Communication between chemoattractant and growth factor receptors and downstream Rho family small GTPases initiates cellular events important for the motile responses of leukocytes and fibroblasts. Dr. Bokoch and his colleagues will investigate one such receptor-regulated pathway, the Rac/Cdc42-mediated activation of the target protein Pak (p21-activated kinase). The proposed studies address the biological pathways used by receptors to regulate the actin cytoskeleton, and will identify novel mechanisms by which Pak activity is controlled. In their first specific aim, the molecular interactions through which Pak regulates the actin cytoskeleton will be determined structurally and mechanistically in a well-defined fibroblast model system. Domains in Pak important for cytoskeletal regulation will be identified using a combination of molecular and cellular approaches. Target proteins which interact with these domains will be isolated and characterized. They will extend their studies of cytoskeletal regulation by Pak in the second aim, which will evaluate a novel GTPase-independent mechanism for Pak regulation. The molecular basis for Pak activation by membrane targeting will be investigated. The investigators will examine the significance of Pak activation by membrane association in cytoskeletal regulation of neurite development using the PC12 cell as a model system. The role of the Pak-binding Nck adapter protein in this process will also be probed. Receptors appear to control Pak activity by mechanisms distinct from direct activation via Rac or Cdc42 which include regulatory dephosphorylation reactions critical to the Pak activation process.
The third aim will thus focus upon identification and purification of phosphatases that act upon Pak. Using molecular and biochemical means, they will probe the mechanisms by which phosphatases control Pak activity, and will initiate studies on the relevance of such regulatory phosphatases to receptor-mediated Pak activation.
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