Calcium-independent activation of extracellularly regulated kinases 1 and 2 by angiotensin II in hepatic C9 cells: roles of protein kinase Cdelta, Src/proline-rich tyrosine kinase 2, and epidermal growth receptor trans-activation.

Agonist activation of endogenous angiotensin II (Ang II) AT(1) receptors expressed in hepatic C9 cells markedly stimulated inositol phosphate production, phosphorylation of the proline-rich tyrosine kinase PyK-2, and ERK activation. Ang II caused activation of protein kinase C delta (PKCdelta) in C9 cells, and its stimulatory actions on Pyk2 and extracellularly regulated kinase (ERK) phosphorylation were abolished by PKC depletion and selective inhibition of PKCdelta by rottlerin, but not by Ca(2+)-chelators. These effects, and the similar actions of the Src kinase inhibitor PP2 indicate the involvement of PKCdelta and Src kinase in ERK activation. In C9 cells, phorbol-12-myristate-13-acetate (PMA) caused much greater phosphorylation of Pyk2 and ERK than the Ca(2+) ionophore ionomycin, and the effects of PMA and Ang II were abolished in PKC-depleted cells. Ang II increased the association of Pyk2 with Src and with the epidermal growth factor receptor (EGF-R). EGF caused much greater tyrosine phosphorylation of the EGF-R than Ang II and PMA. Ang II-induced activation of ERK, but not Pyk2, was prevented by inhibition of EGF receptor phosphorylation by AG 1478 and of Src kinase by PP1. Ang II also increased the association of the adaptor protein Grb2 with the EGF-R. These findings indicate that Src and Pyk2 act upstream of the EGF-R and that the majority of Ang II-induced ERK phosphorylation is dependent on trans-activation of the EGF-R. Ang II-induced ERK activation in C9 cells is initiated by a PKCdelta-dependent but Ca(2+)-independent mechanism and is mediated by the Src/Pyk2 complex through trans-activation of the EGF-R.