PHOSPHORYLATION SPECIFICITIES OF PROTEIN-KINASE-C ISOZYMES FOR BOVINECARDIAC TROPONIN-I AND TROPONIN-T AND SITES WITHIN THESE PROTEINS ANDREGULATION OF MYOFILAMENT PROPERTIES

Protein kinase C (PKC) isozymes alpha, delta, epsilon, and zeta, shown to be expressed in adult rat cardiomyocytes, displayed distinct subst rate specificities in phosphorylating troponin I and troponin T subuni ts in the bovine cardiac troponin complex, Thus, because they have dif ferent substrate affinities, PKC-alpha, -delta, and -epsilon phosphory lated troponin I more than troponin T, but PKC-zeta conversely phospho rylated the latter more than the former, Furthermore, PKC isozymes exh ibited discrete specificities in phosphorylating distinct sites in the se proteins as free subunits or in the troponin complex, Unlike other isozymes, PKC-delta was uniquely able to phosphorylate Ser-23/Ser-24 i n 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, PK C-delta, like PKC-alpha, readily phosphorylated Ser-43/Ser-45 (sites c ommon 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. Phosphoryl ation of troponin T by PKC-alpha resulted in decreases in both Ca2+ se nsitivity and maximal activity, whereas phosphorylation by PKC-zeta re sulted 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 ra t cardiomyocytes. The present study has demonstrated for the first tim e distinct specificities of PKC isozymes for phosphorylation of two ph ysiological substrates in the myocardium, with functional consequences .