ESCRT proteins restrict constitutive NF-κB signaling by trafficking cytokine receptors

Abstract

Because signaling mediated by the transcription factor nuclear factor κB (NF-κB) is initiated by ligands and receptors that can undergo internalization, we investigated how endocytic trafficking regulated this key physiological pathway. We depleted all of the ESCRT (endosomal sorting complexes required for transport) subunits, which mediate receptor trafficking and degradation, and found that the components Tsg101, Vps28, UBAP1, and CHMP4B were essential to restrict constitutive NF-κB signaling in human embryonic kidney 293 cells. In the absence of exogenous cytokines, depletion of these proteins led to the activation of both canonical and noncanonical NF-κB signaling, as well as the induction of NF-κB-dependent transcriptional responses in cultured human cells, zebrafish embryos, and fat bodies in flies. These effects depended on cytokine receptors, such as the lymphotoxin β receptor (LTβR) and tumor necrosis factor receptor 1 (TNFR1). Upon depletion of ESCRT subunits, both receptors became concentrated on and signaled from endosomes. Endosomal accumulation of LTβR induced its ligand-independent oligomerization and signaling through the adaptors TNFR-associated factor 2 (TRAF2) and TRAF3. These data suggest that ESCRTs constitutively control the distribution of cytokine receptors in their ligand-free state to restrict their signaling, which may represent a general mechanism to prevent spurious activation of NF-κB.

ESCRT-III Acts Downstream of MLKL to Regulate Necroptotic Cell Death and Its Consequences

“…The alternative explanation, that disruption of ESCRT-III alters TNFR signaling to promote necroptosis, is unlikely, as TNF-induced IκBα degradation or apoptosis induced by TNF plus CHX were unaffected when ESCRT-III components were silenced, and no differences in the levels of TNF-induced phospho-MLKL were detected (Figure S6C to S6F). This is consistent with a report (Maminska et al, 2016) that although ESCRT-III functions to suppress spontaneous NF-κB activation in 293 cells as a consequence of intracellular TNFR accumulation and signaling, ESCRT-III does not affect NF-κB activation by extracellular TNF addition. While another report (Mahul-Mellier et al, 2008) suggested that the ESCRT components Alix and ALG-2 promote caspase-8 activity and apoptosis upon treatment with TNF, we were unable to find a role for these ESCRT components in necroptosis.…”

The non-canonical NF-κB pathway in immunity and inflammation

“…RNA interference- mediated knockdown of major subunits of ESCRT (endosomal sorting complexes required for transport) causes endosomal accumulation of LTβR, which leads to non-canonical NF-κB pathway activation in a ligand-independent manner. These findings suggest that ESCRT prevents the aberrant sorting of LTβR and constitutive activation of the non-canonical NF-κB pathway 71 . Whether other non-canonical NF-κB pathway-stimulating receptors are also subject to ESCRT regulation remains to be examined.…”

“…Non-canonical NF-κB signalling is also regulated at the level of receptor trafficking and degradation, as demonstrated for LTβR 71 . RNA interference- mediated knockdown of major subunits of ESCRT (endosomal sorting complexes required for transport) causes endosomal accumulation of LTβR, which leads to non-canonical NF-κB pathway activation in a ligand-independent manner.…”

A Pseudomonas aeruginosa TIR effector mediates immune evasion by targeting UBAP1 and TLR adaptors

“…While we were conducting this work, another study reported UBAP1 participates in control of TNFR1 and other cytokine receptor trafficking (Mamińska et al , 2016). Our data along with this recently published study thus suggest that PumA interaction with UBAP1 results in inhibition of the TNF receptor‐mediated pathway.…”

“…UBAP1 has been shown to control endosomal sorting of ubiquitinated EGFR (Stefani et al , 2011) as well as ubiquitin‐dependent degradation of antiviral surface proteins (Agromayor et al , 2012) and integrins (Kharitidi et al , 2015). More recently, UBAP1 was shown to modulate steady‐state trafficking of cytokine receptors in non‐stimulated cells (Mamińska et al , 2016). UBAP1 is expressed in a wide range of tissues, but when deleted in mice, it is lethal for embryos (Agromayor et al , 2012).…”