Myofilament anchoring of protein kinase C-epsilon in cardiac myocytes Available to Purchase

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 (ϵ-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 (δ-PKC) did not bind to cardiac myofibrils regardless of the activator used, and the alpha isoform (α-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 β′-COP were each tested for their involvement in cytoskeletal anchoring. F-actin bound ϵ-PKC selectively over δ-PKC and α-PKC, but this interaction was readily distinguishable from cardiac myofilament binding in two ways. First, the F-actin/ϵ-PKC interaction was independent of PKC activation, and second, the synthetic hexapeptide LKKQET derived from the C1 region of ϵ-PKC effectively blocked ϵ-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 ϵ-PKC, despite its presence in intact cardiac myocytes. The ϵ-PKC translocation inhibitor peptide EAVSLKPT reduced activated ϵ-PKC binding to cardiac myofibrils in a concentration dependent manner, suggesting that a RACK2 or a similar protein plays a role in ϵ-PKC anchoring in cardiac myofilaments.