Abstract

Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKCepsilon and zeta and troponin T (cTnT) associated with PKC alpha, delta, and epsilon. Based on its association with cTnI, we hypothesized that PKCzeta is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKCzeta: the pseudo-substrate region (A119E), 3'-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKCzeta using PKC phospho-motif antibodies to determine the phosphorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKCzeta T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKCzeta. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKCzeta A119E. Both PKCzeta A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKCzeta exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKCzeta is a novel regulator of myofilament protein phosphorylation.