Determination of the phosphorylation sites of smooth muscle caldesmon by protein kinase C.
Smooth muscle caldesmon was phosphorylated by protein kinase C up to 1.90 mol P/mol caldesmon. Phosphorylated caldesmon was completely digested by trypsin and the produced phosphopeptides were purified by C-8 and C-18 reverse phase chromatography. Four phosphopeptides were determined and two phosphoserines were identified. Both were localized in the C-terminal domain at serine-587 and serine-726. By following the time course of phosphorylation, serine-587 was found to be the preferred site. Effects of the phosphorylation of caldesmon by protein C on the inhibition of acto-H-meromyosin ATPase activity was also examined. While unphosphorylated caldesmon inhibited the ATPase activity by 60%, phosphorylated caldesmon hardly inhibited the ATPase activity. Therefore, it was concluded that the phosphorylation at serine-726 and serine-587 reverses the inhibitory activity of caldesmon.
Ikebe
M
M
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106.
Hornick
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits
D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D011493:Protein Kinase C