Two tandem verprolin homology domains are necessary for a strong activation of Arp2/3 complex-induced actin polymerization and induction of microspike formation by N-WASP

All WASP family proteins share a common C terminus that consists of the ver prolin homology domain (V), cofilin homology domain (C), and acidic region (A), through which they activate Arp2/3 complex-induced actin polymerizatio n. In this study, we characterized the Arp2/3 complex-mediated actin polyme rization activity of VCA fragments of all of the WASP family proteins: WASP , N-WASP, WAVE1, WAVE2, and WAVE3. All of the VCA fragments stimulated the nucleating activity of Arp2/3 complex. Among them, N-WASP VCA, which posses ses two tandem V motifs, had a more potent activity than other VCA proteins . The chimeric protein experiments revealed that the V motif was more impor tant to the activation potency than the CA region; two V motifs were requir ed for full activity of N-WASP. COS7 cells overexpressing N-WASP form micro spikes in response to epidermal growth factor. However, when a chimeric pro tein in which the VCA region of N-WASP is replaced with WAVE1 VCA was overe xpressed, microspike formation was suppressed. Interestingly, when the N-WA SP VCA region was replaced with WAVE1 VCA, having two V motifs, this chimer ic protein could induce microspike formation. These results indicate that s trong activation of Arp2/3 complex by N-WASP is mainly caused by its two ta ndem V motifs, which are essential for actin microspike formation.