title: Translocation of diacylglycerol kinase theta from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C.
abstract: Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. We previously identified DGK as one of nine mammalian DGK isoforms and reported on its regulation by interaction with RhoA and by translocation to the plasma membrane in response to noradrenaline. Here, we have investigated how the localization of DGK, fused to green fluorescent protein, is controlled upon activation of G protein-coupled receptors in A431 cells. Extracellular ATP, bradykinin, or thrombin induced DGK translocation from the cytoplasm to the plasma membrane within 2-6 min. This translocation, independent of DGK activity, was preceded by protein kinase C (PKC) translocation and was blocked by PKC inhibitors. Conversely, activation of PKC by 12-O-tetradecanoylphorbol-13-acetate induced DGK translocation. Membrane-permeable DAG (dioctanoylglycerol) also induced DGK translocation but in a PKC (staurosporin)-independent fashion. Mutations in the cysteine-rich domains of DGK abrogated its hormone- and DAG-induced translocation, suggesting that these domains are essential for DAG binding and DGK recruitment to the membrane. We show that DGK interacts selectively with and is phosphorylated by PKCepsilon and -eta and that peptide agonist-induced selective activation of PKCepsilon directly leads to DGK translocation. Our data are consistent with the concept that hormone-induced PKC activation regulates the intracellular localization of DGK, which may be important in the negative regulation of PKCepsilon and/or PKCeta activity.
authors.affiliation: Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam 1066 CX, The Netherlands.
authors.forename: Jürgen
authors.initials: J
authors.lastname: van Baal
authors.forename: John
authors.initials: J
authors.lastname: de Widt
authors.forename: Nullin
authors.initials: N
authors.lastname: Divecha
authors.forename: Wim J
authors.initials: WJ
authors.lastname: van Blitterswijk
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001920:Bradykinin / Q000378:metabolism; D019556:COS Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D019852:Diacylglycerol Kinase / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D008954:Models, Biological; D009154:Mutation; D009638:Norepinephrine / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019311:Staurosporine / Q000494:pharmacology; D013347:Subcellular Fractions; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D015533:Transcriptional Activation
chemical_list: D018076:DNA, Complementary; D010455:Peptides; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D019852:Diacylglycerol Kinase; D011493:Protein Kinase C; D013917:Thrombin; D019204:GTP-Binding Proteins; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D005990:Glycerol; D001920:Bradykinin; D009638:Norepinephrine