Understanding how intracellular signals are specifically relayed from the membranes to distinct intracellular targets still remains a daunting challenge, given the large number of polypeptides devoted to the process of signal transduction within a eukaryotic cell. It is now apparent that the restriction of these polypeptides to localized sites of action is a regulatory process that functions to influence the specificity pf these signaling enzymes. While the overall focus of the program project is to understand the subcellular targeting interactions that compartmentalize two key classes of protein kinase: the cAMP dependent protein kinase (PKA) and the Ca2+/phospholipid dependent protein kinase C (PKC), project will focus on the compartmentalize two key classes of protein kinase: the cAMP dependent protein kinase (PKA) and the Ca2+/phospholipid dependent protein kinase C (PKC), project 2 will focus on the compartmentalization of both kinases through association with a common anchoring protein called gravin. Gravin contains enzyme binding sites that associate with PKA and PKC, and several targeting motifs that direct the gravin signaling scaffold to the membrane-cytoskeleton.
Aim 1 focuses on gravin PKC interaction and inhibit kinase activity. In collaboration with Dr. Newton (project) we will establish whether both regions participate in enzyme binding or whether there are multiple PKC-binding sites on gravin. Surface plasmon resonance and spin fluorescence techniques will be used to measure the binding affinity of interaction and establish the mechanism of gravin/PKC interaction by screening a family of PKC mutants developed by Dr. Newton (Project 3). Collaborative ventures with Dr. Jennings (Project 4) will employ multi-dimensional NMR to determine the solution structure of the PKC binding peptides when complexed with PKCbetaII.
Aim 2 focuses on the gravin targeting domains. Immunofluorescence data suggest that gravin is targeted to the membrane-cytoskeleton and is enriched in the filopodia of adherent cells, biochemical and co-sedimentation approaches will be utilized to determine whether gravin is an actin binding protein and if it associates with other cyoskeletal components. Induction of gravin expression is concomitant with the onset of an adherent phenotype in certain cell-types. Functional studies (with Dr. Adams and Ellisman, imaging core) will be initiated to determine whether correct PKA or PKC anchoring and/or membrane-cytoskeleton targeting of gravin is necessary to maintain an adherent phenotype.
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