cAMP-Dependent Protein Kinases: Mediators of Receptors

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Many hormones and neurotransmitters exert their action through the mobilization of the ubiquitous second messenger cAMP. Intracellular transduction systems receive these signals and transmit them quickly and precisely resulting in the amplification of appropriate biological responses. Specificity is achieved by the assembly of multi-protein signaling complexes that create focal points of enzyme activity. Accordingly, the spatiotemporal activation of protein kinases and/orphosphatases is important in controlling where and when substrate phosphorylation occurs. Anchoring proteins and targeting subunits prove a molecular framework that orients protein kinases and phosphatases towards selected substrates. Prototypic examples of these "signal directing molecules" are A-kinases Anchoring Proteins (AKAPs) which sustain multi-component signaling complexes of the cAMP-dependent protein kinase (PKA) and other enzymes. This proposal focuses on understanding the functional ramifications of AKAP-mediated targeting of PKA to substrates that participate in the control of actin reorganization events and cell movement. These experiments have developed from a recent finding that WAVE-1, a member of the Wiskott-Aldrich Syndrome Protein (WASP) family of scaffolding proteins, is an AKAP. WAVE coordinates actin reorganization events by coupling the GTPase Rac to the mobilization of the Arp2/3 complex. Preliminary studies have demonstrated that WAVE-1 also binds to two PKA substrates" a novel GTPase activating protein called WARM and Mena, a protein involved in the control of cell motility. There are two specific aims of this proposal. Aim 1 will map the WARP binding site on WAVE-1, establish if WARP is a Rac selective GAP and determine if WARP functions to attenuate Rac-mediated actin remodeling events. Aim 2 will map the Mena binding site on WAVE-1, establish whether Mena is preferentially phosphorylated by anchored PKA and determine if WAVE-1 functions to recruit Mena at the leading edg3e of migrating fibroblasts to control the rate of cell motility.