title: Myofilament anchoring of protein kinase C-epsilon in cardiac myocytes.
abstract: 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 (epsilon-PKC) activated by arachidonic acid (AA) binds reversibly to cardiac myofibrils with an EC(50) of 86 nM. Binding occurred near the Z-lines giving rise to a striated staining pattern. The delta isoform of PKC (delta-PKC) did not bind to cardiac myofibrils regardless of the activator used, and the alpha isoform (alpha-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 beta'-COP were each tested for their involvement in cytoskeletal anchoring. F-actin bound epsilon-PKC selectively over delta-PKC and alpha-PKC, but this interaction was readily distinguishable from cardiac myofilament binding in two ways. First, the F-actin/epsilon-PKC interaction was independent of PKC activation, and second, the synthetic hexapeptide LKKQET derived from the C1 region of epsilon-PKC effectively blocked epsilon-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 epsilon-PKC, despite its presence in intact cardiac myocytes. The epsilon-PKC translocation inhibitor peptide EAVSLKPT reduced activated epsilon-PKC binding to cardiac myofibrils in a concentration dependent manner, suggesting that a RACK2 or a similar protein plays a role in epsilon-PKC anchoring in cardiac myofilaments.
authors.affiliation: Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL 33431, USA. xhuang@fau.edu
authors.forename: Xupei
authors.initials: X
authors.lastname: Huang
authors.forename: Jeffery W
authors.initials: JW
authors.lastname: Walker
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D018398:Homeodomain Proteins / Q000378:metabolism; D015151:Immunoblotting; D007313:Insecta; D007700:Kinetics; D060588:LIM Domain Proteins; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011817:Rabbits; D051381:Rats
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018398:Homeodomain Proteins; D060588:LIM Domain Proteins; C485639:Ldb3 protein, mouse; D010455:Peptides; D020033:Protein Isoforms; D016718:Arachidonic Acid; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon