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


Tumour-necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key component in NF-κB signalling triggered by TNF-α1,2. Genetic evidence indicates that TRAF2 is necessary for the polyubiquitination of receptor interacting protein 1 (RIP1)3 that then serves as a platform for recruitment and stimulation of IκB kinase, leading to activation of the transcription factor NF-κB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyses 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 the production of S1P is necessary for lysine-63-linked polyubiquitination of RIP1, phosphorylation of IκB kinase and IκBα, and IκBα degradation, leading to NF-κB activation. These responses were mediated by intracellular S1P independently of its cell surface G-protein-coupled receptors. S1P specifically binds to TRAF2 at the amino-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, markedly increased recombinant TRAF2-catalysed lysine-63-linked, but not lysine-48-linked, polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data show that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing cofactor for TRAF2 E3 ubiquitin ligase activity, indicating a new paradigm for the regulation of lysine-63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-α signalling and the canonical NF-κB activation pathway important in inflammatory, antiapoptotic and immune processes.

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Figure 1: SphK1 and intracellular S1P are necessary for NF-κB activation by TNF-α independently of S1P receptors.
Figure 2: SphK1 is required for TNF-α-induced Lys-63-linked polyubiquitination of RIP1.
Figure 3: S1P is required for TRAF2-mediated Lys-63-linked polyubiquitination of RIP1 in vitro.
Figure 4: Specific binding of S1P to TRAF2.


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We thank Z. J. Chen, B. Darnay and M. Karin for HA–ubiquitin and TRAF constructs, and R. Proia for the Sphk1−/− mice. This work was supported by grants from the National Institute of Health (R37GM043880, R01CA61774, R01AI50094, U19AI077435 to S.S.) and in part by the Ministry of Scientific and Technology of China (2009CB918502 to C.L.).

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Authors and Affiliations



S.E.A. and K.B.H. planned and performed most experiments, with assistance from N.C.H., G.M.S., E.Y.K., J.A. and M.M.; C.L. and H.J. performed molecular docking; T.K. contributed to the planning of the experiments; S.M. and S.S. conceived the study, contributed to planning of the experiments and wrote the manuscript.

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Correspondence to Sarah Spiegel.

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The authors declare no competing financial interests.

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Alvarez, S., Harikumar, K., Hait, N. et al. Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2. Nature 465, 1084–1088 (2010).

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