CR16 forms a complex with N-WASP in brain and is a novel member of a conserved proline-rich actin-binding protein family

The Neuronal Wiskott–Aldrich syndrome protein (N-WASP) has emerged as a central regulator of the actin cytoskeleton with abilities to integrate multiple upstream signal inputs and transmit them to the Arp2/3 complex. Here, we demonstrate that native N-WASP is present in a tight complex with a proline-rich protein, CR16, which shares ≈25% identity with WASP interacting protein. CR16 is encoded by a gene previously cloned as a glucocorticoid-regulated mRNA from a rat hippocampal cDNA library. Although N-WASP is expressed ubiquitously, full-length CR16 protein is found predominately in the brain. CR16 and N-WASP colocalize in primary hippocampal neurons and at the tips of their growth cone filopodia. In vitro , CR16 directly binds both monomeric and filamentous actin but does not affect the kinetics of actin polymerization mediated by N-WASP and the Arp2/3 complex. Sequence homologues of CR16 are found not only in other vertebrates but also in the invertebrate Caenorhabditis elegans and in yeast. Thus, CR16 and WASP interacting protein belong to a family of N-WASP-binding proteins.