WIP provides an essential link between Nck and N-WASP during Arp2/3-dependent actin polymerization.

Nck links phosphotyrosine-based signaling to Arp2/3-dependent actin polymerization during many different cellular processes as well as actin-based motility of enteropathogenic Escherichia coli (EPEC), vaccinia, and other vertebrate poxviruses by interacting with N-WASP/WASP. Nck also binds WASP-interacting protein (WIP), which inhibits the ability of N-WASP to activate the Arp2/3 complex until it receives an appropriate signaling input. Using mouse embryonic fibroblasts (MEFs) lacking Nck, WIP, or N-WASP, we have investigated whether an interaction of Nck with both WIP and N-WASP is required for their recruitment to vaccinia during Arp2/3-dependent actin assembly. We find that WIP or its homolog WIRE is required for N-WASP recruitment and actin-based motility of the virus. WIP contains two Nck-binding sites and is recruited to the virus, bound to N-WASP, by interacting with the second SH3 domain of Nck. N-WASP also contains two Nck-binding sites, but its recruitment is dependent on its interaction with WIP rather than Nck. The first and third SH3 domains of Nck are not required to recruit the WIP:N-WASP complex but are essential to stimulate actin assembly. We have established that WIP acts as an essential link between Nck and N-WASP. Our observations provide important insights into the hierarchy and connections in one of the major cellular signaling networks stimulating Arp2/3 complex-dependent actin polymerization.