Role of the Diacylglycerol Kinase α-Conserved Domains in Membrane Targeting in Intact T Cells

Ernesto Merino,Miguel A Sanjuán,Ignacio Moraga,Angel Ciprés,Isabel Mérida

doi:10.1074/jbc.m702085200

Ernesto Merino, Miguel A Sanjuán + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m702085200

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Abstract

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGKalpha is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGKalpha domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr(335). This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.

Highlights

The diacylglycerol kinases (DGK)5 are a family of enzymes that phosphorylate diacylglycerol (DAG) to produce phospha
DGK activity is found in organisms from bacteria to mammals, the protein identified as a DGK in bacteria is an integral membrane protein [2] and has little structural homology with the DGK characterized in multicellular organisms [3]
Ten DGK isoforms have been identified in mammals and grouped into five subtypes based on the presence of various domains in their primary sequences, which define distinct regulatory motifs

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The J-HM1–2.2 cell line was generated by stable transfection of the human muscarinic subtype-1 receptor in the Jurkat T cell line [22]. Fractionation Studies—T lymphocytes were harvested, washed in PBS, resuspended in TES buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.25 M succors) with protein inhibitors, and lysed by 10 passages of the cell suspension through a 25-gauge needle. Western Blot—To determine expression of transfected proteins, cells were lysed in 1 ml of lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM sodium pyrophosphate, 1 mM Na3VO4, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 10 ␮g/ml each aprotinin and leupeptin) for 30 min on ice. After centrifugation (20,000 ϫ g, 10 min, 4 °C), supernatants were analyzed by SDS-PAGE. CD69 expression on the cell surface was analyzed 6 h after stimulation gating for GFP-positive cells) using a phosphatidylethanolamine-conjugated anti-human CD69 monoclonal antibody (Pharmingen). Images were captured each 15 s by confocal microscopy and processed using ImageJ software

RESULTS

B TLC PA

DISCUSSION