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Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity

Nature Chemical Biology volume 8, pages 57–64 (2012)Cite this article

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Abstract

Protein sulfenylation is a post-translational modification of emerging importance in higher eukaryotes. However, investigation of its diverse roles remains challenging, particularly within a native cellular environment. Herein we report the development and application of DYn-2, a new chemoselective probe for detecting sulfenylated proteins in human cells. These studies show that epidermal growth factor receptor–mediated signaling results in H2O2 production and oxidation of downstream proteins. In addition, we demonstrate that DYn-2 has the ability to detect differences in sulfenylation rates within the cell, which are associated with differences in target protein localization. We also show that the direct modification of epidermal growth factor receptor by H2O2 at a critical active site cysteine (Cys797) enhances its tyrosine kinase activity. Collectively, our findings reveal sulfenylation as a global signaling mechanism that is akin to phosphorylation and has regulatory implications for other receptor tyrosine kinases and irreversible inhibitors that target oxidant-sensitive cysteines in proteins.

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Figure 1: Cellular redox status affects EGF-mediated signaling.
Figure 2: Development and validation of probes for detecting sulfenic acid.
Figure 3: EGF-mediated ROS production and protein sulfenylation.
Figure 4: Differential sulfenylation of PTPs in EGF-treated cells.
Figure 5: EGF-mediated sulfenylation of EGFR Cys797 in cells.
Figure 6: Model for redox regulation of EGFR signaling.

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References

  1. Finkel, T. Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7–15 (2011).

    Article  CAS  Google Scholar 

  2. Dickinson, B.C. & Chang, C.J. Chemistry and biology of reactive oxygen species in signaling or stress responses. Nat. Chem. Biol. 7, 504–511 (2011).

    Article  CAS  Google Scholar 

  3. Miller, E.W., Tulyathan, O., Isacoff, E.Y. & Chang, C.J. Molecular imaging of hydrogen peroxide produced for cell signaling. Nat. Chem. Biol. 3, 263–267 (2007); erratum 3, 349 (2007).

    Article  CAS  Google Scholar 

  4. Woo, H.A. et al. Inactivation of peroxiredoxin I by phosphorylation allows localized H2O2 accumulation for cell signaling. Cell 140, 517–528 (2010).

    Article  CAS  Google Scholar 

  5. Paulsen, C.E. & Carroll, K.S. Orchestrating redox signaling networks through regulatory cysteine switches. ACS Chem. Biol. 5, 47–62 (2010).

    Article  CAS  Google Scholar 

  6. Winterbourn, C.C. & Hampton, M.B. Thiol chemistry and specificity in redox signaling. Free Radic. Biol. Med. 45, 549–561 (2008).

    Article  CAS  Google Scholar 

  7. Reddie, K.G. & Carroll, K.S. Expanding the functional diversity of proteins through cysteine oxidation. Curr. Opin. Chem. Biol. 12, 746–754 (2008).

    Article  CAS  Google Scholar 

  8. Roos, G. & Messens, J. Protein sulfenic acid formation: from cellular damage to redox regulation. Free Radic. Biol. Med. 51, 314–326 (2011).

    Article  CAS  Google Scholar 

  9. Charles, R.L. et al. Protein sulfenation as a redox sensor: proteomics studies using a novel biotinylated dimedone analogue. Mol. Cell. Proteomics 6, 1473–1484 (2007).

    Article  CAS  Google Scholar 

  10. Depuydt, M. et al. A periplasmic reducing system protects single cysteine residues from oxidation. Science 326, 1109–1111 (2009).

    Article  CAS  Google Scholar 

  11. Leonard, S.E., Reddie, K.G. & Carroll, K.S. Mining the thiol proteome for sulfenic acid modifications reveals new targets for oxidation in cells. ACS Chem. Biol. 4, 783–799 (2009).

    Article  CAS  Google Scholar 

  12. Takanishi, C.L. & Wood, M.J. A genetically encoded probe for the identification of proteins that form sulfenic acid in response to H2O2 in Saccharomyces cerevisiae. J. Proteome. Res. 10, 2715–2724 (2011).

    Article  CAS  Google Scholar 

  13. Wani, R. et al. Isoform-specific regulation of Akt by PDGF-induced reactive oxygen species. Proc. Natl. Acad. Sci. USA 108, 10550–10555 (2011).

    Article  CAS  Google Scholar 

  14. Leonard, S.E. & Carroll, K.S. Chemical 'omics' approaches for understanding protein cysteine oxidation in biology. Curr. Opin. Chem. Biol. 15, 88–102 (2011).

    Article  CAS  Google Scholar 

  15. Bae, Y.S. et al. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation. J. Biol. Chem. 272, 217–221 (1997).

    Article  CAS  Google Scholar 

  16. Paulsen, C.E. & Carroll, K.S. Chemical dissection of an essential redox switch in yeast. Chem. Biol. 16, 217–225 (2009).

    Article  CAS  Google Scholar 

  17. Michalek, R.D. et al. The requirement of reversible cysteine sulfenic acid formation for T cell activation and function. J. Immunol. 179, 6456–6467 (2007).

    Article  CAS  Google Scholar 

  18. Benitez, L.V. & Allison, W.S. The inactivation of the acyl phosphatase activity catalyzed by the sulfenic acid form of glyceraldehyde 3-phosphate dehydrogenase by dimedone and olefins. J. Biol. Chem. 249, 6234–6243 (1974).

    CAS  PubMed  Google Scholar 

  19. Poole, L.B. et al. Fluorescent and affinity-based tools to detect cysteine sulfenic acid formation in proteins. Bioconjug. Chem. 18, 2004–2017 (2007).

    Article  CAS  Google Scholar 

  20. Reddie, K.G., Seo, Y.H., Muse, W.B. III, Leonard, S.E. & Carroll, K.S. A chemical approach for detecting sulfenic acid-modified proteins in living cells. Mol. Biosyst. 4, 521–531 (2008).

    Article  CAS  Google Scholar 

  21. Seo, Y.H. & Carroll, K.S. Profiling protein thiol oxidation in tumor cells using sulfenic acid-specific antibodies. Proc. Natl. Acad. Sci. USA 106, 16163–16168 (2009).

    Article  CAS  Google Scholar 

  22. Charron, G. et al. Robust fluorescent detection of protein fatty-acylation with chemical reporters. J. Am. Chem. Soc. 131, 4967–4975 (2009).

    Article  CAS  Google Scholar 

  23. Bedard, K. & Krause, K.H. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87, 245–313 (2007).

    Article  CAS  Google Scholar 

  24. Lee, S.R., Kwon, K.S., Kim, S.R. & Rhee, S.G. Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor. J. Biol. Chem. 273, 15366–15372 (1998).

    Article  CAS  Google Scholar 

  25. Meng, T.C., Fukada, T. & Tonks, N.K. Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol. Cell 9, 387–399 (2002).

    Article  CAS  Google Scholar 

  26. Tanner, J.J., Parsons, Z.D., Cummings, A.H., Zhou, H. & Gates, K.S. Redox regulation of protein tyrosine phosphatases: structural and chemical aspects. Antioxid. Redox Signal. 15, 77–97 (2011).

    Article  CAS  Google Scholar 

  27. Singh, J., Petter, R.C., Baillie, T.A. & Whitty, A. The resurgence of covalent drugs. Nat. Rev. Drug Discov. 10, 307–317 (2011).

    Article  CAS  Google Scholar 

  28. Singh, J., Petter, R.C. & Kluge, A.F. Targeted covalent drugs of the kinase family. Curr. Opin. Chem. Biol. 14, 475–480 (2010).

    Article  CAS  Google Scholar 

  29. Go, Y.M. & Jones, D.P. Redox compartmentalization in eukaryotic cells. Biochim. Biophys. Acta 1780, 1273–1290 (2008).

    Article  CAS  Google Scholar 

  30. Nelson, K.J. et al. Use of dimedone-based chemical probes for sulfenic acid detection methods to visualize and identify labeled proteins. Methods Enzymol. 473, 95–115 (2010).

    Article  CAS  Google Scholar 

  31. Cuddihy, S.L., Winterbourn, C.C. & Hampton, M.B. Assessment of redox changes to hydrogen peroxide-sensitive proteins during EGF signaling. Antioxid. Redox Signal. 15, 167–174 (2011).

    Article  CAS  Google Scholar 

  32. Chen, C.Y., Willard, D. & Rudolph, J. Redox regulation of SH2-domain-containing protein tyrosine phosphatases by two backdoor cysteines. Biochemistry 48, 1399–1409 (2009).

    Article  CAS  Google Scholar 

  33. Denu, J.M. & Tanner, K.G. Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry 37, 5633–5642 (1998).

    Article  CAS  Google Scholar 

  34. Chen, K., Kirber, M.T., Xiao, H., Yang, Y. & Keaney, J.F. Jr. Regulation of ROS signal transduction by NADPH oxidase 4 localization. J. Cell Biol. 181, 1129–1139 (2008).

    Article  CAS  Google Scholar 

  35. Lee, C. et al. Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nat. Struct. Mol. Biol. 11, 1179–1185 (2004).

    Article  CAS  Google Scholar 

  36. Lee, J.W., Soonsanga, S. & Helmann, J.D. A complex thiolate switch regulates the Bacillus subtilis organic peroxide sensor OhrR. Proc. Natl. Acad. Sci. USA 104, 8743–8748 (2007).

    Article  CAS  Google Scholar 

  37. Giannoni, E., Buricchi, F., Raugei, G., Ramponi, G. & Chiarugi, P. Intracellular reactive oxygen species activate Src tyrosine kinase during cell adhesion and anchorage-dependent cell growth. Mol. Cell. Biol. 25, 6391–6403 (2005).

    Article  CAS  Google Scholar 

  38. Tang, H., Hao, Q., Rutherford, S.A., Low, B. & Zhao, Z.J. Inactivation of SRC family tyrosine kinases by reactive oxygen species in vivo. J. Biol. Chem. 280, 23918–23925 (2005).

    Article  CAS  Google Scholar 

  39. Cunnick, J.M. et al. Role of tyrosine kinase activity of epidermal growth factor receptor in the lysophosphatidic acid-stimulated mitogen-activated protein kinase pathway. J. Biol. Chem. 273, 14468–14475 (1998).

    Article  CAS  Google Scholar 

  40. Kemble, D.J. & Sun, G. Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation. Proc. Natl. Acad. Sci. USA 106, 5070–5075 (2009).

    Article  CAS  Google Scholar 

  41. Leonard, S.E., Garcia, F.J., Goodsell, D.S. & Carroll, K.S. Redox-based probes for protein tyrosine phosphatases. Angew. Chem. Int. Edn Engl. 50, 4423–4427 (2011).

    Article  CAS  Google Scholar 

  42. Dickinson, B.C., Huynh, C. & Chang, C.J. A palette of fluorescent probes with varying emission colors for imaging hydrogen peroxide signaling in living cells. J. Am. Chem. Soc. 132, 5906–5915 (2010).

    Article  CAS  Google Scholar 

  43. Dickinson, B.C., Peltier, J., Stone, D., Schaffer, D.V. & Chang, C.J. Nox2 redox signaling maintains essential cell populations in the brain. Nat. Chem. Biol. 7, 106–112 (2011).

    Article  CAS  Google Scholar 

  44. Truong, T.H., Garcia, F.J., Seo, Y.H. & Carroll, K.S. Isotope-coded chemical reporter and acid-cleavable affinity reagents for monitoring protein sulfenic acids. Bioorg. Med. Chem. Lett. 21, 5015–5020 (2011).

    Article  CAS  Google Scholar 

  45. Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010).

    Article  CAS  Google Scholar 

  46. Seo, Y.H. & Carroll, K.S. Quantification of protein sulfenic acid modifications using isotope-coded dimedone and iododimedone. Angew. Chem. Int. Edn Engl. 50, 1342–1345 (2011).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors acknowledge funding from the Camille Henry Dreyfus Teacher Scholar Award (to K.S.C.) and the American Heart Association Scientist Development Award (0835419N to K.S.C.). The authors also wish to thank R. Petter for helpful discussions.

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Authors and Affiliations

  1. Chemical Biology Graduate Program, University of Michigan, Ann Arbor, Michigan, USA

    Candice E Paulsen & Stephen E Leonard

  2. Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA

    Thu H Truong

  3. Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, USA

    Francisco J Garcia, Arne Homann, Vinayak Gupta & Kate S Carroll

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  1. Candice E Paulsen
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Contributions

C.E.P., T.H.T. and A.H. performed cell culture and immunostaining experiments. F.J.G. performed mass spectrometry experiments. V.G. and S.E.L. performed synthetic experiments. K.S.C. designed experimental strategies with help from C.E.P. and V.G. K.S.C. and C.E.P. wrote the paper with input from all coauthors.

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Correspondence to Kate S Carroll.

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Paulsen, C., Truong, T., Garcia, F. et al. Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity. Nat Chem Biol 8, 57–64 (2012). https://doi.org/10.1038/nchembio.736

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