Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2

Abstract

TNF receptor-associated factor 2 (TRAF2) is a key component in NF-κB signaling triggered by TNF–α 1,2. Genetic evidence indicates that TRAF2 is necessary for polyubiquitination of receptor interacting protein 1 (RIP1) 3 that then serves as a platform for recruitment and stimulation of IκB kinase (IKK) leading to activation of the transcription factor NF-κB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyzes the ubiquitination of RIP1 is lacking. TRAF2 binds to sphingosine kinase 1 (SphK1) 4, one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. Here we show that SphK1 and production of S1P is necessary for Lys 63-linked polyubiquitination of RIP1, phosphorylation of IKK and IκBα, and IκBα degradation, leading to NF-κB activation. Surprisingly, these responses were mediated by intracellular S1P independently of its cell surface G protein-coupled receptors. S1P specifically binds to TRAF2 at the N-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, dramatically increased recombinant TRAF2-catalyzed Lys 63- but not Lys 48-linked polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data reveal that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing co-factor for TRAF2 E3 ubiquitin ligase activity, suggesting a new paradigm for regulation of Lys 63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-α signaling and the canonical NF-κB activation pathway important in inflammatory, anti-apoptotic, and immune processes.

Sphingosine‐1‐phosphate signaling in physiology and diseases

“…SphK1-derived S1P regulates pro-inflammatory signaling pathways, including activation of nuclear factor-κB [55]. S1P 1 regulates endothelial barrier integrity as stated above [49][50][51][52]56]; cytokine and adhesion molecule expression, lymphocyte maturation, differentiation and trafficking, and mast cell migration.…”

“…The activation of PAR1 in turn stimulates SphK1, S1P export to the cell exterior, 24 and S1P 3 activation, which induces amplification of inflammation by stimulating the production of IL-1 and tissue factor from dendritic cells and disrupting EC barrier function. SphK1 is also implicated in the actions of tumor necrosis factor (TNF) and other cytokines, in which intracellular S1P produced by SphK1 binds to TRAF2 and thereby activates NE-B [55]. Disruption of SphK1 gene alleviates inflammatory diseases including colitis and arthritis, providing further support for the involvement of SphK1 in inflammatory responses [12].…”

RIP kinases: key decision makers in cell death and innate immunity

“…This is followed by the recruitment of a number of E3 ubiquitin ligases to RIP1, including TNF receptor-associated factor 2 (TRAF2) or TRAF5 and the cellular inhibitor of apoptosis proteins (cIAPs) cIAP1 and cIAP2 resulting in the formation of Complex I. [21][22][23][24] TRAF2 25,26 and cIAPs [27][28][29][30] catalyse the polyubiquitination of RIP1. The ubiquitin-decorated RIP1 is recognized by ubiquitin-binding domain containing proteins in the IkB kinase (IKK) 31 and TAK1 kinase complexes [32][33][34] thus facilitating TAK-1-mediated phosphorylation and activation of IKKs.…”

De-ubiquitinating protease USP2a targets RIP1 and TRAF2 to mediate cell death by TNF

“…2-5 The E3 ubiquitin ligase TRAF2 was the first to be described as being responsible for K63 ubiquitination of RIP1 5-7 together with the lipid shingosine-1-phosphate. 8 Other publications have shown that cIAP1/2 can attach K63 ubiquitin chains to RIP1 [9][10][11][12] or that TRAF2 and cIAPs cooperate for the ubiquitination of RIP1 6,13,14 . These ubiquitin chains bind and associate the TAB/TAK (TAK1/TAB2/TAB3) and NEMO/IKK (IKKa, b, g) kinases complex.…”