Identification of phosphorylation sites in βPIX and PAK1

doi:10.1242/jcs.008177 PIX (PAK1-interacting exchange factor beta, encoded by ARHGEF7) and PAK1 (p21 activated kinase) are interacting proteins that form a signaling module that mediates a number of cellular activities pertinent to cell migration (Kim et al., 2001; Koh et al., 2001; Webb et al., 2002). The interaction of PIX with GIT1 provides a signaling anchor through a GIT1-paxillin interaction; this signaling anchor serves to sequester the complex to adhesions and to the leading edge of migrating cells (Brown et al., 2002; Manabe et al., 2002; West et al., 2001). This complex plays a pivotal role in adhesion turnover and lamellipodial protrusion during cell migration (Nayal et al., 2006; Webb et al., 2006). It is thought that the sequestration of PAK1 and PIX to adhesions places the two proteins in proximity to membrane-anchored RAC1/CDC42, such that PIX can activate RAC1/CDC42, and activated RAC1/CDC42 can activate PAK1 (Rosenberger and Kutsche, 2006; ten Klooster et al., 2006). Recent studies have also linked PAK1 to centrosomes, in which it is anchored by the GIT1-PIX complex, induces Aurora A phosphorylation, and leads to a crucial check point in mitosis (Zhao et al., 2005). Although much is known about the functions of these proteins, the phosphorylations that regulate them have not been addressed in a systematic manner. PIX is a member of the DBL (diffuse B-cell lymphoma) family of Rho-GEFs (guanine nucleotide exchange factors) for RAC1/CDC42. The gene encoding PIX, ARHGEF7, located on chromosome 13, has six known splice variants. They are: PIX-a, PIX-b, PIX-bL, PIX-c, PIX-d, and p50 (Kim and Park, 2001). The primary 85 kD transcript, PIX-a, is ubiquitously expressed and was first identified as a component of adhesions and a direct binding partner of PAK1 (Bagrodia et al.