Specific phosphorylation of a COOH-terminal site on the full-length form of the alpha 1 subunit of the skeletal muscle calcium channel by cAMP-dependent protein kinase

J Biol Chem. 1992 Aug 15;267(23):16100-5.

Abstract

The primary (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive calcium channels is present in full-length (212 kDa) and truncated (190 kDa) forms which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. In the present study, phosphorylation of the purified calcium channel by cA-PK followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed differential phosphorylation of the related 190- and 212-kDa forms. The 190-kDa form of the alpha 1 subunit was phosphorylated on three major and three minor tryptic phosphopeptides; the 212-kDa form was phosphorylated on all six of these phosphopeptides plus two that were unique. Time course experiments showed that a single site on the COOH-terminal portion of the full-length form of the alpha 1 subunit is most intensely and rapidly (within 10 s) phosphorylated. Phosphorylation occurs almost exclusively on this COOH-terminal site unless harsh conditions such as treatment with denaturing detergents are employed to expose phosphorylation sites within the 190-kDa segment of the molecule. Elution of phosphopeptides from the second dimension chromatograph followed by immunoprecipitation with an anti-peptide antibody (anti-CP1) directed against the COOH-terminal amino acid sequence enabled us to identify this major phosphorylation site as serine 1854. The nearby consensus sites for cA-PK phosphorylation at serines 1757 and 1772 were phosphorylated only after denaturation or proteolytic cleavage. Phosphorylation of serine 1854 may play a pivotal role in the regulation of calcium channel function by cA-PK.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Calcium Channels / metabolism*
  • Chromatography, Affinity
  • Electrophoresis, Gel, Two-Dimensional
  • Electrophoresis, Polyacrylamide Gel
  • Macromolecular Substances
  • Membrane Proteins / isolation & purification
  • Membrane Proteins / metabolism
  • Microsomes / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • Muscles / metabolism*
  • Peptide Mapping
  • Phosphopeptides / isolation & purification
  • Phosphorylation
  • Protein Kinases / metabolism*

Substances

  • Calcium Channels
  • Macromolecular Substances
  • Membrane Proteins
  • Phosphopeptides
  • Protein Kinases