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.

was combined with the inorganic phosphate assay of Itaya and Ui (12) and the ashing procedure of Ames (13) for measurement of protein-bound phosphate. All materials were previously washed in 6 N HCl to remove contaminating phosphate.
Protein samples containing 0.2 to 1.5 nmol of phosphate were added to borosilicate glass tubes (6 x 50 mm) and were precipitated with 10% trichloroacetic acid at 0". The supernatant solution was carefully removed after centrifugation and the protein was washed twice with 5% trichloroacetic acid. The prote-in was suspended in 16% trichloroacetic acid, heated for 20 min at 90", and centrifuged after cooling to 0". The protein pellet was solubilized quickly in 0.1 N NaOH, 0", and immediately precipitated with 20% trichloroacetic acid. After two more washes in 5% trichloroacetic acid, the protein was dissolved in 25 ~1 of 10% MgNO, .6H,O in 95% ethanol and gently heated to dryness. The samples were then ashed over an intense flame. After dissolving the residue in 150 ~1 of 1.2 N HCl, 50 ~1 of filtered phosphate reagent was added, and after 5 minutes, the developed color was measured at 660 nm. The phosphate reagent contained 1 volume of 10% (NH&Moo,, .4H,O in 4 N HCl to 3 volumes of 0.2% malachite green. Phosphate standards (0.2 to 1.5 nmol of KH,PO,) were also ashed. Recovery of protein-bound phosphate in "'P-phosphorylase a and J'P-troponin previously phosphorylated by phosphorylase kinase and cyclic AMP-dependent protein kinase, respectively, was generally 97%. The variation on multiple assays of the same protein sample was 5% or less. Polyacrylamide Gel Electrophoresis -The purity and apparent molecular weight of protein samples were estimated bv nolvacrvlamide gel electrochore& in the presence of SDS as descr"ided "by *eber et al. (14)  Chromatography on CM-agarose in the presence of 6 M urea and EDTA was used to separate the individual protein components of the troponin . tropomyosin complex (Fig. 2). The protein in the first peak was not adsorbed to the CM-agarose and was identified as a mixture of tropomyosin and TN-C by gel electrophoresis (Fig. 1, Gels ZJ and c). Occasionally this fraction was contaminated with a small amount of TN-T. The tropomyosin was separated from TN-C by adjusting the pH to 4.6 in the presence of 1 M KC1 and collecting the precipitated tropomyosin by centrifugation. The subsequent two peaks obtained by elution with KC1 were identified as TN-T and TN-I, respectively, by their electrophoretic mobilities on polyacrylamide gels (Fig. 1, Gels d and e). Quantitative densitometry of the purified protein fractions showed a purity of 99% for tropomyosin, 93% for TN-C, 97% for TN-T, and 97% for TN-I. Sulfopropyl Sephadex was also used to separate the protein components, but the distinct separation of TN-T and TN-I was not as consistently successful as when CM-agarose was used. These results show that the troponin subunits may be purified without the prior separation of tropomyosin from the troponin complex.  7). Actin at a molar ratio to troponin . tropomyosin of 7:l did not appear to affect the rate or extent of phosphorylation of troponin by cyclic AMP-dependent protein kinase. Identical results were obtained with molar ratios of 3.5:l and 14:l (data not shown).
The presence or absence of Ca'+ did not significantly alter the phosphorylation reaction (Fig. 7) in the presence of actin.

DISCUSSION
Beef cardiac troponin is a phosphoprotein of the myotibrils whose function may be regulated by phosphorylation and dephosphorylation reactions. Although skeletal muscle troponin also contains protein-bound phosphate (18, differences between the cardiac and skeletal muscle proteins suggest different roles for the phosphorylation. The subunit of skeletal muscle troponin which contains most of the phosphate was identified as 20). This is in contrast to results reported here and observations by others (3) where the TN-I subunit from cardiac muscle contained most of the phosphate. A significant portion of the phosphate in cardiac troponin was found in TN-T as well. Skeletal muscle troponin is not phosphorylated in response to tetanic contractions (5, 21) or after be an important criteria for indicating a possible role for a phosphorylation reaction in a particular cellular process.
We have found that TN-I is specifically phosphorylated by cyclic AMP-dependent protein kinase in the heterogeneous protein complex of troponin which is in agreement with others (3). In addition we found tropomyosin added to cardiac troponin was not phosphorylated and did not alter the maximal amount of :VzP incorporated into TN-I. The maximal rate of phosphorylation of cardiac troponin by cyclic AMP-dependent protein kinase was about 3.5 times greater than the rate of phosphorylation of unfractionated 3 In the experiments described in this report, cyclic AMP-dependent protein kinase purified from porcine skeletal muscle was used. Cyclic AMP-dependent protein kinase has been shown to exist in two general isozymic forms (28-30). The relative amounts of the two forms may vary within a specific tissue, but the properties of any one form isolated from many tissues appear to be similar.
Both isozymic forms of cyclic AMP-dependent protein kinase contain a dimeric regulatory subunit and two catalytic subunits which dissociate when cyclic AMP binds to the regulatory subunits. Differences in physical properties have been identified between regulatory subunits, but no differences between the catalytic subunits of either enzyme have been noted (28, 31, 321. The catalytic subunits demonstrate identical specificities and relative rates of phosphorylation with a variety of protein substrates. Likewise no differences were noted in regard to the phosphorylation of beef cardiac troponin by either isozymic form. Therefore "cyclic AMP-dependent protein kinase" has been used in this report without qualification and refers to either isozymic form.