title: Prostate-apoptosis response-4 phosphorylation in vascular smooth muscle.
abstract: The protein prostate-apoptosis response (Par)-4 has been implicated in the regulation of smooth muscle contraction, based largely on studies with the A7r5 cell line. A mechanism has been proposed whereby Par-4 binding to MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase, MLCP) blocks access of zipper-interacting protein kinase (ZIPK) to Thr697 and Thr855 of MYPT1, whose phosphorylation is associated with MLCP inhibition. Phosphorylation of Par-4 at Thr155 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition and contraction. We tested this "padlock" hypothesis in a well-characterized vascular smooth muscle system, the rat caudal artery. Par-4 was retained in Triton-skinned tissue, suggesting a tight association with the contractile machinery, and indeed Par-4 co-immunoprecipitated with MYPT1. Treatment of Triton-skinned tissue with the phosphatase inhibitor microcystin (MC) evoked phosphorylation of Par-4 at Thr155, but did not induce its dissociation from the contractile machinery. Furthermore, analysis of the time courses of MC-induced phosphorylation of MYPT1 and Par-4 revealed that MYPT1 phosphorylation at Thr697 or Thr855 preceded Par-4 phosphorylation. Par-4 phosphorylation was inhibited by the non-selective kinase inhibitor staurosporine, but not by inhibitors of ZIPK, Rho-associated kinase or protein kinase C. In addition, Par-4 phosphorylation did not occur upon addition of constitutively-active ZIPK to skinned tissue. We conclude that phosphorylation of Par-4 does not regulate contraction of this vascular smooth muscle tissue by inducing dissociation of Par-4 from MYPT1 to allow phosphorylation of MYPT1 and inhibition of MLCP.
authors.affiliation: Smooth Muscle Research Group and Department of Biochemistry & Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, AB, Canada T2N 4Z6. jmacdo@ucalgary.ca
authors.forename: Justin A
authors.initials: JA
authors.lastname: MacDonald
authors.forename: Lori D
authors.initials: LD
authors.lastname: Moffat
authors.forename: Abdulhameed
authors.initials: A
authors.lastname: Al-Ghabkari
authors.forename: Cindy
authors.initials: C
authors.lastname: Sutherland
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
mesh_terms: D000577:Amides / Q000494:pharmacology; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001158:Arteries / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D064550:Death-Associated Protein Kinases; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D052998:Microcystins / Q000494:pharmacology; D009119:Muscle Contraction; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D017830:Octoxynol / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
chemical_list: D000577:Amides; D051017:Apoptosis Regulatory Proteins; D007211:Indoles; D008301:Maleimides; D052998:Microcystins; D011725:Pyridines; D011994:Recombinant Proteins; C101296:prostate apoptosis response-4 protein; C108830:Y 27632; D013912:Threonine; C078588:microcystin; D017830:Octoxynol; D064550:Death-Associated Protein Kinases; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D019311:Staurosporine; C070515:bisindolylmaleimide I