Linker for Activation of T Cells Integrates Positive and Negative Signaling in Mast Cells

Abstract

The transmembrane adapter linker for activation of T cells (LAT) is thought to couple immunoreceptors to intracellular signaling pathways. In mice, its intracytoplasmic domain contains nine tyrosines which, when phosphorylated upon receptor aggregation, recruit Src-homology 2 domain-containing cytosolic enzymes and adapters. The four distal tyrosines are critical for both TCR and FcεRI signaling. Unexpectedly, knock-in mice expressing LAT with a point mutation of the first or of the last three of these tyrosines exhibited an abnormal T cell development characterized by a massive expansion of TH2-like αβ or γδ T cells, respectively. This phenotype suggests that, besides positive signals, LAT might support negative signals that normally regulate terminal T cell differentiation and proliferation. We investigated here whether LAT might similarly regulate mast cell activation, by generating not only positive but also negative signals, following FcR engagement. To this end, we examined IgE- and/or IgG-induced secretory and intracellular responses of mast cells derived from knock-in mice expressing LAT with combinations of tyrosine mutations (Y136F, Y(175, 195, 235)F, or Y(136, 175, 195, 235)F). A systematic comparison of pairs of mutants enabled us to dissect the respective roles played by the five proximal and the four distal tyrosines. We found that LAT tyrosines differentially contribute to exocytosis and cytokine secretion and differentially regulate biological responses of mucosal- and serosal-type mast cells. We also found that, indeed, both positive and negative signals may emanate from distinct tyrosines in LAT, whose integration modulates mast cell secretory responses.

Non-T Cell Activation Linker Promotes Mast Cell Survival by Dampening the Recruitment of SHIP1 by Linker for Activation of T Cells

“…NTAL deletion, however, also enhances LAT-dependent negative signals which are mediated by SHIP1 and affect primarily cell survival. Finally, our results provide a molecular mechanism to the LAT-dependent negative effects previously observed in mast cells from LAT knockin mice (16). These can indeed be explained by the presence of two SHIP1-binding sites in LAT (Fig.…”

“…We found in a previous work conducted in mast cells from knockin mice, in which individual LAT tyrosines were mutated into phenylalanines (14,15), that the four distal tyrosines of LAT could generate not only positive, but also negative, signals in mast cells. Indeed, whereas IgE-induced responses of bone marrow-derived mast cells (BMMC) from Y136F or of Y(175,195,235)F mice were markedly reduced, the responses of BMMC from Y(136,175,195,235)F mice were almost as intense as those of wild-type (WT) BMMC (16). Molecular mechanisms responsible for LAT-dependent negative signaling were not elucidated.…”

Peritoneal Cell-Derived Mast Cells: An In Vitro Model of Mature Serosal-Type Mouse Mast Cells

“…They can be separated from other peritoneal cells by techniques based on centrifugation through high-density medium (28), but in very low numbers (Յ1 ϫ 10 5 /mouse) only, and with a variable purity. We found recently that significant numbers of mast cells can be generated in culture from mouse peritoneal cells (29), which might be useful for studying mast cell-dependent IgE-and IgG-induced tissue inflammation. They indeed respond both to IgE and to IgG Abs and we show here that this property is due to mild SHIP1-dependent negative regulation.…”

Negative Signaling in Fc Receptor Complexes

“…A systematic comparison of biologic responses observed in pairs of mutants enabled us to dissect the respective roles played by LAT tyrosines in mast cells (Malbec et al, 2004). As expected, Y136 and the three distal tyrosines differentially contributed to exocytosis and the secretion of cytokines, on the one hand, and to the generation or the activation of major cytosolic effectors such as intracellular Ca 2+ and the terminal MAP kinases of the ras pathway, Erk1/2, on the other hand.…”