Phosphorylation of cardiac troponin by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.
The purpose of this investigation was to characterize the phosphorylation of bovine cardiac troponin by cyclic AMP-dependent protein kinase. The purified troponin-tropomyosin complex from beef heart contained 0.78 +/- 0.15 mol of phosphate per mol of protein. Analysis of the isolated protein components indicated that the endogenous phosphate was predominately in the inhibitory subunit (TN-I) and the tropomyosin-binding subunit (TN-T) of troponin. When cardiac troponin or the troponin-tropomyosin complex was incubated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, the rate of phosphorylation was stimulated by cyclic AMP and inhibited by the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase. The 32P was incorporated specifically into the TN-I subunit with a maximal incorporation of 1 mol of phosphate per mol of protein. The maximal amount of phosphate incorporated did not vary significantly between troponin preparations that contained low or high amounts of endogenous phosphate. The Vmax of the initial rates of phosphorylation with troponin or troponin-tropomyosin as substrates was 3.5-fold greater than the value obtained with unfractionated histones. The rate or extent of phosphorylation was not altered by actin in the presence or absence of Ca2+. The maximal rate of phosphorylation occurred between pH 8.5 and 9.0. At pH 6.0 and 7.0 the maximal rates of phosphorylation were 13 and 45% of that observed at pH 8.5, respectively. These results indicate that cyclic AMP formation in cardiac muscle may be associated with the rapid and specific phosphorylation of the TN-I subunit of troponin. The presence of endogenous phosphate in TN-T and TN-I suggests that kinases other than cyclic AMP-dependent protein kinase may also phosphorylate troponin in vivo.
D000199:Actins / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D009124:Muscle Proteins / Q000378:metabolism / Q000502:physiology; D009132:Muscles / Q000201:enzymology; D009206:Myocardium; D006656:Protamine Kinase / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine; D014335:Tropomyosin; D014336:Troponin / Q000378:metabolism
D000199:Actins; D046911:Macromolecular Substances; D009124:Muscle Proteins; D014335:Tropomyosin; D014336:Troponin; D000242:Cyclic AMP; D011494:Protein Kinases; D006656:Protamine Kinase; D002118:Calcium