Translocation of Diacylglycerol Kinase θ from Cytosol to Plasma Membrane in Response to Activation of G Protein-coupled Receptors and Protein Kinase C*

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. We previously identified DGKθ 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 DGKθ, fused to green fluorescent protein, is controlled upon activation of G protein-coupled receptors in A431 cells. Extracellular ATP, bradykinin, or thrombin induced DGKθ 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 DGKθ translocation. Membrane-permeable DAG (dioctanoylglycerol) also induced DGKθ translocation but in a PKC (staurosporin)-independent fashion. Mutations in the cysteine-rich domains of DGKθ abrogated its hormone- and DAG-induced translocation, suggesting that these domains are essential for DAG binding and DGKθ recruitment to the membrane. We show that DGKθ interacts selectively with and is phosphorylated by PKCϵ and -η and that peptide agonist-induced selective activation of PKCϵ directly leads to DGKθ translocation. Our data are consistent with the concept that hormone-induced PKC activation regulates the intracellular localization of DGKθ, which may be important in the negative regulation of PKCϵ and/or PKCη activity.