Influence of the c terminus of Wiskott-Aldrich syndrome protein (WASp) andthe Arp2/3 complex on actin polymerization

The 70 C-terminal amino acids of Wiskott-Aldrich syndrome protein (WASp WA) activate the actin nucleation activity of the Arp2/3 complex. WASp WA bind s both the Arp2/3 complex and actin monomers, but the mechanism by which it activates the Arp2/3 complex is not known. We characterized the effect of WASp WA on actin polymerization in the absence and presence of the human Ar p2/3 complex. WASp WA binds actin monomers with an apparent K-d of 0.4 mu M , inhibiting spontaneous nucleation and subunit addition to pointed ends, b ut not addition to barbed ends. A peptide containing only the WASp homology 2 motif behaves similarly but with a 10-fold lower affinity. In contrast t o previously published results, neither WASp WA nor a similar region of the protein Scar1 significantly depolymerizes actin filaments under a variety of conditions. WASp WA and the Arp2/3 complex nucleate actin filaments, and the rate of this nucleation is a function of the concentrations of both WA Sp WA and the Arp2/3 complex, With excess WASp WA and <10 nM Arp2/3 complex , there is a 1:1 correspondence between the Arp2/3 complex and the concentr ation of filaments produced, but the filament concentration plateaus at an Arp2/3 complex concentration far below the cellular concentration determine d to be 9.7 mu M in human neutrophils. Preformed filaments increase the rat e of nucleation by WASp WA and the Arp2/3 complex but not the number of fil aments that are generated. We propose that filament side binding by the Arp 2/3 complex enhances its activation by WASp WA.