Noland T A, Raynor R L, Kuo J F
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.
J Biol Chem. 1989 Dec 5;264(34):20778-85.
As an extension of our previous reports that cardiac and skeletal muscle troponin I (Tn-I) and troponin T (Tn-T) are excellent substrates for protein kinase C (PKC) (Katoh, N., Wise, B. C., and Kuo, J. F. (1983) Biochem. J. 209, 189-195; Mazzei, G. J., and Kuo, J. F. (1984) Biochem. J. 218, 361-369), we have now determined that PKC phosphorylated serine 43 (and/or serine 45), serine 78, and threonine 144 in the free Tn-I subunit and threonine 190, threonine 199, and threonine 280 in the free Tn-T subunit of bovine cardiac troponin. PKC appeared to phosphorylate the same sites of the subunits present in the form of the troponin complex, as indicated by the similarity in the two-dimensional phosphopeptide maps. Although some of the phosphorylation sites were shared by other classes of protein kinases, PKC exhibited a distinct substrate specificity. It was also noted that phosphorylated serine and threonine residues in Tn-I and Tn-T had neighboring basic amino acid residues separated by 1 or 2 other residues both at the amino and carboxyl termini, in agreement with the conclusion of House et al. (House, C., Wettenhall, R. E. H., and Kemp, B. E. (1987) J. Biol. Chem. 262, 772-777) based upon their studies on other substrate proteins. Several peptides having sequences around the phosphorylating sites have been synthesized. The phosphorylation experiments indicated that these peptides were substrates for PKC, and their relative substrate activity (determined by the ratios of Vmax/Km) compared with other proteins, in descending order, was Tn-I = Tn-I(134-154) greater than Tn-T much greater than histone H1 greater than Tn-I(33-35) approximately Tn-T(268-284) greater than Tn-T(179-198) approximately Tn-T(191-209). It is suggested that PKC phosphorylation of Tn-I and Tn-T could be biologically significant in terms of possible modifications in interactions among the individual contractile protein components as well as the Ca2+ sensitivity and activity of actomyosin ATPase.
作为我们之前报道的延伸，即心肌和骨骼肌肌钙蛋白I（Tn-I）和肌钙蛋白T（Tn-T）是蛋白激酶C（PKC）的优良底物（加藤，N.，怀斯，B.C.，和国，J.F.（1983年）《生物化学杂志》209，189 - 195；马泽伊，G.J.，和国，J.F.（1984年）《生物化学杂志》218，361 - 369），我们现在已经确定PKC使牛心肌肌钙蛋白游离Tn-I亚基中的丝氨酸43（和/或丝氨酸45）、丝氨酸78和苏氨酸144以及游离Tn-T亚基中的苏氨酸190、苏氨酸199和苏氨酸280发生磷酸化。二维磷酸肽图谱的相似性表明，PKC似乎使以肌钙蛋白复合物形式存在的亚基的相同位点发生磷酸化。尽管一些磷酸化位点为其他类别的蛋白激酶所共有，但PKC表现出独特的底物特异性。还注意到，Tn-I和Tn-T中磷酸化的丝氨酸和苏氨酸残基在氨基和羧基末端都有相邻的碱性氨基酸残基，它们被1个或2个其他残基隔开，这与豪斯等人（豪斯，C.，韦滕哈尔，R.E.H.，和肯普，B.E.（1987年）《生物化学杂志》262，772 - 777）基于对其他底物蛋白的研究得出的结论一致。已经合成了几种在磷酸化位点周围具有序列的肽。磷酸化实验表明这些肽是PKC的底物，并且它们与其他蛋白质相比的相对底物活性（由Vmax/Km的比值确定），从高到低依次为Tn-I = Tn-I(134 - 154)大于Tn-T远大于组蛋白H1大于Tn-I(33 - 35)约等于Tn-T(268 - 284)大于Tn-T(179 - 198)约等于Tn-T(191 - 209)。有人提出，就单个收缩蛋白成分之间相互作用的可能改变以及肌动球蛋白ATP酶的Ca2 +敏感性和活性而言PKC对Tn-I和Tn-T的磷酸化可能具有生物学意义。
