Angiotensin II-induced stimulation of p21-activated kinase and c-Jun NH2-terminal kinase is mediated by Rac1 and Nck

p21-activated kinase (PAK) has been shown to be an upstream mediator of JNK in angiotensin II (AngII) signaling. Little is known regarding other signaling molecules involved in activation of PAR and JNK by AngII. Rho family GTPases Pac and Cdc42 have been shown to enhance PAR activity by binding to pal-binding domain of PAR (PAK-PBD), In vascular smooth muscle cells (VSMC) AngII stimulated Rad binding to GST-PAK-PBD fusion protein. Pretreatment of VSMC by genistein inhibited AngII-induced Rad activation, whereas Src inhibitor PP1 had no effect. Inhibition of protein kinase C by phorbol 12,13-dibutyrate pretreatment also decreased AngII-mediated activation of Rac1. The adaptor molecule Nck has been shown previously to mediate PAR activation by facilitating translocation of PAR to the plasma membrane. In VSMC AngII stimulated translocation of Nck and PAK to the membrane fraction. Overexpression of dominant-negative Nck in Chinese hamster ovary (CHO) cells, stably expressing the AngII type I receptor (CHO-AT1), inhibited both PAK and JNK activation by AngII, whereas it did not affect ERK1/2. Finally, dominant-negative Nck inhibited AngII-induced DNA synthesis in CHO-AT1 cells. Our data provide evidence for Rad and Nck as upstream mediators of PAR and JNK in AngII signaling and implicate JNK in AngII-induced growth responses.