Phosphorylation of the purified receptor for calcium channel blockers by cAMP kinase and protein kinase C

Abstract

The dihydropyridine receptor purified from rabbit skeletal muscle contains three proteins of 165, 55 and 32 kDa. cAMP kinase and protein kinase C phosphorylate the 165-kDa and the 55-kDa proteins. At identical concentrations of each protein kinase, cAMP kinase phosphorylates the 165-kDa protein faster than the 55-kDa protein. Protein kinase C phosphorylates preferentially the 55-kDa protein. cAMP kinase incorporates up to 1.6 mol phosphate/mol protein into the 165-kDa protein and 1 mol/mol into the 55-kDa protein upon prolonged incubation. At a physiological concentration of cAMP kinase 1 mol phosphate is incorporated/mol 165-kDa protein within 10 min, suggesting a physiological role of this phosphorylation. Protein kinase C incorporates up to 1 mol phosphate/mol into the 55-kDa protein and less than 1 mol/mol into the 165-kDa protein. Tryptic phosphopeptide analysis reveals that cAMP kinase phosphorylates two distinct peptides in the 165-kDa protein, whereas protein kinase C phosphorylates a single peptide in the 165-kDa protein. cAMP kinase and protein kinase C phosphorylate three and two peptides in the 55-kDa protein, respectively. Mixtures of the tryptic phosphopeptides derived from the 165-kDa and 55-kDa proteins elute according to the composite of the two elution profiles.These results suggest that the 165-kDa protein, which contains the binding sites for each class of calcium channel blockers and the basic calcium-conducting structure, is a specific substrate for cAMP kinase. The 55-kDa protein apparently contains sites preferentially phosphorylated by protein kinase C.The dihydropyridine receptor has been purified from rabbit skeletal muscle transverse tubulus [l -31. The receptor contains three proteins of 165, 55 and 32 kDa which copurify in stoichiometric concentrations [4, 51. The 165-kDa peptide has been isolated [4, 51 and photoaffinity labeling experiments suggest that it contains the high-affinity binding sites for dihydropyridines, phenylalkylamines and benzothiazepines [4 -71. This large protein also contains the calciumconducting unit of the L-type calcium channel, the openstate probability of which is increased by CAMP-dependent phosphorylation [8]. The phosphorylated protein has not been conclusively identified, so far. Originally it was reported that cAMP kinase phosphorylates preferentially the 55-kDa protein of the dihydropyridine receptor at a slow rate although some radioactivity was associated with the large protein [9]. Later work suggested [ 101 that cAMP kinase phosphorylates 140 -160-kDa and 60 -50-kDa proteins in t-tubular membranes. It was further suggested that the 60-50-kDa phosphoprotein was derived proteolytically from the large protein since identical V8 protease peptides were obtained from both proteins [lo]. These conclusions were supported by the finding that cAMP kinase phosphorylated preferentially the large 165-kDa protein of a dihydropyridine receptor prepCorrespondence to F. Hofmann, Physiologische Chemie, Medizinische Fakultat der Universitit des Saarlandes, D-6650...

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