Identification of three cAMP-dependent protein kinase (PKA) phosphorylation sites within the major intracellular domain of neuronal nicotinic receptor α4 subunits

Abstract

This study determined whether all protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites on the α4 subunit of rat α4β2 neuronal nicotinic receptors could be localized to the M3/M4 cytoplasmic domain of the protein, and investigated specific amino acid substrates for the kinases through two-dimensional phosphopeptide mapping and site-directed mutagenesis. Experiments were conducted using α4β2 receptors expressed in Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of α4 (α4333-594). When oocytes expressing α4β2 receptors were incubated with [32P]orthophosphate in order to label endogenous ATP stores, phosphorylation of α4 subunits was evident. Incubation of either immunoprecipitated receptors or the fusion protein with [32P]ATP and either PKA or PKC followed by trypsinization of the samples demonstrated that the kinases phosphorylated α4 subunits on multiple phosphopeptides, and that the phosphorylated full-length α4 protein and fusion protein produced identical phosphopeptide maps. Site-directed mutagenesis of Ser365, Ser472 and Ser491 to alanines in the fusion protein eliminated phosphopeptides phosphorylated by PKA, but not by PKC. Other mutations investigated, Ser470, Ser493, Ser517 and Ser590, did not alter the phosphopeptide maps. Results indicate that Ser365, Ser472 and Ser491 on neuronal nicotinic receptor α4 subunits are phosphorylated by PKA and are likely to represent post-translational regulatory sites on the receptor.