Human phagocytic leukocytes play important roles in host defense against a variety of infectious agents. Upon encountering a microorganism, they undergo morphological and functional alterations dependent upon the cytoskeleton, and usually accompanied by membrane respiratory burst activity that initiates the generation of toxic oxygen metabolites; both processes are regulated by the GTPase, Rac. The investigators have identified a novel class of Rac effector proteins which are activated by chemotactic stimuli in human neutrophils, the p21-activated protein kinases (PAKs). Recent evidence indicates that these kinases play important roles in neutrophil signaling as it relates to immune functions. This project will investigate whether certain Rac-dependent neutrophil responses are mediated through PAK by elucidating the PAK activation process in more detail and by identifying PAK target proteins in neutrophils. They will investigate mechanisms of PAK activation by GTPases through mutational analysis of Rac1, Rac2, Cdc42 and PAK itself. Using purified components, intact neutrophils and established cell lines, the role of upstream signals in addition to activated Rac/Cdc42 will be elucidated through studies of regulatory lipid second messengers and of novel PAK binding proteins. To define how PAK mediated cellular responses to Rac or CdC42, they will pursue the identification of downstream effectors. Initially, their studies will focus on NADPH oxidase components as PAK phosphorylation targets and will be extended to identify novel PAK-activated or interacting neutrophil proteins. They will characterize these proteins biochemically and evaluate their role in PAK-mediated signalling pathways using cellular approaches. These studies should enable us to gain further insight in Rac/PAK regulated events in host defense and inflammatory diseases.
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