Biochemical dissection of a signaling pathway that controls actin assembly

To engage in complex behaviors such as movement, polarization, morphogenesis, and cell division, cells must use signaling pathways to direct actin assembly with a high degree of spatial and temporal resolution. Two types of signaling intermediates, Rho family small GTPases and phosphoinositides, are thought to be particularly important components of pathways that control actin assembly. Taking a biochemical approach, we have fractionated bovine brain and Xenopus egg extracts to identify the minimal set of components required to propagate a signal from Cdc42, a Rho family member, and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) to actin assembly. Three components are required: the actin nucleating Arp2/3 complex, the Wiskott Aldrich Syndrome Protein (WASP) family member N-WASP, and a 140kDa, Cdc42-binding protein that has been purified nearly 2000 fold but remains unidentified. Purified PI(4,5)P₂, Cdc42, N-WASP, and the Arp2/3 complex are sufficient to reconstitute signal dependent actin nucleation, which is the initiating step in actin assembly. In the scheme that emerges, Cdc42 and PI(4,5)P₂ bind to and synergistically activate N-WASP, and activated N-WASP, in turn, potently stimulates the actin nucleation activity of the Arp2/3 complex. PI(4,5)P₂ plays a second role in Xenopus extracts, indirectly leading to the activation of Cdc42 by a poorly understood mechanism. We have dissected the N-WASP activation step in some detail. At rest, N-WASP exists in an autoinhibited conformation, stabilized by intramolecular interactions between its N- and C-termini. The upstream activators Cdc42 and PI(4,5)P₂ bind to the N-terminal regulatory domain of N-WASP, the former through a previously described interaction with the G-protein binding domain and the latter through an adjacent basic stretch conserved amongst WASP family proteins. As a result of these interactions, the affinity between the N- and C-termini is reduced, and the C-terminal effector domain, now exposed, binds to the Arp2/3 complex and G-actin to potently catalyze actin nucleation. In summary, PI(4,5)P₂, Cdc42, N-WASP, and the Arp2/3 complex comprise a core signaling module for stimulating actin nucleation at the plasma membrane.