Cyclic AMP-dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of alpha 4 neuronal nicotinic receptor subunits

To determine whether alpha4 subunits of alpha4 beta2 neuronal nicotinic rec eptors are phosphorylated within the M3/M4 intracellular region by cyclic A MP-dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecip itated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(336-597)) were incubated wi th ATP and either PKA or PKC. Both (alpha4 and (alpha4(336-597) were phosph orylated by PKA and PKC, providing the first direct biochemical evidence th at the M3/M4 cytoplasmic domain of neuronal nicotinic receptor alpha4 subun its is phosphorylated by both kinases. When the immunoprecipitated receptor s and the alpha4(336-597) fusion protein were phosphorylated and the labele d proteins subjected to phosphoamino acid analysis, results indicated that alpha4 and (alpha4(336-597) were phosphorylated on the same amino acid resi dues by-each kinase. Furthermore, PKA phosphorylated serines exclusively, w hereas PKC phosphorylated both serines and threonines. To determine whether Ser(368) was a substrate for both kinases, a peptide corresponding to amin o acids 356-371 was synthesized (alpha4(356-371)) and incubated with ATP an d the kinases. The phosphorylation of (alpha4(356-371) by both PKA and PKC was saturable with K(m)s of 15.3 +/- 3.3 muM and 160.8 +/- 26.8 muM, respec tively, suggesting that Ser368 was a better substrate for PKA than PKC.