Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle.
Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites. Stimulation of cultured tracheal smooth muscle cells with ACh or platelet-derived growth factor increased caldesmon phosphorylation at Ser789 by about twofold. Inhibiting ERK MAP kinase activation with 50 microM PD-98059 blocked agonist-induced caldesmon phosphorylation completely. Inhibiting p38 MAP kinases with 25 microM SB-203580 had no effect on ACh-induced caldesmon phosphorylation. Carbachol stimulation increased caldesmon phosphorylation at Ser789 in intact tracheal smooth muscle, which was blocked by the M(2) antagonist AF-DX 116 (1 microM). AF-DX 116 inhibited carbachol-induced isometric contraction by 15 +/- 1.4%, thus dissociating caldesmon phosphorylation from contraction. Activation of M(2) receptors leads to activation of ERK MAP kinases and phosphorylation of caldesmon with little or no functional effect on isometric force. P38 MAP kinases are also activated by muscarinic agonists, but they do not phosphorylate caldesmon in vivo.
Hedges
J C
JC
Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557-0046, USA.
Oxhorn
B C
BC
Carty
M
M
Adam
L P
LP
Yamboliev
I A
IA
Gerthoffer
W T
WT
D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000378:metabolism; D015398:Signal Transduction; D014132:Trachea / Q000166:cytology / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D002148:Calmodulin-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D020033:Protein Isoforms; D011725:Pyridines; D011976:Receptors, Muscarinic; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one