RESUMO
Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. Here we identified a tenth member of the DGK family designated DGK kappa. The kappa-isozyme (1271 amino acids, calculated molecular mass, 142 kDa) contains a pleckstrin homology domain, two cysteine-rich zinc finger-like structures, and a separated catalytic region as have been found commonly for the type II isozymes previously cloned (DGKdelta and DGKeta). The new DGK isozyme has additionally 33 tandem repeats of Glu-Pro-Ala-Pro at the N terminus. Reverse transcriptase-PCR showed that the DGK kappa mRNA is most abundant in the testis, and to a lesser extent in the placenta. DGK kappa, when expressed in HEK293 cells, was persistently localized at the plasma membrane even in the absence of cell stimuli. Deletion analysis revealed that the short C-terminal sequence (amino acid residues 1199-1268) is necessary and sufficient for the plasma membrane localization. Interestingly, DGK kappa, but not other type II DGKs, was specifically tyrosine-phosphorylated at Tyr78 through the Src family kinase pathway in H2O2-treated cells. Moreover, H2O2 selectively inhibited DGK kappa activity in a Src family kinase-independent manner, suggesting that the isozyme changes the balance of signaling lipids in the plasma membrane in response to oxidative stress. The expression patterns, subcellular distribution, and regulatory mechanisms of DGK kappa are distinct from those of DGKdelta and DGKeta despite high structural similarity, suggesting unique functions of the individual type II isozymes.
Assuntos
Diacilglicerol Quinase/química , Diacilglicerol Quinase/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Sanguíneas/química , Western Blotting , Células COS , Catálise , Domínio Catalítico , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Chlorocebus aethiops , Clonagem Molecular , Cisteína/química , DNA Complementar/metabolismo , Diacilglicerol Quinase/metabolismo , Diglicerídeos/química , Relação Dose-Resposta a Droga , Deleção de Genes , Humanos , Peróxido de Hidrogênio/farmacologia , Immunoblotting , Imunoprecipitação , Lipídeos/química , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Estresse Oxidativo , Ácidos Fosfatídicos/química , Fosfoproteínas/química , Fosforilação , Plasmídeos/metabolismo , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Transdução de Sinais , Suínos , Testículo/metabolismo , Distribuição Tecidual , Tirosina/química , Dedos de ZincoRESUMO
Diacylglycerol kinase (DGK) regulates the level of the second messenger diacylglycerol and produces phosphatidic acid (PA), another signaling molecule. The Arabidopsis thaliana genome encodes seven putative diacylglycerol kinase isozymes (named AtDGK1 to -7), structurally falling into three major clusters. So far, enzymatic activity has not been reported for any plant Cluster II DGK. Here, we demonstrate that a representative of this cluster, AtDGK7, is biochemically active when expressed as a recombinant protein in Escherichia coli. AtDGK7, encoded by gene locus At4g30340, contains 374 amino acids with an apparent molecular mass of 41.2 kDa. AtDGK7 harbors an N-terminal catalytic domain, but in contrast to various characterized DGKs (including AtDGK2), it lacks a cysteine-rich domain at its N terminus, and, importantly, its C-terminal DGK accessory domain is incomplete. Recombinant AtDGK7 expressed in E. coli exhibits Michaelis-Menten type kinetics with 1,2-dioleoyl-sn-glycerol as substrate. AtDGK7 activity was affected by pH, detergents, and the DGK inhibitor R59022. We demonstrate that both AtDGK2 and AtDGK7 phosphorylate diacylglycerol molecular species that are typically found in plants, indicating that both enzymes convert physiologically relevant substrates. AtDGK7 is expressed throughout the Arabidopsis plant, but expression is strongest in flowers and young seedlings. Expression of AtDGK2 is transiently induced by wounding. R59022 at approximately 80 mum inhibits root elongation and lateral root formation and reduces plant growth, indicating that DGKs play an important role in plant development.