Myofilament anchoring of protein kinase C-epsilon in cardiac myocytes

Regulatory proteins on muscle filaments are substrates for protein kinase C (PKC) but mechanisms underlying activation and translocation of PKC to this non-membrane compartment are poorly understood. Here we demonstrate that the epsilon isoform of PKC (-PKC) activated by arachidonic acid (AA) binds reversibly to cardiac myofibrils with an EC₅₀ of 86 nM. Binding occurred near the Z-lines giving rise to a striated staining pattern. The delta isoform of PKC (-PKC) did not bind to cardiac myofibrils regardless of the activator used, and the alpha isoform (-PKC) bound only under strong activating conditions. Three established PKC anchoring proteins, filamentous actin (F-actin), the LIM domain protein Cypher-1, and the coatamer protein '-COP were each tested for their involvement in cytoskeletal anchoring. F-actin bound -PKC selectively over -PKC and -PKC, but this interaction was readily distinguishable from cardiac myofilament binding in two ways. First, the F-actin/-PKC interaction was independent of PKC activation, and second, the synthetic hexapeptide LKKQET derived from the C1 region of -PKC effectively blocked -PKC binding to F-actin, but was without effect on its binding to cardiac myofilaments. Involvement of Cypher-1 was ruled out on the basis of its absence from detergent-skinned myofibrils that bound -PKC, despite its presence in intact cardiac myocytes. The -PKC translocation inhibitor peptide EAVSLKPT reduced activated -PKC binding to cardiac myofibrils in a concentration dependent manner, suggesting that a RACK2 or a similar protein plays a role in -PKC anchoring in cardiac myofilaments.