The mammalian Verprolin, WIRE induces filopodia independent of N-WASP through IRSp53

The mammalian verprolin family of proteins, WIP (WASP Interacting Protein), CR16 (Corticoid Regulated) and WIRE (WIp-RElated) regulate the actin cytoskeleton through WASP/N-WASP (Wiskott Aldrich Syndrome Protein and Neural-WASP). In order to characterize the WASP/N-WASP-independent function of WIRE, we screened and identified IRSp53 (Insulin Receptor Substrate) as a WIRE interacting protein. Expression of IRSp53 with WIRE in N-WASP−/− mouse fibroblast cells induced filopodia while co-expression of IRSp53 with WIP did not. The induction of filopodia is dependent on WIRE–IRSp53 interaction as mutation in the SH3 domain of IRSp53 abolished WIRE–IRSp53 interaction as well as the ability to induce filopodia. Similarly, the Verprolin (V)-domain of WIRE is critical for IRSp53–WIRE interaction and for filopodia formation. The interaction between WIRE and IRSp53 is regulated by Cdc42 as mutations which abolish Cdc42-IRSp53 interaction lead to loss of IRSp53–WIRE interaction as shown by pull down assay. The plasma membrane localization of IRSp53 is dependent on Cdc42 and WIRE. Expression of Cdc42G12V (active mutant) with WIRE–IRSp53 caused significant increase in the number of filopodia per cell. Thus our results show that Cdc42 regulates the activity of IRSp53 by regulating the IRSp53–WIRE interaction as well as localization of the complex to plasma membrane to generate filopodia.