T cell activation in vivo targets diacylglycerol kinase alpha to the membrane: a novel mechanism for Ras attenuation.
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid, leading to decreased and increased levels, respectively, of these two lipid messengers that play a central role in T cell activation. Nine 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. In this study, we have researched in vivo the regulation of DGK alpha, using a transgenic mouse model in which injection of an antigenic peptide activates the majority of peripheral T cells. We demonstrate that DGK alpha, highly expressed in resting T lymphocytes, is subject to complex control at the mRNA and protein levels during in vivo T cell activation. Subcellular fractionation of T lymphocytes shortly after in vivo engagement of the TCR shows rapid translocation of cytosolic DGK alpha to the membrane fraction. At early time points, DGK alpha translocation to the membrane correlates with rapid translocation of Ras guanyl nucleotide-releasing protein (RasGRP), a nucleotide exchange activator for Ras that associates to the membrane through a diacylglycerol-binding domain. To demonstrate a causal relationship between DGK alpha activity and RasGRP relocation to the membrane, we determined RasGRP translocation kinetics in a T cell line transiently transfected with constitutive active and dominant-negative DGK alpha mutants. We show that membrane localization of DGK alpha is associated with a negative regulatory signal for Ras activation by reversing RasGRP translocation. This study is the first demonstration of in vivo regulation of DGK alpha, and provides new insight into the functional role of a member of this family of lipid kinases in the regulation of the immune response.
Sanjuán
Miguel A
MA
Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Madrid, Spain.
Pradet-Balade
Bérengère
B
Jones
David R
DR
Martínez-A
Carlos
C
Stone
James C
JC
Garcia-Sanz
Jose A
JA
Mérida
Isabel
I
D000818:Animals; D018106:CD28 Antigens / Q000276:immunology / Q000378:metabolism / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019852:Diacylglycerol Kinase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D021381:Protein Transport / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism / Q000502:physiology; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology*; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000378:metabolism*; D020666:ras-GRF1 / Q000378:metabolism
D018106:CD28 Antigens; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D007527:Isoenzymes; D008565:Membrane Proteins; C491790:RASGRP1 protein, human; C112278:Rasgrp1 protein, mouse; D011948:Receptors, Antigen, T-Cell; D020666:ras-GRF1; D019852:Diacylglycerol Kinase; D018631:ras Proteins