Sites of selective cAMP-dependent phosphorylation of the L-type calcium channel alpha 1 subunit from intact rabbit skeletal muscle myotubes.
The principal (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive (L-type) calcium channels is present in full-length (212 kDa) and COOH-terminal truncated (190 kDa) forms, which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. Immunoprecipitation of the calcium channel from rabbit muscle myotubes in primary cell culture followed by phosphorylation with cA-PK, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed comparable phosphorylation of three COOH-terminal phosphopeptides found in the purified full-length alpha 1 subunit. Stimulation of muscle myotubes with a permeant cAMP analogue, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate, prior to immunoprecipitation of alpha 1 results in a 60-80% reduction of cA-PK catalyzed "back" phosphorylation of each of these sites in vitro in calcium channels purified from the cells, indicating that these sites are phosphorylated in vivo in response to increased intracellular cAMP. Serine 687, the most rapidly phosphorylated site in the truncated 190-kDa alpha 1 subunit, was observed as a minor phosphopeptide whose level of phosphorylation was not significantly affected by stimulation of endogenous cA-PK in the myotubes. The COOH-terminal sites, designated tryptic phosphopeptides 4, 5, and 6, were identified as serine 1757 (phosphopeptides 4 and 6) and 1854 (phosphopeptide 5) by a combination of protease cleavage, phosphorylation of synthetic peptides and fusion proteins, specific immunoprecipitation, and phosphopeptide mapping. Phosphorylation of serines 1757 and 1854 in the COOH-terminal region of the 212-kDa alpha 1 subunit in intact skeletal muscle cells may play a pivotal role in the regulation of calcium channel function by cA-PK.
Rotman
E I
EI
Department of Pharmacology, University of Washington, Seattle 98195, USA.
Murphy
B J
BJ
Catterall
W A
WA
D000818:Animals; D015220:Calcium Channels / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D010449:Peptide Mapping; D010455:Peptides / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D009124:Muscle Proteins; D010455:Peptides; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases