CALCIUM-DEPENDENT AND PROTEIN-KINASE-C-DEPENDENT ACTIVATION OF THE TYROSINE KINASE PYK2 BY ANGIOTENSIN-II IN VASCULAR SMOOTH-MUSCLE

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) gro wth by activating G(q)-protein-coupled AT(1) receptors, which leads to elevation of cytosolic Ca2+ ([Ca2+](i)) and activation of protein kin ase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II-induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca2+](i) or ac tivate PKC. PYK2 distribution was demonstrated in rat aortic tissue an d in cultured VSMC by immunohistochemistry, revealing a cytosolic dist ribution distinct from smooth muscle alpha-actin, focal adhesion kinas e, or paxillin. The involvement of PYK2 in Ang II signaling was measur ed by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-de pendent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT(1 ) receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca2+](i). Treatment with either phorbol ester or Ca2+ i onophore also increased PYK2 phosphorylation, suggesting that PKC acti vation and/or increased [Ca2+](i) are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-src complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca2+](i) and PK C activation with downstream signaling events such as mitogen-activate d protein kinase activation involved in the regulation of VSMC growth.