Selective coupling of the T cell antigen receptor to phosphoinositide‐derived diacylglycerol production in HPB‐ALL T cells correlates with CD45‐regulated p59^fyn activity

Abstract

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.

Disruption of Lipid Rafts Stimulates Phospholipase D Activity in Human Lymphocytes: Implication in the Regulation of Immune Function

“…However, the role of PLD in T cell activation remains controversial. Although some reports have described an activation of PLD following ligation of the TCR/CD3 complex (6, 7), other studies failed to demonstrate efficient CD3 coupling to PLD, either in normal peripheral lymphocytes (8) or in T cell lines (9). It has been shown that TCR ligation was accompanied by a fast and large increase in PA level occurring in the first minutes of T cell activation (10,11).…”

Regulation of pseudopodia localization in lymphocytes through application of mechanical forces by optical tweezers

“…1 We also point out that HPB-ALL cell line responses to TCR engagement include generation of intracellular signals such as diacylglycerol production and protein kinase C activation, where PKC in turn regulates calcium influx. [41][42][43] The highly improved localized response observed after application of tangential forces resulted in the lymphocyte actively pulling the antibody-coated bead out of the optical trap and, for a small bead, engulfing it in almost half of the cases. TCR-mediated internalization of peptide-MHC ligand present on the surface of APCs has been reported by several investigators.…”

T-Cell Antigen Receptor-Induced Signal-Transduction Pathways. Activation and Function of Protein Kinases C in T Lymphocytes

“…This diacylglycerol production is normally transient and temporally corresponds to the formation of InsP,. It is frequently followed by a more sustained increase in the amount of diacylglycerol (Asaoka et al, 1992;Nishizuka, 1992), although it has been claimed that sustained elevation of diacylglycerol resulted exclusively from phosphatidylinositol phosphate cleavage (Biffen et al, 1993) regulated by p59fy" in stimulated human T lymphocytes. Extensive analysis of fatty acid compositions has indicated that the second phase of diacylglycerol formation probably results from hydrolysis of phosphatidylcholine in various stimulated cells, including T lymphocytes (van Blitterswijk et al , 1991aBoarder, 1994).…”