Identification of PK-A phosphorylation sites in the carboxyl terminus of L-type calcium channel alpha 1 subunits.
Full length L-type calcium channel alpha 1 subunits are rapidly phosphorylated by protein kinase A (PK-A) in vitro and in vivo at sites located in their long carboxyl terminal tails. In skeletal muscle, heart, and brain the majority of biochemically isolated alpha 1 subunits lacks these phosphorylation sites due to posttranslational proteolytic processing. Truncation may therefore modify the regulation of channel activity by PK-A. We combined site-directed mutagenesis and heterologous expression to investigate the extent to which putative cAMP-dependent phosphorylation sites in the C-terminus of alpha 1 subunits from skeletal muscle, heart, and brain are phosphorylated in vitro. The full length size form of wild-type and mutant calcium channel alpha 1 subunits was obtained at high yield after heterologous expression in Saccharomyces cerevisiae. Like in fetal rabbit myotubes [Rotman, E.I., et al. (1995) J. Biol. Chem. 270, 16371-16377], the rabbit skeletal muscle alpha 1 C-terminus was phosphorylated at serine residues 1757 and 1854. In the carboxyl terminus of alpha 1S from carp skeletal muscle and alpha 1C from rabbit heart a single serine residue was phosphorylated by PK-A in vitro. The C-terminus of alpha 1D was phosphorylated at more than one site. Employing deletion mutants, most of the phosphorylation ( > 70%) was found to occur between amino acid residues 1805 and 2072. Serine 1743 was identified as additional phosphorylation site in alpha 1D. We conclude that in class S and C calcium channels the most C-terminal phosphorylation sites are substrate for PK-A in vitro, whereas in class D calcium channels phosphorylation also occurs at a site which is likely to be retained even after posttranslational truncation.
Mitterdorfer
J
J
Institut für Biochemische Pharmakologie, Innsbruck, Austria.
Froschmayr
M
M
Grabner
M
M
Moebius
F F
FF
Glossmann
H
H
Striessnig
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D002347:Carps / Q000378:metabolism; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology; D010766:Phosphorylation; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012694:Serine / Q000378:metabolism
D015220:Calcium Channels; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D012694:Serine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases