The adaptor protein TRADD is essential for TNF-like ligand 1A/death receptor 3 signaling - PubMed (original) (raw)

The adaptor protein TRADD is essential for TNF-like ligand 1A/death receptor 3 signaling

Yelena L Pobezinskaya et al. J Immunol. 2011.

Abstract

TNFR-associated death domain protein (TRADD) is a key effector protein of TNFR1 signaling. However, the role of TRADD in other death receptor (DR) signaling pathways, including DR3, has not been completely characterized. Previous studies using overexpression systems suggested that TRADD is recruited to the DR3 complex in response to the DR3 ligand, TNF-like ligand 1A (TL1A), indicating a possible role in DR3 signaling. Using T cells from TRADD knockout mice, we demonstrate in this study that the response of both CD4(+) and CD8(+) T cells to TL1A is dependent upon the presence of TRADD. TRADD knockout T cells therefore lack the appropriate proliferative response to TL1A. Moreover, in the absence of TRADD, both the stimulation of MAPK signaling and activation of NF-κB in response to TL1A are dramatically reduced. Unsurprisingly, TRADD is required for recruitment of receptor interacting protein 1 and TNFR-associated factor 2 to the DR3 signaling complex and for the ubiquitination of receptor interacting protein 1. Thus, our findings definitively establish an essential role of TRADD in DR3 signaling.

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Figures

Figure 1

Figure 1. TRADD-deficient T cells do not proliferate in response to TL1A treatment

CFSE labeled wild type and knockout T cells were treated with TL1A (50 μg/ml; top panels) or IL-2 (100U/ml; bottom panels) for 72 hours, then stained with anti-CD4 or anti-CD8 antibodies and analyzed by flow cytometry. Histograms are gated on CD4+ (a) and CD8+ cells (b).

Figure 2

Figure 2. TRADD is required for MAP kinase and NFκB activation

a–c, Western blot analysis of lysates of wild type and TRADD knockout CD4+ (a), CD8+ (b) T cells and pan-T cells (c) treated with TL1A (50 μg/ml) (a,b) for the indicated times or PMA (100nM) (c). d, Nuclear extracts of wild type and knockout T cells treated with TNFα (30 ng/ml) and TL1A (50 μg/ml) for indicated times were analyzed by EMSA.

Figure 3

Figure 3. TRADD is required for formation of the functional DR3 signaling complex

Cell extracts from wild type and TRADD knockout T cells treated with TL1A (50 μg/ml) for 5 min were immunoprecipitated with anti-DR3 (ip:DR3) and analyzed by western blotting. Input, 2% of extract before immunoprecipitation. Ub-, ubiquitinated.

Figure 4

Figure 4. TL1A does not induce apoptosis in primary T cells

MTS assay of the viability of wild type and TRADD knockout T cells treated for 24 hours with cycloheximide (C) alone (10μg/ml), cycloheximide and TL1A (50 μg/ml) or cycloheximide and anti-FAS (10ng/ml) (a) and SMAC mimetic (SM) alone (10nM), SMAC mimetic and TL1A (50 μg/ml) or SMAC mimetic and anti-FAS (10ng/ml) (b). Error bars shown represent SEM.

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