Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Nature volume 465, pages 1084–1088 (2010)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

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.

References

  1. Karin, M. & Gallagher, E. TNFR signaling: ubiquitin-conjugated TRAFfic signals control stop-and-go for MAPK signaling complexes. Immunol. Rev. 228, 225–240 (2009)

    Article  CAS  Google Scholar 

  2. Hayden, M. S. & Ghosh, S. Shared principles in NF-κB signaling. Cell 132, 344–362 (2008)

    Article  CAS  Google Scholar 

  3. Lee, T. H., Shank, J., Cusson, N. & Kelliher, M. A. The kinase activity of Rip1 is not required for tumor necrosis factor-α-induced IκB kinase or p38 MAP kinase activation or for the ubiquitination of Rip1 by Traf2. J. Biol. Chem. 279, 33185–33191 (2004)

    Article  CAS  Google Scholar 

  4. Xia, P. et al. Sphingosine kinase interacts with TRAF2 and dissects tumor necrosis factor-α signaling. J. Biol. Chem. 277, 7996–8003 (2002)

    Article  CAS  Google Scholar 

  5. Bhoj, V. G. & Chen, Z. J. Ubiquitylation in innate and adaptive immunity. Nature 458, 430–437 (2009)

    Article  ADS  CAS  Google Scholar 

  6. Paugh, S. W. et al. A selective sphingosine kinase 1 inhibitor integrates multiple molecular therapeutic targets in human leukemia. Blood 112, 1382–1391 (2008)

    Article  CAS  Google Scholar 

  7. Spiegel, S. & Milstien, S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nature Rev. Mol. Cell Biol. 4, 397–407 (2003)

    Article  CAS  Google Scholar 

  8. Kimura, T. et al. Role of scavenger receptor class B type I and sphingosine 1-phosphate receptors in high-density lipoprotein-induced inhibition of adhesion molecule expression in endothelial cells. J. Biol. Chem. 281, 37457–37467 (2006)

    Article  CAS  Google Scholar 

  9. Ki, S. H., Choi, M. J., Lee, C. H. & Kim, S. G. Gα12 specifically regulates COX-2 induction by sphingosine 1-phosphate: Role for JNK-dependent ubiquitination and degradation of IκBα. J. Biol. Chem. 282, 1938–1947 (2007)

    Article  CAS  Google Scholar 

  10. Pitson, S. M. et al. Activation of sphingosine kinase 1 by ERK1/2-mediated phosphorylation. EMBO J. 22, 5491–5500 (2003)

    Article  CAS  Google Scholar 

  11. Zhao, Y. et al. Intracellular generation of sphingosine 1-phosphate in human lung endothelial cells: Role of lipid phosphate phosphatase-1 and sphingosine kinase 1. J. Biol. Chem. 282, 14165–14177 (2007)

    Article  CAS  Google Scholar 

  12. Van Brocklyn, J. R. et al. Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled orphan receptor edg-1 and intracellular to regulate proliferation and survival. J. Cell Biol. 142, 229–240 (1998)

    Article  CAS  Google Scholar 

  13. Giussani, P. et al. Sphingosine-1-phosphate phosphohydrolase regulates endoplasmic reticulum-to-Golgi trafficking of ceramide. Mol. Cell. Biol. 26, 5055–5069 (2006)

    Article  CAS  Google Scholar 

  14. Ea, C. K., Deng, L., Xia, Z. P., Pineda, G. & Chen, Z. J. Activation of IKK by TNFα requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol. Cell 22, 245–257 (2006)

    Article  CAS  Google Scholar 

  15. Wu, C. J., Conze, D. B., Li, T., Srinivasula, S. M. & Ashwell, J. D. Sensing of Lys-63-linked polyubiquitination by NEMO is a key event in NF-κB activation. Nature Cell Biol. 8, 398–406 (2006)

    Article  CAS  Google Scholar 

  16. Wang, H. et al. Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin. Proc. Natl Acad. Sci. USA 105, 20197–20202 (2008)

    Article  ADS  CAS  Google Scholar 

  17. Newton, K. et al. Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies. Cell 134, 668–678 (2008)

    Article  CAS  Google Scholar 

  18. Shi, C. S. & Kehrl, J. H. Tumor necrosis factor (TNF)-induced germinal center kinase-related (GCKR) and stress-activated protein kinase (SAPK) activation depends upon the E2/E3 complex Ubc13-Uev1A/TNF receptor-associated factor 2 (TRAF2). J. Biol. Chem. 278, 15429–15434 (2003)

    Article  CAS  Google Scholar 

  19. Habelhah, H. et al. Ubiquitination and translocation of TRAF2 is required for activation of JNK but not of p38 or NF-κB. EMBO J. 23, 322–332 (2004)

    Article  CAS  Google Scholar 

  20. Li, S., Wang, L. & Dorf, M. E. PKC phosphorylation of TRAF2 mediates IKKα/β recruitment and K63-linked polyubiquitination. Mol. Cell 33, 30–42 (2009)

    Article  CAS  Google Scholar 

  21. Bertrand, M. J. et al. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. Mol. Cell 30, 689–700 (2008)

    Article  CAS  Google Scholar 

  22. Varfolomeev, E. et al. c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor α (TNFα)-induced NF-κB activation. J. Biol. Chem. 283, 24295–24299 (2008)

    Article  CAS  Google Scholar 

  23. Xu, M., Skaug, B., Zeng, W. & Chen, Z. J. A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFα and IL-1β. Mol. Cell 36, 302–314 (2009)

    Article  CAS  Google Scholar 

  24. Yin, Q., Lamothe, B., Darnay, B. G. & Wu, H. Structural basis for the lack of E2 interaction in the RING domain of TRAF2. Biochemistry 48, 10558–10567 (2009)

    Article  CAS  Google Scholar 

  25. Morris, G. M. et al. Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function. J. Comput. Chem. 19, 1639–1662 (1998)

    Article  CAS  Google Scholar 

  26. Hait, N. C. et al. Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate. Science 325, 1254–1257 (2009)

    Article  ADS  CAS  Google Scholar 

  27. Maceyka, M., Alvarez, S. E., Milstien, S. & Spiegel, S. Filamin A links sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 at lamellipodia to orchestrate cell migration. Mol. Cell. Biol. 28, 5687–5697 (2008)

    Article  CAS  Google Scholar 

  28. Allende, M. L. et al. Mice deficient in sphingosine kinase 1 are rendered lymphopenic by FTY720. J. Biol. Chem. 279, 52487–52492 (2004)

    Article  CAS  Google Scholar 

  29. Paugh, B. S. et al. EGF regulates plasminogen activator inhibitor-1 (PAI-1) by a pathway involving c-Src, PKCδ, and sphingosine kinase 1 in glioblastoma cells. FASEB J. 22, 455–465 (2008)

    Article  CAS  Google Scholar 

  30. Theiss, A. L. et al. Prohibitin inhibits tumor necrosis factor α-induced nuclear factor-κB nuclear translocation via the novel mechanism of decreasing importin α3 expression. Mol. Biol. Cell 20, 4412–4423 (2009)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.).

Author information

Author notes

  1. Sergio E. Alvarez

    Present address: Present address: IMIBIO-SL CONICET, Ejército de los Andes 950, San Luis 5700, Argentina.,

  2. Sergio E. Alvarez and Kuzhuvelil B. Harikumar: These authors contributed equally to this work.

Authors and Affiliations

  1. 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,

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

  2. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

    Hualiang Jiang & Cheng Luo

Authors
  1. Sergio E. Alvarez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kuzhuvelil B. Harikumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nitai C. Hait
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jeremy Allegood
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Graham M. Strub
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eugene Y. Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Michael Maceyka
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hualiang Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Cheng Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Tomasz Kordula
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Sheldon Milstien
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Sarah Spiegel
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Sarah Spiegel.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1 and Supplementary Tables 1-10 w (PDF 4949 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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). https://doi.org/10.1038/nature09128

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09128

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing