Pyk2 Is a Redox-Sensitive Tyrosine Kinase in Vascular Smooth Muscle Cells

Abstract

P162 PYK2 is a focal adhesion kinase-related protein tyrosine kinase (PTK) and is implicated in downstream mitogen-activated protein kinase activation. In cultured vascular smooth muscle cells (VSMCs), Ca 2+ and/or protein kinase C- (PKC) dependent PYK2 activation by angiotensin II and platelet-derived growth factor has been reported. Because reactive oxygen species (ROS) have been shown to mediate mitogenic signals by these agonists in VSMCs, we hypothesized that PYK2 represents a redox-sensitive PTK used by vascular mitogens. Activation of PYK2 was assessed by three distinct methods: (1) immunoblotting with phospho-specific antibody against autophosphorylated PYK2 at Tyr 402 ; (2) immunoprecipitation of PYK2 by anti-PYK2 antibody and immunoblotting with anti-phosphotyrosine antibody; (3) measurement of PYK2’s kinase activity towards the tyrosine kinase specific substrate poly-[Glu 80 Tyr 20 ] after immunoprecipitating with anti-PYK2 antibody. In cultured rat VSMCs, H 2 O 2 concentration- and time- dependently induced PYK2 phosphorylation at its autophosphorylation site Tyr 402 . Maximal phosphorylation occurred using 10-20 μM H 2 O 2 at 5-10 min and the phosphorylated protein co-migrated with a major tyrosine-phosphorylated 120 kDa protein induced by H 2 O 2 . Diamide (1 mM), a thiol-oxidizing agent, gave similar results to that of H 2 O 2 . H 2 O 2 also increased tyrosine phosphorylation of PYK2 immunoprecipitated with anti PYK2 antibody. Moreover, treatment of VSMCs with 10 μM H 2 O 2 for 5 min increased PTK activity immunoprecipitated with anti PYK2 antibody. However, 10 μM H 2 O 2 had no effect on intracellular Ca 2+ concentration detected by the fluorescent dye, fura-2 and GF109203X, a PKC inhibitor, failed to inhibit H 2 O 2 -induced PYK2 phosphorylation indicating that ROS-dependent PYK2 activation does not require Ca 2+ or PKC. These data demonstrated for the first time that PYK2 represents a major redox-sensitive PTK in the vessel wall, suggesting its pivotal role in vascular remodeling.