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

Abstract

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 Gαs 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.