Phosphorylation of the purified receptor for calcium channel blockers by cAMP kinase and protein kinase C.
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.
Nastainczyk
W
W
Physiologische Chemie, Medizinische Fakultät der Universität des Saarlandes, Homburg/Saar, Federal Republic of Germany.
Röhrkasten
A
A
Sieber
M
M
Rudolph
C
C
Schächtele
C
C
Marmè
D
D
Hofmann
F
F
D000818:Animals; D015220:Calcium Channels; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000494:pharmacology*; D007700:Kinetics; D008861:Microsomes / Q000032:analysis; D009132:Muscles / Q000032:analysis; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D015220:Calcium Channels; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C; D014357:Trypsin