title: cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor.
abstract: Postsynaptic membranes, rich in the nicotinic acetylcholine receptor, were isolated from the electric organ of Torpedo californica and shown to contain a cAMP-dependent protein kinase and a calcium/calmodulin-dependent protein kinase. The cAMP-dependent protein kinase phosphorylated the gamma and delta subunits of the acetylcholine receptor. The phosphorylated subunits were identified after purification of the acetylcholine receptor by affinity chromatography on a choline carboxymethyl affinity gel. In contrast, the calcium/calmodulin-dependent protein kinase phosphorylated proteins that were separated from the acetylcholine receptor by affinity chromatography. Protein kinase inhibitor, a specific inhibitor of the catalytic subunit of cAMP-dependent protein kinase, abolished the basal endogenous phosphorylation of the gamma and delta subunits of the receptor. cAMP activation of the endogenous phosphorylation of the gamma and delta subunits was dose dependent with a half-maximal response at 25 nM. Studies were also carried out with acetylcholine receptor purified from T. californica and catalytic subunit of cAMP-dependent protein kinase purified from bovine heart. The purified acetylcholine receptor was rapidly and specifically phosphorylated on the gamma and delta subunits by the purified catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.0 and 0.89 mol of (32)P per mol of receptor, respectively. The initial rates of phosphorylation of the gamma and delta subunits of the receptor were comparable to those of histone f2B and synapsin I (protein I), two of the most effective substrates for the catalytic subunit. Under the conditions used, the gamma and delta subunits had K(m) values of 4.0 and 3.3 muM and V(max) values of 2.7 and 2.1 mumol/min per mg, respectively. The results are consistent with the idea that the acetylcholine receptor is phosphorylated in vivo by a cAMP-dependent protein kinase.
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014101:Torpedo
chemical_list: D002147:Calmodulin; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D000242:Cyclic AMP; D011494:Protein Kinases; D002118:Calcium