title: A role for the tyrosine kinase Pyk2 in depolarization-induced contraction of vascular smooth muscle.
abstract: Depolarization of the vascular smooth muscle cell membrane evokes a rapid (phasic) contractile response followed by a sustained (tonic) contraction. We showed previously that the sustained contraction involves genistein-sensitive tyrosine phosphorylation upstream of the RhoA/Rho-associated kinase (ROK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase (MLCP)) and myosin regulatory light chains (LC20). In this study, we addressed the hypothesis that membrane depolarization elicits activation of the Ca(2+)-dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2). Pyk2 was identified as the major tyrosine-phosphorylated protein in response to membrane depolarization. The tonic phase of K(+)-induced contraction was inhibited by the Pyk2 inhibitor sodium salicylate, which abolished the sustained elevation of LC20 phosphorylation. Membrane depolarization induced autophosphorylation (activation) of Pyk2 with a time course that correlated with the sustained contractile response. The Pyk2/focal adhesion kinase (FAK) inhibitor PF-431396 inhibited both phasic and tonic components of the contractile response to K(+), Pyk2 autophosphorylation, and LC20 phosphorylation but had no effect on the calyculin A (MLCP inhibitor)-induced contraction. Ionomycin, in the presence of extracellular Ca(2+), elicited a slow, sustained contraction and Pyk2 autophosphorylation, which were blocked by pre-treatment with PF-431396. Furthermore, the Ca(2+) channel blocker nifedipine inhibited peak and sustained K(+)-induced force and Pyk2 autophosphorylation. Inhibition of Pyk2 abolished the K(+)-induced translocation of RhoA to the particulate fraction and the phosphorylation of MYPT1 at Thr-697 and Thr-855. We conclude that depolarization-induced entry of Ca(2+) activates Pyk2 upstream of the RhoA/ROK pathway, leading to MYPT1 phosphorylation and MLCP inhibition. The resulting sustained elevation of LC20 phosphorylation then accounts for the tonic contractile response to membrane depolarization.
authors.affiliation: From the Smooth Muscle Research Group, Department of Biochemistry and Molecular Biology, Libin Cardiovascular Institute and Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada and.
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Mills
authors.affiliation: the Department of Pharmacodynamics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Mita
authors.affiliation: the Department of Pharmacodynamics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Nakagawa
authors.affiliation: the Department of Pharmacodynamics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Shoji
authors.affiliation: From the Smooth Muscle Research Group, Department of Biochemistry and Molecular Biology, Libin Cardiovascular Institute and Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada and.
authors.forename: Cindy
authors.initials: C
authors.lastname: Sutherland
authors.affiliation: From the Smooth Muscle Research Group, Department of Biochemistry and Molecular Biology, Libin Cardiovascular Institute and Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada and walsh@ucalgary.ca.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism*; D008297:Male; D009119:Muscle Contraction / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000502:physiology*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar
chemical_list: D051418:Focal Adhesion Kinase 2
keywords: Depolarization; Myosin; Myosin Light Chain Phosphorylation; Protein-Tyrosine Kinase (Tyrosine-Protein Kinase); Rho (Rho GTPase); Rho-associated Kinase; Tonic Contraction; Vascular Smooth Muscle