Eur. J. Immunol. 17, 237 (1987

No abstract

Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium-channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase I1 and casein kinase 11 phosphorylated the 165-kDa and the 55-kDa proteins of the purified CaCB receptor. The 130/ 28-kDa and the 32-kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165-kDa subunit with 2-3-fold higher initial rate than the 55-kDa subunit. Casein kinase I1 phosphorylated the 165-kDa and the 55-kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase I1 phosphorylated preferentially the 55-kDa protein. The 55-kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase I1 or casein kinase I1 and about 10 times faster by these enzymes than by cAMP kinase. Two-dimensional peptide maps of the 165-kDa subunit yielded a total of 11 phosphopeptides. Four or fivc peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase I1 and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55-kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165-kDa and the 55-kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase I1 and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor.The calcium current of a variety of cells is modulated by hormones which increase or decrease the current through activation of GTP-binding proteins and/or phosphorylation of the channel protein (see [I] for a recent introduction). cAMP kinase, cGMP kinase and protein kinase C have been identified as in vivo modulators of calcium currents in neuronal and nonneuronal cells [2 -131. Detailed studies in cardiac myocytes [2-41 and neurons [5, 61 show that cAMP kinase increases the calcium current by raising the probability of channel opening and by stabilization of the channel life time. It was even suggested [S] that CAMP-dependent phosphorylation of the channel is a prerequisite for its voltagedependent opening. The importance of the phosphorylation state for the function of calcium channel is further strengthened by the observation that calcium-dependent dephosphorylation of the channel promotes its inactivation [5]. Similar effects to those observed with cAMP b a s e are induced by cGMP kinase in specific neurons of Helix aspera [9]. In contrast, opposing effects of protein kinase C have been observed [lo-131. Protein kinase C increases the number of calcium channels [lo, 1 I] and/or the density of dihydropyridine-bind- ing sites [12] in some cells, whereas in other cells a decrease in calcium current has been reported [13]. These results suggest that, similar to many enzymes involved in energy m...

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...

No abstract