The Notch Regulator Numb Links the Notch and TCR Signaling Pathways

Abstract

Both the Notch and TCR signaling pathways play an important role in T cell development, but the links between these signaling pathways are largely unexplored. The adapter protein Numb is a well-characterized inhibitor of Notch and also contains a phosphotyrosine binding domain, suggesting that Numb could provide a link between these pathways. We explored this possibility by investigating the physical interactions among Notch, Numb, and the TCR signaling apparatus and by examining the consequences of a Numb mutation on T cell development. We found that Notch and Numb cocluster with the TCR at the APC contact during Ag-driven T cell-APC interactions in both immature and mature T cells. Furthermore, Numb coimmunoprecipitates with components of the TCR signaling apparatus. Despite this association, T cell development and T cell activation occur normally in the absence of Numb, perhaps due to the expression of the related protein, Numblike. Together our data suggest that Notch and TCR signals may be integrated at the cell membrane, and that Numb may be an important adapter in this process.

TheO-fucose glycan in the ligand-binding domain of Notch1 regulates embryogenesis and T cell development

“…2), most of which are resorbed by E10.5. lular domain (23) revealed similar patterns of cell surface Notch1 expression in T cell subsets from Notch1 12f/12f and Notch1 ϩ/ϩ littermates (Fig. 3A).…”

Notch ligands Delta-like1, Delta-like4 and Jagged1 differentially regulate activation of peripheral T helper cells

“…Altogether, these data indicate a direct interference of Notch with TCR signaling most likely at a membraneproximal site. This probably involves recruitment of Notch into the immunological synapse, as has been demonstrated recently for Notch1 and the regulator of Notch activity, Numb [30,47].…”

“…Recently, Notch1 was also detected by immunofluorescent staining with a specific antibody [30]. However, for T helper cells the receptor binding capacity of individual Notch ligands has not been analyzed.…”

“…Protein concentration was determined in each extract by Bradford assay and equal amounts of protein were assayed for DNA-binding by EMSA as described [57]. Briefly, 3.5 lg of protein were incubated for 30 min at 30 C with 32 -P-labeled probe (10 000 cpm) in 20 lL binding buffer (20 mM HEPES pH7.9, 60 mM KCl, 2 mM DTT, 4% Ficoll, 2 lg BSA and 2 lg poly dI/dC). Subsequently, the complexes were resolved by electrophoresis on a native 5% polyacrylamide gel (in 0.5xTBE).…”

Up-Regulation of Gene Related to Anergy in Lymphocytes Is Associated with Notch-Mediated Human T Cell Suppression

“…This observation, in addition to the data described, led us to hypothesize that Jagged-1 signaling might abrogate TCR-mediated signaling events such that the T cells behaved as though they had received no signal through the TCR. Indeed, this hypothesis is bolstered by reports that Notch receptors colocalize with TCRs during T cell activation and that coligation of TCR, CD28, and Notch-1 inhibits AKT phosphorylation and activation of several TCR-induced transcription factors (6,8,16). To directly test this hypothesis, we used flow cytometry and phospho-specific Abs to a variety of molecules to determine whether Jagged-1 Fc signaling during T cell stimulation could abrogate or reduce proximal TCR signaling events.…”