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

Jürgen Van Baal,John De Widt,Nullin Divecha,Wim J Van Blitterswijk

doi:10.1074/jbc.m409301200

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.

Highlights

Diacylglycerol kinase (DGK)1 regulates signal transduction by modulating, both temporally and spatially, the balance between two signaling lipids, diacylglycerol (DAG) and phosphatidic acid (PA) [1,2,3,4,5,6,7]
We show that DGK␪ interacts selectively with and is phosphorylated by protein kinase C (PKC)⑀ and -␩ and that peptide agonist-induced selective activation of PKC⑀ directly leads to DGK␪ translocation
Endogenous Expression of DGK␪—Western blotting analysis revealed that DGK␪ is expressed in diverse cell types (Fig. 1), such as epithelial-like cell lines (A431, Mel-Juso, HeLa, U2-OS, MCF-7, and Madin-Darby caning kidney cells), fibroblast-like cell lines (COS-7, human embryonic kidney (HEK293), and Chinese hamster ovary cells), neuronal N1E-115 cells, and Jurkat-J6 lymphocytes

Summary

EXPERIMENTAL PROCEDURES

Materials—Dulbecco’s modified Eagle’s medium and Geneticin (G418) were from Invitrogen. [␥-32P]ATP was from Amersham Biosciences. CDNA Constructs—Fusion proteins between GFP and DGK␪ were generated by using appropriate primers and subcloning the amplified DNA into the EcoRI site of the pEGFP-N3 eukaryotic expression vector (Clontech). GST Pull-down Assay—Cytosols from COS-7 cells transfected with pcDNA3 vector encoding human PKC␣, -␤2, -␦, -⑀, -␩, -␨, or -␭ were incubated with beads containing 5–10 ␮g of DGK␪-GST fusion protein for 2 h at 4 °C. HDAPIGYD (pseudo-⑀RACK; ␺⑀RACK), which is derived from the regulatory V1 region of PKC⑀ (amino acid residues 85–92) and was previously shown to selectively activate the translocation of PKC⑀ [48, 49] To introduce this peptide into cells, it was coupled NH2-terminally to the peptide YARAAARQARAG, which is an optimized version of the protein transduction domain of the TAT protein of human immunodeficiency virus (HIV-1) [50]. It was added to cells at a concentration of 5 ␮M to induce PKC⑀-GFP translocation effectively within 3 min

RESULTS

DISCUSSION

PKC released back to cytosol