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RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation

Nature Cell Biology volume 10, pages 538546 (2008) | Download Citation

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Abstract

In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor α (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.

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References

  1. 1.

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

  2. 2.

    , & SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J. Biol. Chem. 276, 12654–12659 (2001).

  3. 3.

    et al. Polymeric chains of sumo-2 and sumo-3 are conjugated to protein substrates by sae1/sae2 and ubc9. J. Biol. Chem. 276, 35368–35374 (2001).

  4. 4.

    et al. In vivo identification of human SUMO polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy. Mol. Cell Proteomics 1 ,132–144 (2007).

  5. 5.

    , , , & Specification of SUMO1 and SUMO2 interacting motifs. J. Biol. Chem. 23, 16117–16127 (2006).

  6. 6.

    , , , . & Identification of a SUMO-binding motif that recognizes SUMO-modified proteins. Proc. Natl Acad. Sci. USA 101, 14373–14378 (2004).

  7. 7.

    et al. SUMO-targeted ubiquitin ligases in genome stability. EMBO J. 26, 4089–101 (2007).

  8. 8.

    , & Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins. EMBO J. 26, 4102–4112 (2007).

  9. 9.

    et al. Ubiquitin-dependent proteolytic control of SUMO conjugates. J. Biol. Chem. 47, 34167–34175 (2007).

  10. 10.

    et al. The yeast HEX3–SLX8 heterodimer is a ubiquitin ligase stimulated by substrate sumoylation. J. Biol. Chem. 47, 34176–34184 (2007).

  11. 11.

    et al. Identification of a novel RING finger protein as a co-regulator in steroid receptor-mediated gene transcription. Mol. Cell Biol. 18, 5128–5139 (1998).

  12. 12.

    , , , & Transcriptional co-regulator SNURF (RNF4) possesses ubiquitin E3 ligase activity. FEBS Lett. 560, 56–62 (2004).

  13. 13.

    , & Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC). J. Proteome Res. 2, 173–181 (2003).

  14. 14.

    et al. In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML–RAR α/PML proteins. Blood 88, 1052–1061 (1996).

  15. 15.

    et al. Role of promyelocytic leukemia (PML) sumolation in nuclear body formation, 11S proteasome recruitment, and As2O3-induced PML or PML/retinoic acid receptor alpha degradation. J. Exp. Med. 193, 1361–1371 (2001).

  16. 16.

    , & Trivalent antimonials induce degradation of the PML–RAR oncoprotein and reorganization of the promyelocytic leukemia nuclear bodies in acute promyelocytic leukemia NB4 cells. Blood 92, 4308–4316 (1998).

  17. 17.

    et al. Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR α protein in acute promyelocytic leukemia cells. J. Natl Cancer Inst. 90, 124–133 (1998).

  18. 18.

    et al. PIC-1/SUMO-1-modified PML–retinoic acid receptor α mediates arsenic trioxide-induced apoptosis in acute promyelocytic leukemia. Mol. Cell Biol. 19, 5170–5178 (1999).

  19. 19.

    et al. Arsenic-induced PML targeting onto nuclear bodies: implications for the treatment of acute promyelocytic leukemia. Proc. Natl Acad. Sci. USA 94, 3978–3983 (1997).

  20. 20.

    et al. SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation. J. Cell Sci. 112, 381–393 (1999).

  21. 21.

    et al. Identification of three major sentrinization sites in PML. J. Biol. Chem. 273, 26675–26682 (1998).

  22. 22.

    C., , , & The slx5–slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination. Mol. Cell Biol. 27, 6153–6162 (2007).

  23. 23.

    , , & Fission yeast Rnf4 homologs are required for DNA repair. J. Biol. Chem. 282, 20388–20394 (2007).

  24. 24.

    , & Genetic analysis connects SLX5 and SLX8 to the SUMO pathway in Saccharomyces cerevisiae. Genetics 172, 1499–1509 (2006).

  25. 25.

    , , & SUMO-1 promotes association of SNURF (RNF4) with PML nuclear bodies. Exp. Cell Res. 304, 224–233 (2005).

  26. 26.

    , , & An improved PCR-based method for site directed mutagenesis using megaprimers. Mol. Cell Probes 12, 345–348 (1998).

  27. 27.

    et al. SUMO protease SENP1 induces isomerization of the scissile peptide bond. Nature Struct. Mol. Biol. 13, 1069–1077 (2006).

  28. 28.

    , & Ubch9 conjugates SUMO but not ubiquitin. FEBS Lett. 417, 297–300 (1997).

  29. 29.

    , , , & The structure of SENP1–SUMO-2 complex suggests a structural basis for discrimination between SUMO paralogues during processing. Biochem. J. 397, 279–288 (2006).

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Acknowledgements

This work was supported by Cancer Research UK and the RUBICON EU Network of Excellence. A. P was supported by a Wellcome Trust Studentship. We would like to thank Hugues de The for helpful discussions and providing the chicken anti-PML antibody, and Douglas Lamont, manager of the Fingerprints Proteomics Facility, University of Dundee, for generating the Orbitrap mass spectrometry data. The provision of critical reagents by Roel van Driel and Dan Bailey is gratefully acknowledged.

Author information

Author notes

    • Michael H. Tatham
    • , Marie-Claude Geoffroy
    •  & Linnan Shen

    These authors made an equal contribution to the work.

Affiliations

  1. Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

    • Michael H. Tatham
    • , Marie-Claude Geoffroy
    • , Linnan Shen
    • , Anna Plechanovova
    • , Neil Hattersley
    • , Ellis G. Jaffray
    •  & Ronald T. Hay
  2. Department of Medical Biochemistry, University of Kuopio, P.O. Box 1627, FI-70211, Kuopio, Finland.

    • Jorma J. Palvimo

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Contributions

M. H. T. carried out the biochemical and proteomic analyses and participated in the writing of the manuscript; M. C. G. carried out the in vivo analysis of PML degradation; L. S. generated and assayed expressed proteins and mutants of RNF4; A. P. carried out ubiquitin site-mapping on poly-SUMO chains modified in vitro; N. H. was involved in initial immunofluorescence microscopy studies; E. J. G. established conditions for siRNA depletion of RNF4; J. J. P. generated expression constructs and antibodies to RNF4; R. T. H. conceived the project and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Ronald T. Hay.

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https://doi.org/10.1038/ncb1716