Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C alpha and delta isoforms.
Histamine stimulus triggers inhibition of myosin phosphatase-enhanced phosphorylation of myosin and contraction of vascular smooth muscle. In response to histamine stimulation of intact femoral artery, a smooth muscle-specific protein called CPI-17 (for protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa) is phosphorylated and converted to a potent inhibitor for myosin phosphatase. Phosphorylation of CPI-17 is diminished by pretreatment with either or GF109203x, suggesting involvement of multiple kinases (Kitazawa, T., Eto, M., Woodsome, T. P., and Brautigan, D. L. (2000) J. Biol. Chem. 275, 9897--9900). Here we purified and identified CPI-17 kinases endogenous to pig artery that phosphorylate CPI-17. DEAE-Toyopearl column chromatography of aorta extracts separated two CPI-17 kinases. One kinase was protein kinase C (PKC) alpha, and the second kinase was purified to homogeneity as a 45-kDa protein, and identified by sequencing as PKC delta. Purified PKC delta was 3-fold more reactive with CPI-17 compared with myelin basic protein, whereas purified PKC alpha and recombinant RhoA-activated kinases (Rho-associated coiled-coil forming protein Ser/Thr kinase and protein kinase N) showed equal activity with CPI-17 and myelin basic protein. inhibited CPI-17 phosphorylation by purified PKC delta with IC(50) of 0.6 microm (in the presence of 0.1 mm ATP) or 14 microm (2.0 mm ATP). significantly suppressed CPI-17 phosphorylation in smooth muscle cells, and the contraction of permeabilized rabbit femoral artery induced by stimulation with phorbol ester. GF109203x inhibited phorbol ester-induced contraction of rabbit femoral artery by 80%, whereas a PKC alpha/beta inhibitor, Go6976, reduced contraction by 47%. The results imply that histamine stimulation elicits contraction of vascular smooth muscle through activation of PKC alpha and especially PKC delta to phosphorylate CPI-17.
Eto
M
M
Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA. me2h@virginia.edu
Kitazawa
T
T
Yazawa
M
M
Mukai
H
H
Ono
Y
Y
Brautigan
D L
DL
D000577:Amides / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000502:physiology; D002227:Carbazoles / Q000494:pharmacology; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology*; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D009124:Muscle Proteins / Q000378:metabolism*; D009125:Muscle Relaxants, Central / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D004676:Myelin Basic Protein / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D010446:Peptide Fragments / Q000737:chemistry; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D011725:Pyridines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology*
D000577:Amides; C491521:CPI-17 protein, Oryctolagus cuniculus; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D009124:Muscle Proteins; D009125:Muscle Relaxants, Central; D004676:Myelin Basic Protein; D010446:Peptide Fragments; D010750:Phosphoproteins; D011725:Pyridines; D011994:Recombinant Proteins; C081021:Go 6976; C108830:Y 27632; D015240:Phorbol 12,13-Dibutyrate; D006632:Histamine; D017886:Durapatite; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; C070515:bisindolylmaleimide I; C064758:Ro 31-8220