Protein kinase C-ζ mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells

Abstract

Activation of 44 and 42 kDa extracellular signal-regulated kinases (ERK)I/2 by angiotensin II (angII) plays an important role in vascular smooth muscle cell (VSMC) function. The dual specificity mitogen-actived protein (MAP) kinase/ERK kinase (MEK) activates ERK1/2 in response to angII, but the MEK activating kinases remain undefined. Raf is a candidate MEK kinase. However, a kinase other than Raf appears responsible for angII-mediated signal transduction because we showed previously that treatment with 1 μM phorbol 12,13-dibutyrate (PDBU) for 24 h completely blocked Raf-Ras association in VSMC but did not inhibit activation of MEK and ERK1/2 by angII. We hypothesized that an atypical protein kinase C (PKC) isoform, which lacks a phorbol ester binding domain, mediated ERK1/2 activation by angII. Western blot analysis of rat aortic VSMC with PKC isoform-specific antibodies showed PKC-α, -β1, -δ, -ε, and -ζ in relative abundance. All isoforms except PKC-ζ were down-regulated by 1 μM PDBU for 24 h suggesting that PKC- ζ was responsible for angII-mediated ERKI/2 activation. In response to angII, PKC-ζ associated with Ras as shown by co-precipitation of PKC-ζ with anti-H-Ras antibody. To characterize further the role of PKC-ζ, PKC-ζ protein was depleted specifically by transfection with antisense PKC-ζ oligonucleotides. Antisense PKC-ζ oligonucleotide treatment significantly decreased PKC-ζ protein expression (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 not significantly inhibited. These results demonstrate an important difference in signal transduction by angII compared with PDGF and phorbol ester in VSMC, and suggest a critical role for PKC-ζ and Ras in angII stimulation of ERK1/2.