title: Expression of a catalytically inactive form of diacylglycerol kinase alpha induces sustained signaling through RasGRP.
abstract: Regulating the generation and clearance of lipid second messengers, such as diacylglycerol (DAG), is critical for the correct propagation of intracellular signaling pathways. DAGK type alpha acts as a negative modulator of the DAG levels generated during T cell activation, which is initiated by triggering of the endogenous T cell receptor (TCR), as well as by stimulation of an ectopically expressed human muscarinic type 1 receptor. Here we show that stimulation of either of these receptors causes rapid, transient membrane translocation of the recently discovered Ras guanyl nucleotide release protein (RasGRP), followed by activation of mitogen-activated protein kinase (MAPK). When cells expressing a catalytically inactive form of DAGKalpha were analyzed, however, similar agonist stimulation resulted in sustained signaling through RasGRP and MAPK. Biochemical analysis showed that expression of kinase-dead diacylglycerol kinase a (DGKalpha) led to a greater, more sustained, DAG accumulation following receptor stimulation. These results suggest that, in T cells, agonist-stimulated DAG generation is the key factor controlling activation of the Ras/MAPK signaling pathway through membrane translocation of RasGRP. Moreover, we demonstrate that through the modulation of the intracellular level of agonist-stimulated DAG, DGKalpha alters Ras activation and downstream signaling dramatically, a process of utmost importance for sound immunological function.
authors.affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Cantoblanco, E-28049 Madrid, Spain.
authors.forename: David R
authors.initials: DR
authors.lastname: Jones
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Sanjuán
authors.forename: James C
authors.initials: JC
authors.lastname: Stone
authors.forename: Isabel
authors.initials: I
authors.lastname: Mérida
mesh_terms: D002217:Carbachol / Q000494:pharmacology; D002384:Catalysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019852:Diacylglycerol Kinase / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007202:Indicators and Reagents / Q000032:analysis; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009154:Mutation; D021381:Protein Transport; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology*
chemical_list: D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; C491790:RASGRP1 protein, human; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002217:Carbachol; D019852:Diacylglycerol Kinase; D020928:Mitogen-Activated Protein Kinases