Regulation of actin polymerization by Arg and N-WASp

Cellular locomotion, cell shape change, and other important cellular processes require selective cell membrane protrusion in response to extracellular signaling cues. The Abl family non-receptor tyrosine kinase Arg is stimulated by extracellular cues to promote rearrangements in the actin cytoskeleton necessary for cellular protrusion and morphogenesis. Several Arg-interacting proteins have been identified as potential links to actin cytoskeletal regulation, but exactly how Arg stimulates actin polymerization and cellular protrusion has not yet been fully elucidated. In this dissertation, I will describe the methods I used to identify the Arp2/3 complex-interacting protein N-WASp as a novel effector of Arg.;Our lab used an affinity purification approach to identify N-WASp as potential Arg-interacting protein. I demonstrated that direct binding to N-WASp is mediated by the Arg SH3 domain. Arg phosphorylates N-WASp on Y256, leading to a modest increase in Arg-N-WASp affinity that does not require the Arg SH2 domain. I also found that the Arg SH3 domain stimulates N-WASp-dependent actin polymerization in vitro and that N-WASp phosphorylation weakly stimulates this effect. My studies reveal that Arg interacts with N-WASp through both binding and phosphorylation events to stimulate N-WASp-dependent actin polymerization and point to this interaction as a potential mechanism through which Arg-mediated signaling acts on the actin cytoskeleton to generate stimulus-dependent protrusion.