FIG. 4.

Wave-1 interacts with Abi-1 through a region overlapping the putative leucine zipper region. (A) The amino terminus of Wave-1 is necessary for binding to Abi-1. Myc-tagged Wave-1 full-length and Flag-Abi-1 forms were coexpressed in 293T cells, and Flag-Abi-1 forms were immunoprecipitated (IP) with anti-Flag antibodies. Coimmunoprecipitated Wave-1 forms were detected with anti-Myc antibody (upper panel). Expression levels of the Myc-Wave forms are shown in the middle panel, and the levels of Flag-Abi-1 are shown in the lower panel. The asterisk indicates the immunoglobulin G (IgG) heavy-chain band. IB, immunoblot. (B) The region between amino acids 34 to 92 of Wave-1 is necessary to bind Abi-1. Coimmunoprecipitation was performed as described for panel A. The upper panel shows the binding between Flag-Abi-1 and the wild-type and mutant forms of Myc-Wave-1. Wave-1 and Abi-1 levels were visualized by immunoblotting with Myc and Flag antibodies, respectively. (C) Wave-1 amino acids 1 to 96 are sufficient to bind Abi-1. Myc-tagged Wave-1 and Wave-1-1-96 were coexpressed with Flag-Abi-1, and Abi-1 was immunoprecipitated as described for panel A. IgG heavy and light chains are indicated. (D) Schematic representation of the Abi-1-binding region of Wave-1. L-zipper, leucine zipper; ++, basic region; Pro, proline; V, verprolin homology domain; C, cofilin homology domain; A, acidic region. The Wave-1 diagram is not to scale. Asterisks indicate 100% conservation among Wave family members (Wave-1, Wave-2, Wave-3, D. melanogaster Scar, and Dictyostelium discoideum Scar).