Calcium- and Protein Kinase C–Dependent Activation of the Tyrosine Kinase PYK2 by Angiotensin II in Vascular Smooth Muscle

From the Department of Physiology and the Cardiovascular Institute (A.S., G.G., T.M.G., K.L.B., A.M.S., P.A.L.) and the Department of Medicine (K.L.B., A.M.S.), Loyola University Chicago, Stritch School of Medicine, Maywood, Ill.

Correspondence to Pamela A. Lucchesi, Department of Physiology and Biophysics, University of Alabama at Birmingham, 986 McCallum Basic Health Science Bldg, 1918 University Blvd, Birmingham, AL 35294-0005. E-mail lucchesi{at}phybio.bhs.uab.edu

Abstract—Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating G_q-protein–coupled AT₁ receptors, which leads to elevation of cytosolic Ca²⁺ ([Ca²⁺]_i) and activation of protein kinase 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 activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle -actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT₁ receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca²⁺]_i. Treatment with either phorbol ester or Ca²⁺ ionophore also increased PYK2 phosphorylation, suggesting that PKC activation 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 PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth.

Key Words: PYK2 • vascular smooth muscle • angiotensin II • protein kinase C • Ca²⁺

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