title: Cross-regulation of novel protein kinase C (PKC) isoform function in cardiomyocytes. Role of PKC epsilon in activation loop phosphorylations and PKC delta in hydrophobic motif phosphorylations.
abstract: Recent studies identify conventional protein kinase C (PKC) isoform phosphorylations at conserved residues in the activation loop and C terminus as maturational events that influence enzyme activity and targeting but are not dynamically regulated by second messengers. In contrast, this study identifies phorbol 12-myristoyl 13-acetate (PMA)- and norepinephrine-induced phosphorylations of PKC epsilon (at the C-terminal hydrophobic motif) and PKC delta (at the activation loop) as events that accompany endogenous novel PKC (nPKC) isoform activation in neonatal rat cardiomyocytes. Agonist-induced nPKC phosphorylations are prevented (and the kinetics of PMA-dependent PKC down-regulation are slowed) by pharmacologic inhibitors of nPKC kinase activity. PKC delta is recovered from PMA-treated cultures with increased in vitro lipid-independent kinase activity (and altered substrate specificity); the PMA-dependent increase in PKC delta kinase activity is attenuated when PKC delta activation loop phosphorylation is prevented. To distinguish roles of individual nPKC isoforms in nPKC phosphorylations, wild-type (WT) and dominant negative (DN) PKC delta and PKC epsilon mutants were introduced into cardiomyocyte cultures using adenovirus-mediated gene transfer. WT-PKC delta and WT-PKC epsilon are highly phosphorylated at activation loop and hydrophobic motif sites, even in the absence of allosteric activators. DN-PKC delta is phosphorylated at the activation loop but not the hydrophobic motif; DN-PKC epsilon is phosphorylated at the hydrophobic motif but not the activation loop. Collectively, these results identify a role for PKC epsilon in nPKC activation loop phosphorylations and PKC delta in nPKC hydrophobic motif phosphorylations. Agonist-induced nPKC isoform phosphorylations that accompany activation/translocation of the enzyme contribute to the regulation of PKC delta kinase activity, may influence nPKC isoform trafficking/down-regulation, and introduce functionally important cross-talk for nPKC signaling pathways in cardiomyocytes.
authors.affiliation: Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
authors.forename: Vitalyi O
authors.initials: VO
authors.lastname: Rybin
authors.forename: Abdelkarim
authors.initials: A
authors.lastname: Sabri
authors.forename: Jacob
authors.initials: J
authors.lastname: Short
authors.forename: Julian C
authors.initials: JC
authors.lastname: Braz
authors.forename: Jeffery D
authors.initials: JD
authors.lastname: Molkentin
authors.forename: Susan F
authors.initials: SF
authors.lastname: Steinberg
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000495:Allosteric Site; D020816:Amino Acid Motifs; D000818:Animals; D002478:Cells, Cultured; D017124:Conserved Sequence; D015536:Down-Regulation; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques; D005799:Genes, Dominant; D015151:Immunoblotting; D007700:Kinetics; D008954:Models, Biological; D009206:Myocardium / Q000166:cytology*; D010766:Phosphorylation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection
chemical_list: D020033:Protein Isoforms; C497260:Prkcd protein, rat; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon