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-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.
Alvarez
Sergio E
SE
Department of Biochemistry and Molecular Biology and the Massey Cancer Center, Virginia Commonwealth University School of Medicine, 1101 E. Marshall Street, Richmond, Virginia 23298, USA.
Harikumar
Kuzhuvelil B
KB
Hait
Nitai C
NC
Allegood
Jeremy
J
Strub
Graham M
GM
Kim
Eugene Y
EY
Maceyka
Michael
M
Jiang
Hualiang
H
Luo
Cheng
C
Kordula
Tomasz
T
Milstien
Sheldon
S
Spiegel
Sarah
S
D000818:Animals; D055162:Biocatalysis; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008246:Lysophospholipids / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D047992:TNF Receptor-Associated Factor 2 / Q000737:chemistry / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects; D000099157:Sphingosine Kinase
D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D008239:Lysine; D008246:Lysophospholipids; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D017853:Phosphotransferases (Alcohol Group Acceptor); D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D013110:Sphingosine; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D000099157:Sphingosine Kinase; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; C060506:sphingosine 1-phosphate