PROTEIN-KINASE C-ZETA MEDIATES ANGIOTENSIN-II ACTIVATION OF ERK1 2 INVASCULAR SMOOTH-MUSCLE CELLS/

Activation of 44 and 42 kDa extracellular signal-regulated kinases (ER K)1/2 by angiotensin II (angII) plays an important role in vascular sm ooth muscle cell (VSMC) function, The dual specificity mitogen-actived protein (MAP) kinase/ERK kinase (MEK) activates ERK1/S in response to angII, but the MEK activating kinases remain undefined, Raf is a cand idate MEK kinase, However, a kinase other than Raf appears responsible for angII-mediated signal transduction because we showed previously t hat treatment with 1 mu M phorbol 12,13-dibutyrate (PDBU) for 24 h com pletely blocked Raf-Ras association in VSMC but did not inhibit activa tion of MEK and ERK1/2 by angII. We hypothesized that an atypical prot ein kinase C (PKC) isoform, which lacks a phorbol ester binding domain , mediated ERK1/2 activation by angII, Western blot analysis of rat ao rtic VSMC with PKC isoform-specific antibodies showed PKC-alpha, -beta 1, delta, -epsilon, and -zeta in relative abundance. All isoforms exc ept PKC-zeta were down regulated by 1 mu M PDBU for 24 h suggesting th at PKC zeta was responsible for angII-mediated ERK1/2 activation. In r esponse to angII, PKC-zeta associated with Pas as shown by co precipit ation of PKC-zeta with anti H-Ras antibody, To characterize further th e role of PKC-zeta, PKC-zeta protein was depleted specifically by tran sfection with antisense PKC-zeta oligonucleotides. Antisense PKC-zeta oligonucleotide treatment significantly decreased PKC-zeta protein exp ression (without effect on other PKC isoforms) and angII-mediated ERK1 /2 activation in a concentration dependent manner. In contrast, ERK1/2 activation by platelet-derived growth factor and phorbol ester was no t significantly inhibited, These results demonstrate an important diff erence in signal transduction by angII compared with PDGF and phorbol ester in VSMC, and suggest a critical role for PKC-zeta and Ras in ang II stimulation of ERK1/2.