Phosphorylation specificities of protein kinase C isozymes for bovine cardiac troponin I and troponin T and sites within these proteins and regulation of myofilament properties.

Protein kinase C (PKC) isozymes alpha, delta, epsilon, and zeta, shown to be expressed in adult rat cardiomyocytes, displayed distinct substrate specificities in phosphorylating troponin I and troponin T subunits in the bovine cardiac troponin complex. Thus, because they have different substrate affinities, PKC-alpha, -delta, and -epsilon phosphorylated troponin I more than troponin T, but PKC-zeta conversely phosphorylated the latter more than the former. Furthermore, PKC isozymes exhibited discrete specificities in phosphorylating distinct sites in these proteins as free subunits or in the troponin complex. Unlike other isozymes, PKC-delta was uniquely able to phosphorylate Ser-23/Ser-24 in troponin I, the bona fide phosphorylation sites for protein kinase A (PKA); and consequently, like PKA, it reduced Ca2+ sensitivity of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1. In addition, PKC-delta, like PKC-alpha, readily phosphorylated Ser-43/Ser-45 (sites common for all PKC isozymes) and reduced maximal activity of MgATPase. In this respect, PKC-delta functioned as a hybrid of PKC-alpha and PKA. In contrast to PKC-alpha, -delta, and -epsilon, PKC-zeta exclusively phosphorylated two previously unknown sites in troponin T. Phosphorylation of troponin T by PKC-alpha resulted in decreases in both Ca2+ sensitivity and maximal activity, whereas phosphorylation by PKC-zeta resulted in a slight increase of the Ca2+ sensitivity without affecting the maximal activity of MgATPase. Most of the in vitro phosphorylation sites in troponin I and troponin T were confirmed in situ in adult rat cardiomyocytes. The present study has demonstrated for the first time distinct specificities of PKC isozymes for phosphorylation of two physiological substrates in the myocardium, with functional consequences.