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

Abstract

Tumour-necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key component in NF-kappaB signalling triggered by TNF-alpha. Genetic evidence indicates that TRAF2 is necessary for the polyubiquitination of receptor interacting protein 1 (RIP1) that then serves as a platform for recruitment and stimulation of IkappaB kinase, leading to activation of the transcription factor NF-kappaB. 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), 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 IkappaB kinase and IkappaBalpha, and IkappaBalpha degradation, leading to NF-kappaB 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-alpha signalling and the canonical NF-kappaB activation pathway important in inflammatory, antiapoptotic and immune processes.

Figure 1. SphK1 and intracellular S1P are necessary for NF-κB activation by TNF-α independently of S1P receptors

a, HEK 293 cells transfected with siControl or siSphK1 were treated with TNF-α and analyzed by immunoblotting. b, A7 cells were pretreated with SK1-I (10 µM) and stimulated with TNF-α or S1P (100 nM). c, sphk1^+/+ and sphk1^−/− MEFs were stimulated with TNF-α. d, sphk1^+/+ or sphk1^−/− MEFs transfected with V5-SphK1 or catalytically-inactive SphK1^G82D (CI-SphK1) were treated with TNF-α, stained with Hoechst (blue), and p65 (red) and V5 (green) antibodies, and visualized by confocal microscopy. Bar, 20 µm. Percentages of cells with p65 positive nuclei are shown. * P < 0.01. e, NF-κB reporter activity was determined in A7 cells stimulated with TNF-α, 100 nM S1P or dihydro-S1P (DHS1P). f, A7 cells were stimulated with TNF-α (1 ng/ml) or S1P (100 nM or 10 µM) for 10 min. g, HeLa cells were stimulated with TNF-α (1 ng/ml), 10 µM S1P or DHS1P. h, Sphingolipids were analyzed by LC-ESI-MS/MS. i, NF-κB reporter activity was determined in HeLa cells stimulated with TNF-α (1 ng/ml), S1P (10 µM), or both. * P < 0.01 compared to None. n=3.

Figure 2. SphK1 is required for TNF-α-induced Lys 63-linked polyubiquitination of RIP1

a, A7 cells transfected with siControl or siSphK1 were stimulated with TNF-α (10 ng/ml). Proteins were immunoblotted with anti-RIP1 or anti-p65 antibodies. b, Lysates were immunoprecipitated with anti-RIP1 antibody and analyzed with anti-ubiquitin antibody. c, Lysates from cells transfected with HA-Ub and stimulated with TNF-α were immunoprecipitated with anti-RIP1 antibody and analyzed with HA antibody. d, Lysates were immunoprecipitated with anti-RIP1 antibody and proteins analyzed with Lys 63-specific polyubiquitin antibody. e, HEK 293 cells transfected with siControl or siSphK1 were transfected with Flag-TRAF2 and stimulated with TNF-α. Proteins were pulled down with anti-Flag beads and analyzed with anti-TRAF2 antibody.

Figure 3. S1P is required for TRAF2-mediated Lys 63-linked polyubiquitination of RIP1 in Vitro

a, In vitro ubiquitination of purified RIP1 was carried out with ATP, E1, Ubc13/Uev1a, ubiquitin, and TRAF2 with the indicated lipids (100 nM) and examined with anti-RIP1 antibody. b,c, Ubiquitination reactions were carried out with purified WT-TRAF2 or ΔRING-TRAF2 in the presence of UbcH13/Uev1a (b) or UbcH5a/Uev1a (c) as E2s and ubiquitin proteins (WT, Lys 63 only, or Lys 48 only), without or with 100 nM S1P. RIP1 ubiquitination was determined with anti-RIP1 antibody and TRAF2 input with anti-TRAF2 antibody.

Figure 4. Specific binding of S1P to TRAF2

a, Lysates from HEK 293 cells transfected with Flag-TRAF2 were incubated with control (no lipid), S1P, LPA, or sphingosine affinity matrices. b, Lysates from naïve cells were pre-treated with 10 µM S1P, followed by pulldown with control (no lipid), S1P, or sphingosine affinity matrices and bound proteins analyzed by immunoblotting. c, Lysates from vector or Flag-TRAF2 transfected cells were incubated with anti-Flag agarose beads. Beads were washed and incubated with [³²P]S1P (0.1 nM) in the absence or presence of 1 µM unlabeled S1P, dihydro-S1P, sphingosine or LPA, and [³²P]S1P bound to TRAF2 was eluted with Flag peptide and radioactivity determined. Insert, blot of eluted TRAF2. d, Naïve cells were stimulated with TNF-α. Lysates were immunoprecipitated with anti-TRAF2 antibody or control IgG and bound sphingolipids determined by LC-ESI-MS/MS. Of all of the sphingolipids present in these cells (Supplementary Table 1), only S1P was detected in the immunocomplexes. e,f, Cells transfected with vector, Flag-TRAF2, or Flag-ΔRING-TRAF2 were stimulated with TNF-α. Lysates were immunoprecipitated with anti-Flag antibody. (e) Bound S1P determined by LC-ESI-MS/MS. (f) Immunoblot with anti-Flag antibody. g, Docking of S1P into the pocket of the RING domain of TRAF2. Surface contour of the binding site with S1P was colored by electrostatic potential.