Phosphorylation and Dephosphorylation of Skeletal Muscle Troponin

Abstract

The purified inhibitory subunit of rabbit skeletal muscle troponin, TN-I (M.W. 24,000) was phosphorylated by phosphorylase kinase in the presence of Mg− ATP2− and Ca2+, and by protein kinase in the presence of Mg−ATP2− and cyclic AMP2. Phosphorylase phosphatase removed all the phosphate incorporated by either kinase. The primary site of phosphorylation of TN-I by phosphorylase kinase was a threonine residue, as compared to a serine residue in phosphorylase a. Comparison of kinetic parameters for both kinases and the phosphatase with TN-I and other protein substrates showed it to be a favorable substrate. The tropomyosin binding subunit of troponin, TN-T (M.W. 38,000) was also phosphorylated by phosphorylase kinase, but not protein kinase, and dephosphorylated by phosphorylase phosphatase. Incubation of troponin with Mg−ATP2− and either phosphorylase kinase or protein kinase resulted in incorporation of phosphate into both TN-I and TN-T with both kinases. All of the phosphate incorporated was removed by phosphorylase phosphatase.