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

Abstract

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating Gq-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 alpha-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.

Carbachol-stimulated Transactivation of Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase in T84 Cells Is Mediated by Intracellular Ca2+, PYK-2, and p60

“…Furthermore, that p60 src is involved in an antisecretory signaling pathway is supported by our findings that, similar to inhibition of the EGFR or ERK (5), PP2 also potentiates and prolongs secretory responses to both CCh and TG. Since CCh increases co-immunoprecipitation of PYK-2 with p60 src , it is reasonable to assume that, as in other cell types, PYK-2 activation in response to elevations in intracellular Ca 2ϩ leads to recruitment of p60 src to form a PYK-2⅐p60 src complex (18,29,43,52). It is interesting to note that the formation of PYK-2⅐p60 src complexes in response to CCh appear to occur in a biphasic fashion with an initial association occurring within 30 s that temporally corresponds with p60 src phosphorylation and precedes EGFR activation, and a later association that occurs 2-5 min after stimulation with CCh and is not associated with phosphorylation of p60 src (cf.…”

Molecular Biology of Atherosclerosis

“…The tyrosine kinase PYK2 (proline-rich tyrosine kinase 2, also known as FAK2 or PTK2B for protein tyrosine kinase 2, beta) was found to be the primary mediator of these effects in many cells (426,1011,1520). While PYK2 is highly analogous to FAK, it is primarily cytosolic while FAK is mainly associated with integrin signaling and is activated by different signals.…”

CRNK gene transfer improves function and reverses the myosin heavy chain isoenzyme switch during post-myocardial infarction left ventricular remodeling

“…PYK2 coordinates integrin-, Ca 2+ -, and PKC-dependent signal transduction in a number of tissues (for review, see (7)). PYK2 expression and phosphorylation are regulated by intracellular Ca 2+ and protein kinase C (PKC) in cardiomyocytes and vascular smooth muscle cells (8)(9)(10). As examined in various cell lines, PYK2 serves as an "activatable" scaffolding protein, and transduces signals from G-protein coupled receptors to the mitogen-activated protein kinases (MAPK) and the phosphoinositol-3-kinase (PI-(3)K)-PDK1-Akt signaling pathway depending upon which adaptor proteins bind to the phosphorylated enzyme (6,(11)(12)(13).…”