p21-activated kinase (Pak)-interacting exchange factor (Pix), a Rho family guanine nucleotide exchange factor (GEF), has been shown to co-localize with Pak and form activated Cdc42- and Rac1-driven focal complexes. In this study we have presented evidence that treatment of human mesangial cells (HMC) with endothelin 1 (ET-1) and stimulation of adenylate cyclase with either forskolin or with the cAMP analog 8-Br-cAMP activated the GTP loading of Cdc42. Transient expression of constitutively active G alpha(s) also stimulated Cdc42. In addition, overexpression of beta(1)Pix enhanced ET-1-induced Cdc42 activation, whereas the expression of beta(1)Pix SH3m(W43K), which lacks the ability to bind Pak, and beta(1)PixDHm(L238R/L239S), which lacks GEF activity, decreased ET-1-induced Cdc42 activation. Furthermore, ET-1 stimulation induced beta(1)Pix translocation to focal complexes. Interestingly, pretreatment of HMC with protein kinase A (PKA) inhibitors blocked both Cdc42 activation and beta(1)Pix translocation induced by ET-1, indicating the involvement of the PKA pathway. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assay, we have shown that beta(1)Pix is phosphorylated by PKA. Using purified recombinant beta(1)Pix(wt) and beta(1)Pix mutants, we have identified Ser-516 and Thr-526 as the major phosphorylation sites by PKA. beta(1)Pix(S516A/T526A), in which both phosphorylation sites are replaced by alanine, blocks beta(1)Pix translocation and Cdc42 activation. Our results have provided evidence that stimulation of PKA pathway by ET-1 or cAMP analog results in beta(1)Pix phosphorylation, which in turn controls beta(1)Pix translocation to focal complexes and Cdc42 activation.