Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4,5-bisphosphate

Neuronal Wiskott-Aldrich Syndrome protein (N-WASP) transmits signals from C dc42 to the nucleation of actin filaments by Arp2/3 complex. Although full- length N-WASP is a weak activator of Arp2/3 complex, its activity can be en hanced by upstream regulators such as Cdc42 and PI(4,5)P-2. We dissected th is activation reaction and found that the previously described physical int eraction between the NH2-terminal domain and the COOH-terminal effector dom ain of N-WASP is a regulatory interaction because it can inhibit the actin nucleation activity of the effector domain by occluding the Arp2/3 binding site. This interaction between the NH2- and COOH termini must be intramolec ular because in solution N-WASP is a monomer. Phosphatidylinositol 4,5-bisp hosphate (PI(4,5)P-2) influences the activity of N-WASP through a conserved basic sequence element located near the Cdc42 binding site rather than thr ough the WASp homology domain 1. Like Cdc42, PI(4,5)P-2 reduces the affinit y between the NH2- and COOH termini of the molecule. The use of a mutant N- WASP molecule lacking this basic stretch allowed us to delineate a signalin g pathway in Xenopus extracts leading from PI(4,5)P-2 to actin nucleation t hrough Cdc42, N-WASP, and Arp2/3 complex. In this pathway, PI(4,5)P-2 serve s two functions: first, as an activator of N-WASP; and second, as an indire ct activator of Cdc42.