Phosphorylation of the nicotinic acetylcholine receptor by an endogenous tyrosine-specific protein kinase.

Abstract

Postsynaptic membranes from the electric organ of Torpedo californica, rich in the nicotinic acetylcholine receptor, were shown to contain an endogenous tyrosine protein kinase. This endogenous kinase phosphorylated 3 major proteins with molecular masses corresponding to 50 kDa [kilodalton], 60 kDa and 65 kDa. The phosphorylation of these 3 proteins occurred exclusively on tyrosine residues under the experimental conditions used, and was abolished by 0.1% Nonidet P-40 and stimulated by Mn2+. The 50 kDa, 60 kDa and 65 kDa phosphoproteins were demonstrated to be the .beta., .gamma. and .delta. subunits, respectively, of the nicotinic acetylcholine receptor by purification of the phosphorylated receptor using affinity chromatography. The endogenous tyrosine kinase specifically phosphorylated the .beta., .gamma. and .delta. subunits rapidly to a final stoichiometry of .apprxeq. 0.5 mol of phosphate per mol of subunit. Two-dimensional phosphopeptide mapping of the phosphorylated .beta., .gamma. and .delta. subunits, after limit proteolysis with trypsin or thermolysin, indicated that each subunit was phosphorylated on a single site. Locations are proposed for the amino acid residues phosphorylated on the receptor by the tyrosine-specific protein kinase, and by 2 other protein kinases (cAMP-dependent protein kinase and protein kinase C) which phosphorylate the receptor.