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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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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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  2. Shelly M. Wuerzberger-Davis
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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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