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Site-specific incorporation of phosphotyrosine using an expanded genetic code


Access to phosphoproteins with stoichiometric and site-specific phosphorylation status is key to understanding the role of protein phosphorylation. Here we report an efficient method to generate pure, active phosphotyrosine-containing proteins by genetically encoding a stable phosphotyrosine analog that is convertible to native phosphotyrosine. We demonstrate its general compatibility with proteins of various sizes, phosphotyrosine sites and functions, and reveal a possible role of tyrosine phosphorylation in negative regulation of ubiquitination.

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Figure 1: Site-specific incorporation of phosphotyrosine into proteins.
Figure 2: Phosphorylation of Tyr59 in Ub impacts its conformation and function.


  1. Hunter, T. Curr. Opin. Cell Biol. 21, 140–146 (2009).

    CAS  Article  Google Scholar 

  2. Tarrant, M.K. & Cole, P.A. Annu. Rev. Biochem. 78, 797–825 (2009).

    CAS  Article  Google Scholar 

  3. Lim, W.A. & Pawson, T. Cell 142, 661–667 (2010).

    CAS  Article  Google Scholar 

  4. Julien, S.G., Dubé, N., Hardy, S. & Tremblay, M.L. Nat. Rev. Cancer 11, 35–49 (2011).

    CAS  Article  Google Scholar 

  5. Blume-Jensen, P. & Hunter, T. Nature 411, 355–365 (2001).

    CAS  Article  Google Scholar 

  6. Tan, C.S. et al. Sci. Signal. 2, ra39 (2009).

    Article  Google Scholar 

  7. Humphrey, S.J., James, D.E. & Mann, M. Trends Endocrinol. Metab. 26, 676–687 (2015).

    CAS  Article  Google Scholar 

  8. Hornbeck, P.V. et al. Nucleic Acids Res. 40, D261–D270 (2012).

    CAS  Article  Google Scholar 

  9. Ottesen, J.J., Huse, M., Sekedat, M.D. & Muir, T.W. Biochemistry 43, 5698–5706 (2004).

    CAS  Article  Google Scholar 

  10. Oza, J.P. et al. Nat. Commun. 6, 8168 (2015).

    Article  Google Scholar 

  11. Jbara, M., Maity, S.K., Morgan, M., Wolberger, C. & Brik, A. Angew. Chem. Int. Edn. Engl. 55, 4972–4976 (2016).

    CAS  Article  Google Scholar 

  12. Serwa, R. et al. Angew. Chem. Int. Edn. Engl. 48, 8234–8239 (2009).

    CAS  Article  Google Scholar 

  13. Chen, Z. & Cole, P.A. Curr. Opin. Chem. Biol. 28, 115–122 (2015).

    CAS  Article  Google Scholar 

  14. Wang, L., Brock, A., Herberich, B. & Schultz, P.G. Science 292, 498–500 (2001).

    CAS  Article  Google Scholar 

  15. Xie, J., Supekova, L. & Schultz, P.G. ACS Chem. Biol. 2, 474–478 (2007).

    CAS  Article  Google Scholar 

  16. Park, H.S. et al. Science 333, 1151–1154 (2011).

    CAS  Article  Google Scholar 

  17. Pirman, N.L. et al. Nat. Commun. 6, 8130 (2015).

    Article  Google Scholar 

  18. George, S. et al. FEBS Lett. 590, 1530–1542 (2016).

    CAS  Article  Google Scholar 

  19. Fan, C., Ip, K. & Söll, D. FEBS Lett. 590, 3040–3047 (2016).

    CAS  Article  Google Scholar 

  20. Rogerson, D.T. et al. Nat. Chem. Biol. 11, 496–503 (2015).

    CAS  Article  Google Scholar 

  21. Chao, H.-G. et al. J. Org. Chem. 60, 7710–7711 (1995).

    CAS  Article  Google Scholar 

  22. Takimoto, J.K., Dellas, N., Noel, J.P. & Wang, L. ACS Chem. Biol. 6, 733–743 (2011).

    CAS  Article  Google Scholar 

  23. Hoppmann, C. et al. Angew. Chem. Int. Edn. Engl. 53, 3932–3936 (2014).

    CAS  Article  Google Scholar 

  24. Lacey, V.K., Louie, G.V., Noel, J.P. & Wang, L. ChemBioChem 14, 2100–2105 (2013).

    CAS  Article  Google Scholar 

  25. Yau, R. & Rape, M. Nat. Cell Biol. 18, 579–586 (2016).

    CAS  Article  Google Scholar 

  26. Wauer, T. et al. EMBO J. 34, 307–325 (2015).

    CAS  Article  Google Scholar 

  27. Chong, R.A. et al. Proc. Natl. Acad. Sci. USA 111, 8434–8439 (2014).

    CAS  Article  Google Scholar 

  28. Johnson, D.B. et al. Nat. Chem. Biol. 7, 779–786 (2011).

    CAS  Article  Google Scholar 

  29. Xiang, Z. et al. Nat. Methods 10, 885–888 (2013).

    CAS  Article  Google Scholar 

  30. Santoro, S.W., Wang, L., Herberich, B., King, D.S. & Schultz, P.G. Nat. Biotechnol. 20, 1044–1048 (2002).

    CAS  Article  Google Scholar 

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We thank M. Kelly (UCSF NMR Core) for help with NMR measurements and helpful discussions. L.W. acknowledges the support of the NIH (R01GM118384).

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Authors and Affiliations



C.H. conducted experiments and characterized data; A.W. assigned data for NMR; B.Y. prepared UBE2D3; S.L., T.H., and K.M.S. provided helpful discussions; L.W. conceived and directed the project; and C.H. and L.W. wrote the manuscript with inputs from S.L., T.H., and K.M.S.

Corresponding author

Correspondence to Lei Wang.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Table 1, Supplementary Figures 1–12 and Supplementary Note 2 (PDF 2942 kb)

Supplementary Note 1

Synthesis and characterization of Uaa 1 including experimental procedure, NMR and MS spectra. (PDF 267 kb)

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Hoppmann, C., Wong, A., Yang, B. et al. Site-specific incorporation of phosphotyrosine using an expanded genetic code. Nat Chem Biol 13, 842–844 (2017).

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