Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit.
Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology; D004557:Electric Organ / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D007118:Immunoassay; D007942:Leukemia, Experimental / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011950:Receptors, Cholinergic / Q000378:metabolism*; D012697:Serine Endopeptidases; D014101:Torpedo
D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase