Nakamura F, Mino T, Yamamoto J, Naka M, Tanaka T
Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Japan.
J Biol Chem. 1993 Mar 25;268(9):6194-201.
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.
F-肌动蛋白和原肌球蛋白抑制了蛋白激酶C对钙调蛋白的磷酸化作用，而这种磷酸化作用降低了钙调蛋白与F-肌动蛋白和原肌球蛋白的结合。来自[γ-32P]ATP的标记磷酸盐保留在胰凝乳蛋白酶作用产生的NH2末端22 kDa片段（包含肌动蛋白、原肌球蛋白和钙调蛋白结合区域）以及COOH末端12 kDa片段上。通过高效液相色谱法对胰蛋白酶作用产生的32P标记肽段进行分级分离，可分离出三种磷酸肽（分别命名为T1、T2和T3），每种磷酸肽都位于钙调蛋白的三个重复氨基酸基序中。磷酸化的相对初始速率和程度均按T2>T3>T1的顺序降低。T1（GASQAGMTAPGTK）中的丝氨酸和苏氨酸残基均被磷酸化，而T2（FASQQGMTAYGTR）和T3（GASQQGMTVYGLPR）中只有一个苏氨酸残基被磷酸化。由于22 kDa片段仅包含T2，T2中的磷酸化位点似乎调节了钙调蛋白与F-肌动蛋白和原肌球蛋白的结合。T2的氨基酸序列表明蛋白激酶C使Thr184磷酸化。因此，Thr184是磷酸化的首选位点，在功能上是蛋白激酶C在平滑肌钙调蛋白中磷酸化的位点中最重要的。
