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

Sergio E. Alvarez, Kuzhuvelil B. Harikumar, Nitai C. Hait, Jeremy Allegood, Graham M. Strub, Eugene Y. Kim,
Michael Maceyka, Hualiang Jiang, Cheng Luo, Tomasz Kordula, Sheldon Milstien & Sarah Spiegel

Tumour-necrosis factor (TNF) receptor-associated factor 2
(TRAF2) is a key component in NF-kB signalling triggered by
TNF-a1,2. 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
IkB kinase, leading to activation of the transcription factor NF-kB.
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 IkB kinase and IkBa, and IkBa
degradation, leading to NF-kB 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 recombinantTRAF2-
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-a signalling and the canonical NF-kB
activation pathway important in inflammatory, antiapoptotic and
immune processes.