Sphingosine-1-phosphate activates the E3 ligase activity of TRAF2 to promote Lys-63 linked ubiquitination of RIP1 and NF-kB activation.

HARIKUMAR KB*; ALVAREZ SE*; HAIT NC; ALLEGOOD J; STRUB GM; KIM EY; MACEYCKA M; KORDULA T; MILSTIEN S; SPIEGEL S

Saxton Rivers, VE, USA

Congreso; FASEB Summer Research Conference. Ubiquitin and Cellular Regulation.; 2010

Background: TNF receptor-associated factor 2 (TRAF2) is a key component in NF-kB signaling triggered by TNFa. Genetic evidence indicates that TRAF2 is necessary for polyubiquitination of receptor interacting protein 1 (RIP1) that then serves as a platform for recruitment and stimulation of the downstream IKK complex which in turn leads to activation of the transcription factor NF-kB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyzes the ubiquitination of RIP1 is still lacking. TRAF2 binds to sphingosine kinase 1 (SphK1), one of the isoenzymes that generate the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. We have now examined the role of SphK1 and S1P in NF-kB signaling. Methods: In vitro ubiquitination assays were performed as described (Varfolomeev, E. et al J. Biol. Chem. 283: 24295, 2008). S1P and sphingolipids were quantified by LC-ESI-MS/MS (Hait, N. C. et al. Science 325: 1254, 2009). Results: When SphK1 expression and activity were decreased using genetic and pharmacological approaches, TNF-mediated phosphorylation of IKK and IƒÛBa and IƒÛBa degradation were abolished, as well as translocation of p65 and p50 from the cytosol to the nucleus. However, binding of S1P to its cell surface G protein-coupled receptors did not activate NF-kB. Moreover using a variety of approaches, we demonstrated that SphK1 and production of intracellular S1P was indispensable for TRAF2-mediated Lys 63-linked polyubiquitination of RIP1, leading to NF-kB activation. Importantly, S1P dramatically and specifically increased TRAF2-catalyzed Lys 63- but not Lys 48-linked polyubiquitination of RIP1 in vitro, and the structurally related lipids, dihydro-S1P and LPA, did not mimic the effects of S1P. Moreover, using S1P affinity beads, immunoprecipitation, and mass spectrometry, we showed that S1P physically interacts with TRAF2 at the N-terminal RING domain and that this association is required for S1P to stimulate its E3 ligase activity. Conclusions: TRAF2 is a novel intracellular target of S1P indicating that SphK1 and its product S1P have key roles in TNF-a signaling and the canonical NF-kB pathway. Our study establishes the functional role of TRAF2 in the NF-kB pathway and reveals that S1P is the missing co-factor for TRAF2 E3 ubiquitin ligase activity, leading to a new paradigm for regulation of Lys 63-linked polyubiquitination.