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
Article first published online: 7 JUL 2008
Journal of Neurochemistry
Volume 76, Issue 3, pages 711–720, February 2001
How to Cite
Wecker, L., Guo, X., Rycerz, A. M. and Edwards, S. C. (2001), 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. Journal of Neurochemistry, 76: 711–720. doi: 10.1046/j.1471-4159.2001.00041.x
- Issue published online: 7 JUL 2008
- Article first published online: 7 JUL 2008
- Received July 17, 2000; revised manuscript received August 29, 2000; accepted August 30, 2000.
- α4β2 receptors;
- neuronal nicotinic receptors;
- protein kinase A;
- protein kinase C;
- receptor regulation
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.