Cyclic AMP-dependent protein kinase (PKA) phosphorylates Ser362 and 467 and protein kinase C phosphorylates Ser550 within the M3/M4 cytoplasmic domain of human nicotinic receptor alpha4 subunits.
Studies have suggested that the expression, translocation, and function of alpha4beta2 nicotinic receptors may be modulated by alpha4 subunit phosphorylation, but little direct evidence exists to support this idea. The objective of these experiments was to identify specific serine/threonine residues on alpha4 subunits that are phosphorylated in vivo by cAMP-dependent protein kinase and protein kinase C (PKC). To accomplish this, DNAs coding for human alpha4 subunits containing alanines in place of serines/threonines predicted to represent phosphorylation sites were constructed, and transiently transfected with the DNA coding for wild-type beta2 subunits into SH-EP1 cells. Cells were pre-incubated with (32)Pi and incubated in the absence or presence of forskolin or phorbol 12,13-dibutyrate. Immunoprecipitated alpha4 subunits were subjected to immunoblot, autoradiographic and phosphoamino acid analyses, and two-dimensional phosphopeptide mapping. Results confirmed the presence of two alpha4 protein bands, a major band of 71/75 kDa and a minor band of 80/85 kDa. Phosphoamino acid analysis of the major band indicated that only serine residues were phosphorylated. Phosphopeptide maps demonstrated that Ser362 and 467 on the M3/M4 cytoplasmic domain of the alpha4 subunit represent major cAMP-dependent protein kinase phosphorylation sites, while Ser550 also contained within this major intracellular loop is a major site for protein kinase C phosphorylation.
Pollock
Veronica V
VV
Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, Florida, USA.
Pastoor
Tina E
TE
Wecker
Lynn
L
D001345:Autoradiography; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D010449:Peptide Mapping; D019606:Phosphoamino Acids / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014162:Transfection
D017868:Cyclic AMP-Dependent Protein Kinases; D017931:DNA Primers; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D019606:Phosphoamino Acids; D010748:Phosphopeptides; D011493:Protein Kinase C; D011978:Receptors, Nicotinic; D012694:Serine