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PIASy mediates NEMO sumoylation and NF-κB activation in response to genotoxic stress

Nature Cell Biology volume 8, pages 986–993 (2006)Cite this article

Abstract

Protein modification by SUMO (small ubiquitin-like modifier) is an important regulatory mechanism for multiple cellular processes1,2. SUMO-1 modification of NEMO (NF-κB essential modulator), the IκB kinase (IKK) regulatory subunit, is critical for activation of NF-κB by genotoxic agents3. However, the SUMO ligase, and the mechanisms involved in NEMO sumoylation, remain unknown. Here, we demonstrate that although small interfering RNAs (siRNAs) against PIASy (protein inhibitor of activated STATy) inhibit NEMO sumoylation and NF-κB activation in response to genotoxic agents, overexpression of PIASy enhances these events. PIASy preferentially stimulates site-selective modification of NEMO by SUMO-1, but not SUMO-2 and SUMO–3, in vitro. PIASy–NEMO interaction is increased by genotoxic stress and occurs in the nucleus in a manner mutually exclusive with IKK interaction. In addition, hydrogen peroxide (H2O2) also increases PIASy–NEMO interaction and NEMO sumoylation, whereas antioxidants prevent these events induced by DNA-damaging agents. Our findings demonstrate that PIASy is the first SUMO ligase for NEMO whose substrate specificity seems to be controlled by IKK interaction, subcellular targeting and oxidative stress conditions.

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Figure 1: PIASy is necessary for NF-κB activation by genotoxic agents.
Figure 2: PIASy modulates NEMO sumoylation and NF-κB activation in response to genotoxic stress.
Figure 3: PIASy promotes sumoylation of NEMO in vitro.
Figure 4: NEMO interacts with PIASy in a manner that is mutally exclusive with IKKβ binding.
Figure 5: Oxidative stress seems to be required for NEMO sumoylation in response to VP16.

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References

  1. Gill, G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms? Genes Dev. 18, 2046–2059 (2004).

    Article  CAS  Google Scholar 

  2. Hay, R. T. SUMO: a history of modification. Mol. Cell 18, 1–12 (2005).

    Article  CAS  Google Scholar 

  3. Huang, T. T., Wuerzberger-Davis, S. M., Wu, Z. H. & Miyamoto, S. Sequential modification of NEMO/IKKγ by SUMO-1 and ubiquitin mediates NF-κB activation by genotoxic stress. Cell 115, 565–576 (2003).

    Article  CAS  Google Scholar 

  4. Wu, Z. H., Shi, Y., Tibbetts, R. S. & Miyamoto, S. Molecular linkage between the kinase ATM and NF-κB signaling in response to genotoxic stimuli. Science 311, 1141–1146 (2006).

    Article  CAS  Google Scholar 

  5. Pichler, A., Gast, A., Seeler, J.S., Dejean, A. & Melchior, F. The nucleoporin RanBP2 has SUMO1 E3 ligase activity. Cell 108, 109–120 (2002).

    Article  CAS  Google Scholar 

  6. Wuerzberger-Davis, S. M., Chang, P. Y., Berchtold, C. & Miyamoto, S. Enhanced G2–M arrest by NF-κB-dependent p21waf1/cip1 induction. Mol. Cancer Res. 3, 345–353 (2005).

    Article  CAS  Google Scholar 

  7. Bakkenist, C. J. & Kastan, M. B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506 (2003).

    Article  CAS  Google Scholar 

  8. Hochstrasser, M. SP-RING for SUMO: new functions bloom for a ubiquitin-like protein. Cell 107, 5–8 (2001).

    Article  CAS  Google Scholar 

  9. Azuma, Y., Arnaoutov, A., Anan, T. & Dasso, M. PIASy mediates SUMO-2 conjugation of Topoisomerase-II on mitotic chromosomes. EMBO J. 24, 2172–2182 (2005).

    Article  CAS  Google Scholar 

  10. Sampson, D. A., Wang, M. & Matunis, M. J. The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. J. Biol. Chem. 276, 21664–21669 (2001).

    Article  CAS  Google Scholar 

  11. Lin, D. et al. Identification of a substrate recognition site on Ubc9. J. Biol. Chem. 277, 21740–21748 (2002).

    Article  CAS  Google Scholar 

  12. Hayden, M. S. & Ghosh, S. Signaling to NF-κB. Genes Dev. 18, 2195–2224 (2004).

    Article  CAS  Google Scholar 

  13. Riley, P.A. Free radicals in biology: oxidative stress and the effects of ionizing radiation. Int. J. Radiat. Biol. 65, 27–33 (1994).

    Article  CAS  Google Scholar 

  14. Pham, N.A. & Hedley, D.W. Respiratory chain-generated oxidative stress following treatment of leukemic blasts with DNA-damaging agents. Exp. Cell Res. 264, 345–352 (2001).

    Article  CAS  Google Scholar 

  15. Mikkelsen, R.B. & Wardman, P. Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms. Oncogene 22, 5734–5754 (2003).

    Article  CAS  Google Scholar 

  16. England, K., O'Driscoll, C. & Cotter, T.G. Carbonylation of glycolytic proteins is a key response to drug-induced oxidative stress and apoptosis. Cell Death Differ. 11, 252–260 (2004).

    Article  CAS  Google Scholar 

  17. Brummelkamp, T. R., Nijman, S. M., Dirac, A. M. & Bernards, R. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB. Nature 424, 797–801 (2003).

    Article  CAS  Google Scholar 

  18. Liu, B. et al. Negative regulation of NF-κB signaling by PIAS1. Mol. Cell Biol. 25, 1113–1123 (2005).

    Article  CAS  Google Scholar 

  19. Jang, H. D., Yoon, K., Shin, Y. J., Kim, J. & Lee, S. Y. PIAS3 suppresses NF-κB-mediated transcription by interacting with the p65/RelA subunit. J. Biol. Chem. 279, 24873–24880 (2004).

    Article  CAS  Google Scholar 

  20. Janssens, S., Tinel, A., Lippens, S. & Tschopp, J. PIDD mediates NF-κB activation in response to DNA damage. Cell 123, 1079–1092 (2005).

    Article  CAS  Google Scholar 

  21. Karin, M. & Greten, F. R. NF-κB: linking inflammation and immunity to cancer development and progression. Nature Rev. Immunol. 5, 749–759 (2005).

    Article  CAS  Google Scholar 

  22. Aggarwal, B. B. Nuclear factor-κB: the enemy within. Cancer Cell 6, 203–208 (2004).

    Article  CAS  Google Scholar 

  23. Huang, T. T. et al. NF-κB activation by camptothecin. A linkage between nuclear DNA damage and cytoplasmic signaling events. J. Biol. Chem. 275, 9501–9509 (2000).

    Article  CAS  Google Scholar 

  24. Huang, T. T., Feinberg, S. L., Suryanarayanan, S. & Miyamoto, S. The zinc finger domain of NEMO is selectively required for NF-κB activation by UV radiation and topoisomerase inhibitors. Mol. Cell Biol. 22, 5813–5825 (2002).

    Article  CAS  Google Scholar 

  25. Miyamoto, S., Seufzer, B. J. & Shumway, S. D. Novel IκBα proteolytic pathway in WEHI231 immature B cells. Mol. Cell Biol. 18, 19–29 (1998).

    Article  CAS  Google Scholar 

  26. O'Connor, S., Shumway, S. D., Amanna, I. J., Hayes, C. E. & Miyamoto, S. Regulation of constitutive p50/c-Rel activity via proteasome inhibitor-resistant IκBα degradation in B cells. Mol. Cell Biol. 24, 4895–4908 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Y. Azuma for generously providing recombinant purified Xenopus His–PIASy protein and Xenopus pET28a PIASy construct. We thank both M. Dasso and Y. Azuma for providing human PIASy antibody; K. Orth and S. Mukherjee for technical assistance and discussions regarding the development of in vitro sumoylation assays; E. Bresnick for the use of real time PCR equipment; P.-Y. Chang for assistance with quantitative RT–PCR analyses; S. Suryanarayanan and S. Shumway for generation of some NEMO deletion mutants. We also thank S. O'Connor for critical reading of the manuscript, C Berchtold for help with statistical analysis and the members of the Miyamoto lab for helpful discussions. This work is funded by the National Institutes of Health (NIH; T32GM008688) and Department of Defense BC044529 to A.M., Department of Defense BC010767 to S.W.-D., and NIH R01CA77474 and R01CA81065 and a Shaw Scientist Award from the Greater Milwaukee Foundation to S.M.

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  1. Department of Pharmacology, Program in Molecular and Cellular Pharmacology, University of Wisconsin, Madison, 53706, WI, USA

    Angela M. Mabb, Shelly M. Wuerzberger-Davis & Shigeki Miyamoto

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  1. Angela M. Mabb
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  3. Shigeki Miyamoto
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Correspondence to Shigeki Miyamoto.

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Mabb, A., Wuerzberger-Davis, S. & Miyamoto, S. PIASy mediates NEMO sumoylation and NF-κB activation in response to genotoxic stress. Nat Cell Biol 8, 986–993 (2006). https://doi.org/10.1038/ncb1458

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