Phosphorylation of caldesmon in arterial smooth muscle.
We have isolated caldesmon (Mr = 145,000), by immunoprecipitation, from [32P]orthophosphate-loaded porcine carotid arteries. In resting muscles, caldesmon was phosphorylated to 0.45 mol of PO4/mol protein, while the 20,000-dalton myosin regulatory light chain (LC20) was phosphorylated to less than 0.05 mol/mol. After stimulation by KCl (110 mM) for 75 min and phorbol 12,13-dibutyrate (PDBu, 1 microM) for 60 min, caldesmon phosphorylation levels rose to 0.96 and 1.1 mol/mol, respectively. LC20 phosphorylation increased to 0.49 mol/mol at 1 min of stimulation by KCl and decreased to 0.17 mol/mol at 60 min. With PDBu, phosphate incorporation into LC20 rose only slightly, reaching 0.09 mol/mol after 90 min. Muscles contracted with histamine (10 microM) or ouabain (1 microM) also demonstrated elevated levels of phosphate incorporation into caldesmon. In these muscles, LC20 phosphorylation levels were less than 0.05 mol/mol. Three major phosphopeptides of indistinguishable mobility were identified on maps of caldesmon from resting, KCl-stimulated, and PDBu-stimulated muscles. There was, however, little similarity between the phosphopeptide maps of caldesmon phosphorylated in intact tissue and maps of purified caldesmon phosphorylated in vitro by protein kinase C (Ca2+/phospholipid-dependent enzyme) or Ca2+/calmodulin kinase II.
Adam
L P
LP
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Haeberle
J R
JR
Hathaway
D R
DR
D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D009119:Muscle Contraction; D009126:Muscle Relaxation; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011189:Potassium Chloride; D011233:Precipitin Tests; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D002148:Calmodulin-Binding Proteins; D011189:Potassium Chloride; D013755:Tetradecanoylphorbol Acetate