Protein kinase C isoforms differentially phosphorylate Ca(v)1.2 alpha(1c).
The regulation of Ca(2+) influx through the phosphorylation of the L-type Ca(2+) channel, Ca(v)1.2, is important for the modulation of excitation-contraction (E-C) coupling in the heart. Ca(v)1.2 is thought to be the target of multiple kinases that mediate the signals of both the renin-angiotensin and sympathetic nervous systems. Detailed biochemical information regarding the protein phosphorylation reactions involved in the regulation of Ca(v)1.2 is limited. The protein kinase C (PKC) family of kinases can modulate cardiac contractility in a complex manner, such that contractility is either enhanced or depressed and relaxation is either accelerated or slowed. We have previously reported that Ser(1928) in the C-terminus of alpha(1c) was a target for PKCalpha, -zeta, and -epsilon phosphorylation. Here, we report the identification of seven PKC phosphorylation sites within the alpha(1c) subunit. Using phospho-epitope specific antibodies to Ser(1674) and Ser(1928), we demonstrate that both sites within the C-terminus are phosphorylated in HEK cells in response to PMA. Phosphorylation was inhibited with a PKC inhibitor, bisindolylmaleimide. In Langendorff-perfused rat hearts, both Ser(1674) and Ser(1928) were phosphorylated in response to PMA. Phosphorylation of Ser(1674), but not Ser(1928), is PKC isoform specific, as only PKCalpha, -betaI, -betaII, -gamma, -delta, and -theta, but not PKCepsilon, -zeta, and -eta, were able to phosphorylate this site. Our results identify a molecular mechanism by which PKC isoforms can have different effects on channel activity by phosphorylating different residues.
Yang
Lin
L
Division of Cardiology, Department of Medicine, College of Physicians and Surgeons,Columbia University, New York, New York 10032, USA.
Doshi
Darshan
D
Morrow
John
J
Katchman
Alexander
A
Chen
Xiang
X
Marx
Steven O
SO
D000595:Amino Acid Sequence; D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020746:Calcium Channels, L-Type; D007527:Isoenzymes; C419385:L-type calcium channel alpha(1C); D011993:Recombinant Fusion Proteins; D010768:Phosphoserine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate