GRB2 links signaling to actin assembly by enhancing interaction of neural Wiskott-Aldrich syndrome protein (N-WASp) with actin-related protein (ARP2/3) complex

Proteins of the Wiskott-Aldrich Syndrome protein (WASp) family connect sign aling pathways to the actin polymerization-driven cell motility, The ubiqui tous homolog of WASp, N-WASp, is a multidomain protein that interacts with the Arp2/3 complex and G-actin via its C-terminal WA domain to stimulate ac tin polymerization, The activity of N-WASp is enhanced by the binding of ef fecters like Cdc42-guanosine 5'-3-O-(thio)triphosphate, phosphatidylinosito l bisphosphate, or the Shigella IcsA protein. Here we show that the SH3-SH2 -SH3 adaptor Grb2 is another activator of N-WASp that stimulates actin poly merization by increasing the amount of N-WASp Arp2/3 complex, The concentra tion dependence of N-WASp activity, sedimentation velocity and cross-linkin g experiments together suggest that N-WASp is subject to self-association, and Grb2 enhances N-WASp activity by binding preferentially to its active m onomeric form, Use of peptide inhibitors, mutated Grb2, and isolated SH3 do mains demonstrate that the effect of Grb2 is mediated by the interaction of its C-terminal SH3 domain with the proline-rich region of N-WASp. Cdc42 an d Grb2 bind simultaneously to N-WASp and enhance actin polymerization syner gistically, Grb2 shortens the delay preceding the onset of Escherichia coli (IcsA) actin-based reconstituted movement. These results suggest that Grb2 may activate Arp2/3 complex-mediated actin polymerization downstream from the receptor tyrosine kinase signaling pathway.