Selective coupling of the T cell antigen receptor to phosphoinositide-derived diacylglycerol production in HPB-ALL T cells correlates with CD45-regulated p59fyn activity.
In HPB-ALL T-cells the p59fyn tyrosine kinase is regulated by the CD45 phosphotyrosine phosphatase and plays a critical role in coupling the T cell receptor (TCR) to the generation of intracellular signals which include diacylglycerol (DAG) production and protein kinase C activation. The aim of this study was to determine the phospholipid pools from which the DAG is generated and to identify which phospholipase activities are regulated by the TCR. When CD45+ cells were pre-labeled with [3H]arachidonic acid, CD3-antigen cross-linking stimulated negligible increases in both [3H]DAG and [3H]phosphatidic acid (PA). However, CD3 monoclonal antibody (mAb) induced an increase of 300% in [3H]PA when the cells were permeabilized with streptolysin-O, and this correlated with increased levels of protein tyrosine phosphorylation. Stimulation of [3H]PA production upon CD3 cross-linking was 77% lower in permeabilized CD45- cells than in CD45+ cells, consistent with the reduced activity of p59fyn in CD45- cells. The stimulated production of PA was not mediated by activation of phospholipase D (PLD), although the presence of a G-protein-regulated PLD activity was established. The CD3-induced increase in total inositol phosphates (InsP) in permeabilized cells was similar to the stimulated production of [3H]PA production in both CD45+ and CD45- cells. Dose-response curves for InsP and PA production triggered by CD3 mAb were super-imposable and the production of InsP and PA over a range of Ca2+ concentrations was comparable. Differential labeling of phospholipids with 3H-labeled fatty acids revealed that CD3-induced PA production reflected incorporation of label into the phosphatidylinositol pool. Our data suggest that in HPB-ALL cells the production of DAG following CD3-antigen cross-linking can be fully accounted for by the selective coupling of the TCR to breakdown of phosphatidylinositol-(4,5)-bisphosphate as the result of phospholipase C gamma 1 activation. This event correlates with the activity of the CD45-regulated TCR-associated tyrosine kinase, p59fyn.
Biffen
M
M
AFRC Babraham Institute, Cambridge, GB.
Shiroo
M
M
Alexander
D R
DR
D002463:Cell Membrane Permeability; D004075:Diglycerides / Q000096:biosynthesis*; D005227:Fatty Acids / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000276:immunology* / Q000378:metabolism*; D017493:Leukocyte Common Antigens / Q000378:metabolism; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010716:Phosphatidylinositols / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000276:immunology / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D004075:Diglycerides; D005227:Fatty Acids; D007295:Inositol Phosphates; D010712:Phosphatidic Acids; D010716:Phosphatidylinositols; D011518:Proto-Oncogene Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D017493:Leukocyte Common Antigens; D010738:Type C Phospholipases; D010739:Phospholipase D; D019204:GTP-Binding Proteins