Expression of a catalytically inactive form of diacylglycerol kinase alpha induces sustained signaling through RasGRP.
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.
Jones
David R
DR
Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Cantoblanco, E-28049 Madrid, Spain.
Sanjuán
Miguel Angel
MA
Stone
James C
JC
Mérida
Isabel
I
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*
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