title: Protein kinase Cepsilon (PKCepsilon) and Src control PKCdelta activation loop phosphorylation in cardiomyocytes.
abstract: Protein kinase Cdelta (PKCdelta) is unusual among AGC kinases in that it does not require activation loop (Thr(505)) phosphorylation for catalytic competence. Nevertheless, Thr(505) phosphorylation has been implicated as a mechanism that influences PKCdelta activity. This study examines the controls of PKCdelta-Thr(505) phosphorylation in cardiomyocytes. We implicate phosphoinositide-dependent kinase-1 and PKCdelta autophosphorylation in the "priming" maturational PKCdelta-Thr(505) phosphorylation that accompanies de novo enzyme synthesis. In contrast, we show that PKCdelta-Thr(505) phosphorylation dynamically increases in cardiomyocytes treated with phorbol 12-myristate 13-acetate or the alpha(1)-adrenergic receptor agonist norepinephrine via a mechanism that requires novel PKC isoform activity and not phosphoinositide-dependent kinase-1. We used a PKCepsilon overexpression strategy as an initial approach to discriminate two possible novel PKC mechanisms, namely PKCdelta-Thr(505) autophosphorylation and PKCdelta-Thr(505) phosphorylation in trans by PKCepsilon. Our studies show that adenovirus-mediated PKCepsilon overexpression leads to an increase in PKCdelta-Thr(505) phosphorylation. However, this cannot be attributed to an effect of PKCepsilon to function as a direct PKCdelta-Thr(505) kinase, since the PKCepsilon-dependent increase in PKCdelta-Thr(505) phosphorylation is accompanied by (and dependent upon) increased PKCdelta phosphorylation at Tyr(311) and Tyr(332). Further studies implicate Src in this mechanism, showing that 1) PKCepsilon overexpression increases PKCdelta-Thr(505) phosphorylation in cardiomyocytes and Src(+) cells but not in SYF cells (that lack Src, Yes, and Fyn and exhibit a defect in PKCdelta-Tyr(311)/Tyr(332) phosphorylation), and 2) in vitro PKCdelta-Thr(505) autophosphorylation is augmented in assays performed with Src (which promotes PKCdelta-Tyr(311)/Tyr(332) phosphorylation). Collectively, these results identify a novel PKCdelta-Thr(505) autophosphorylation mechanism that is triggered by PKCepsilon overexpression and involves Src-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation.
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: Jianfen
authors.initials: J
authors.lastname: Guo
authors.forename: Zoya
authors.initials: Z
authors.lastname: Gertsberg
authors.forename: Hasnae
authors.initials: H
authors.lastname: Elouardighi
authors.forename: Susan F
authors.initials: SF
authors.lastname: Steinberg
mesh_terms: D000495:Allosteric Site; D000818:Animals; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008954:Models, Biological; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000737:chemistry* / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000737:chemistry* / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D016392:Proto-Oncogene Proteins pp60(c-src); D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon