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 α4 neuronal nicotinic receptor subunits

Authors


Address correspondence and reprint requests to Lynn Wecker, Department of Pharmacology, USFCOM–MDC Box 9, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612-4799, USA. E-mail: lwecker@hsc.usf.edu

Abstract

To determine whether α4 subunits of α4β2 neuronal nicotinic receptors are phosphorylated within the M3/M4 intracellular region by cyclic AMP-dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecipitated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of α4 (α4336−597) were incubated with ATP and either PKA or PKC. Both α4 and α4336−597 were phosphorylated by PKA and PKC, providing the first direct biochemical evidence that the M3/M4 cytoplasmic domain of neuronal nicotinic receptor α4 subunits is phosphorylated by both kinases. When the immunoprecipitated receptors and the α4336−597 fusion protein were phosphorylated and the labeled proteins subjected to phosphoamino acid analysis, results indicated that α4 and α4336−597 were phosphorylated on the same amino acid residues by each kinase. Furthermore, PKA phosphorylated serines exclusively, whereas PKC phosphorylated both serines and threonines. To determine whether Ser368 was a substrate for both kinases, a peptide corresponding to amino acids 356–371 was synthesized (α4356−371) and incubated with ATP and the kinases. The phosphorylation of α4356−371 by both PKA and PKC was saturable with Kms of 15.3 ± 3.3 µm and 160.8 ± 26.8 µm, respectively, suggesting that Ser368 was a better substrate for PKA than PKC.

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