title: ATP7B activity is stimulated by PKCɛ in porcine liver.
abstract: Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)-ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10(-8) M), a PKC activator, increased Cu(I)-ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 μM Ca(2+), whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after phosphatase λ treatment, showing that the ɛ isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced Vmax without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling.
authors.affiliation: Laboratório de Físico-Química Biológica Aída Hassón-Voloch, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, 21941-902 Rio de Janeiro, Brazil.
authors.forename: Luiza H D
authors.initials: LH
authors.lastname: Cardoso
authors.affiliation: Laboratório de Físico-Química Biológica Aída Hassón-Voloch, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, 21941-902 Rio de Janeiro, Brazil; Division of Computational Biology and Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
authors.forename: Thiago
authors.initials: T
authors.lastname: Britto-Borges
authors.affiliation: Laboratório de Físico-Química Biológica Aída Hassón-Voloch, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, 21941-902 Rio de Janeiro, Brazil.
authors.forename: Adalberto
authors.initials: A
authors.lastname: Vieyra
authors.affiliation: Laboratório de Físico-Química Biológica Aída Hassón-Voloch, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, 21941-902 Rio de Janeiro, Brazil. Electronic address: lowe@biof.ufrj.br.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lowe
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology; D000073840:Copper-Transporting ATPases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D007527:Isoenzymes; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013552:Swine
chemical_list: D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D004963:Estrenes; D007527:Isoenzymes; D009281:Naphthalenes; D010703:Phorbol Esters; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C119222:phorbol-12-myristate; D003300:Copper; D011493:Protein Kinase C; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases; C058819:calphostin C
keywords: ATP7B; ATPases; Cell signaling; Copper transport; Protein kinase C ɛ (PKCɛ)