaging experiments in yeast and mammalian cells to address the following Specific Aims: (1) determine the role of phosphoinositides in PKC localization and activation, and (2) determine the molecular basis of PKC activation by novel tool compounds and drugs that target the C1 domains. These studies will deliver functional, structural, thermodynamic, and kinetic information to understand the determinants of potency and isoform selectivity of exogenous PKC agonists and provide key information regarding how PKCs execute coincidence detection of DAG and phosphoinositides. Insights into the PKC regulatory mechanisms obtained from the proposed studies will be impactful in that these will extend the already large problem of PKC regulation into previously unexplored areas. The atomistic information regarding C1 interactions with exogenous ligands and regulatory lipids will be directly applicable to five other large families of essential signaling proteins that rely on C1 domains for regulation of their DAG-dependent activities. This knowledge will also facilitate a more efficient structure-based design of pharmacological agents that modulate PKC activity by direct targeting of its C1 domains.