Identification of the regulatory site in smooth muscle calponin that is phosphorylated by protein kinase C.
F-actin and tropomyosin inhibited the phosphorylation of calponin by protein kinase C, and the phosphorylation reduced the binding of calponin to F-actin and tropomyosin. Labeled phosphate from [gamma-32P]ATP was retained both on the chymotryptic NH2-terminal 22-kDa fragment, which contains the actin-, tropomyosin-, and calmodulin-binding regions, and on the COOH-terminal 12-kDa fragment. Fractionation of tryptic 32P-labeled peptides by high performance liquid chromatography allowed isolation of three phosphopeptides (designated T1, T2, and T3), each of which was located in three repeating amino acid motifs of calponin. Both the relative initial rates and extent of phosphorylation decreased in the order T2 > T3 > T1. Both serine and threonine residues were phosphorylated in T1 (GASQAGMTAPGTK), and only a threonine residue was phosphorylated in T2 (FASQQGMTAYGTR) and in T3 (GASQQGMTVYGLPR). As the 22-kDa fragment contained only T2, the phosphorylation site in T2 appeared to regulate the binding of calponin to F-actin and tropomyosin. The amino acid sequence of T2 indicates that protein kinase C phosphorylates Thr184. Thus Thr184 is the preferred site of phosphorylation and is functionally the most important of the sites phosphorylated by protein kinase C in smooth muscle calponin.
Nakamura
F
F
Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Japan.
Mino
T
T
Yamamoto
J
J
Naka
M
M
Tanaka
T
T
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D014335:Tropomyosin / Q000378:metabolism; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011493:Protein Kinase C; D014335:Tropomyosin; D000096985:Calponins