Phosphorylation of the alpha subunit of rat brain sodium channels by cAMP-dependent protein kinase at a new site containing Ser686 and Ser687.
The alpha subunit of the rat brain sodium channel is phosphorylated by cAMP-dependent protein kinase in vitro and in situ at multiple sites which yield seven tryptic phosphopeptides. Phosphopeptides 1-4 and 7 are derived from phosphorylation sites between residues 554 and 623 in a single large CNBr fragment from the cytoplasmic segment connecting homologous domains I and II of the alpha subunit (Rossie, S., Gordon, D., and Catterall, W. A. (1987) J. Biol. Chem. 262, 17530-17535). In the present work, antibodies were prepared against a synthetic peptide corresponding to residues 676-692 (AbSP15), which contain one additional potential phosphorylation site at Ser686-Ser687 in a different predicted CNBr fragment of this same intracellular segment. AbSP15 recognizes native and denatured sodium channels specifically and immunoprecipitates phosphorylated CNBr fragments of low molecular mass that contain a new site phosphorylated by cAMP-dependent protein kinase. Comparison of tryptic phosphopeptides derived from intact alpha subunits with those derived from the phosphorylated CNBr fragments isolated by immunoprecipitation with AbSP15 indicates that the two previously unidentified phosphopeptides 5 and 6 derived from the intact alpha subunit arise from phosphorylation of the site containing Ser686-Ser687. These results identify a new cAMP-dependent phosphorylation site and show that the major cAMP-dependent phosphorylation sites of the rat brain sodium channel, which are phosphorylated both in vitro and in intact neurons, are all located in a cluster between residues 554 and 687 in the intracellular segment between domains I and II.
Rossie
S
S
Department of Pharmacology, University of Washington, Seattle 98195.
Catterall
W A
WA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010748:Phosphopeptides / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D011863:Radioimmunoassay; D051381:Rats; D012694:Serine / Q000276:immunology / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism*; D014357:Trypsin
D010446:Peptide Fragments; D010748:Phosphopeptides; D015222:Sodium Channels; D012694:Serine; D011494:Protein Kinases; D014357:Trypsin