Endothelin 1 induces β₁Pix translocation and Cdc42 activation via protein kinase A-dependent pathway

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 Gα_s also stimulated Cdc42. In addition, even-expression of β₁Pix enhanced ET-1-induced Cdc42 activation, whereas the expression of β₁Pix SH3m(W43K), which lacks the ability to bind Pak, and β₁PixDHm(L238R/L239S), which lacks GEF activity, decreased ET-1-induced Cdc42 activation. Furthermore, ET-1 stimulation induced β₁Pix translocation to focal complexes. Interestingly, pretreatment of HMC with protein kinase A (PKA) inhibitors blocked both Cdc42 activation and β₁Pix 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 β₁Pix is phosphorylated by PKA. Using purified recombinant β₁Pix(wt) and β₁Pix mutants, we have identified Ser-516 and Thr-526 as the major phosphorylation sites by PKA. β₁Pix(S516A/T526A), in which both phosphorylation sites are replaced by alanine, blocks β₁Pix translocation and Cdc42 activation. Our results have provided evidence that stimulation of PKA pathway by KT-1 or cAMP analog results in β₁Pix phosphorylation, which in turn controls β₁Pix translocation to focal complexes and Cdc42 activation.