Participation of the E3-ligase TRIM13 in NF-κB p65 activation and NFAT-dependent activation of c-Rel upon T-cell receptor engagement.

The nuclear factor κB (NF-κB) family members p65 and c-Rel chiefly orchestrate lymphocytes activation following T-cell receptor (TCR) engagement. In contrast to p65, which is rapidly mobilized, c-Rel activation occurs subsequently as it involves a nuclear factor of activated T-cells (NFAT)-dependent upregulation step. However, how TCR ligation drives p65 and c-Rel activation is not fully understood. Because several ubiquitylated components of NF-κB signaling cascade accumulate in close proximity to membranes, we screened a siRNA library against E3-ligases that contain transmembrane domains on TCR-mediated NF-κB activation. Here, we report the identification of the endoplasmic reticulum resident TRIM13 protein as an enhancer of NF-κB promoter activity. We found that knocking down TRIM13 by RNA interference reduced the activation of p65, while the translocation of c-Rel into the nucleus was blunted. We further observed that c-Rel induction was diminished without TRIM13, as NFAT activation was compromised. These results unveil that TRIM13 is a selective regulator of p65 and of c-Rel activation.

The endoplasmic reticulum acts as a platform for ubiquitylated components of nuclear factor κB signaling.

The innate and adaptive immune responses involve the stimulation of nuclear factor κB (NF-κB) transcription factors through the Lys(63) (K(63))-linked ubiquitylation of specific components of NF-κB signaling pathways. We found that ubiquitylated components of the NF-κB pathway accumulated on the cytosolic leaflet of the endoplasmic reticulum (ER) membrane after the engagement of cell-surface, proinflammatory cytokine receptors or antigen receptors. Through mass spectrometric analysis, we found that the ER-anchored protein metadherin (MTDH) was a partner for these ubiquitylated activators of NF-κB and that it directly bound to K(63)-linked polyubiquitin chains. Knockdown of MTDH inhibited the accumulation of ubiquitylated NF-κB signaling components at the ER, reduced the extent of NF-κB activation, and decreased the amount of proinflammatory cytokines produced. Our observations highlight an unexpected facet of the ER as a key subcellular gateway for NF-κB activation.

Negative regulation of NF-κB signaling in T lymphocytes by the ubiquitin-specific protease USP34.

BACKGROUND: NF-κB is a master gene regulator involved in plethora of biological processes, including lymphocyte activation and proliferation. Reversible ubiquitinylation of key adaptors is required to convey the optimal activation of NF-κB. However the deubiquitinylases (DUBs), which catalyze the removal of these post-translational modifications and participate to reset the system to basal level following T-Cell receptor (TCR) engagement continue to be elucidated. FINDINGS: Here, we performed an unbiased siRNA library screen targeting the DUBs encoded by the human genome to uncover new regulators of TCR-mediated NF-κB activation. We present evidence that knockdown of Ubiquitin-Specific Protease 34 (USP34) selectively enhanced NF-κB activation driven by TCR engagement, similarly to siRNA against the well-characterized DUB cylindromatosis (CYLD). From a molecular standpoint, USP34 silencing spared upstream signaling but led to a more pronounced degradation of the NF-κB inhibitor IκBα, and culminated with an increased DNA binding activity of the transcription factor. CONCLUSIONS: Collectively, our data unveils USP34 as a new player involved in the fine-tuning of NF-κB upon TCR stimulation.