Neuronal specific ss Pix-b stimulates actin-dependent processes via the interaction between its PRD and WH1 domain of N-WASP

Abstract

beta Pix, a Pak-interacting nucleotide exchange factor (Cool-1/p85SPR), is a Cdc42/Rac1-specific guanine nucleotide exchange factor (GEF) involved in various actin-related processes. Many previous studies have focused on ubiquitously expressed beta Pix-a, while the role of the neuronal-specific isoform beta Pix-b is still unknown, especially whether its role is distinct from or similar to beta Pix-a. Here we show that unlike beta Pix-a, overexpression of beta Pix-b stimulates actin-dependent comet formation in BHK21 cells. This effect is attributed to the interaction between its proline-rich domain (PRD) and the WH1 domain of N-WASP. In addition, we show that overexpression of beta Pix-b stimulates actin-dependent dendritic spine formation in rat hippocampal neurons in culture, a formation that is blocked by co-expression of the WH1 domain of N-WASP or the PRD of beta Pix-b. Knocking-down endogenous expression of beta Pix-b by shRNA reduced the number of dendritic spines, which were rescued only by PRD-containing beta Pix-b mutants. GEF activity of beta Pix-b is also required for these effects. The results show that neuronal-specific beta Pix-b stimulates actin-dependent processes in cells via the interaction between its PRD and the WH1 domain of N-WASP. Our results identify N-WASP as the first protein shown to interact with the PRD of beta Pix-b, raising the possibility that, as an N-WASP WH1-binding protein, beta Pix-b may regulate N-WASP's activity in cells. J. Cell. Physiol. 227: 1476-1484, 2012. (C) 2011 Wiley Periodicals, Inc.