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Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function

Nature Structural & Molecular Biology volume 20, pages 46–52 (2013)Cite this article

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Abstract

Cell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity, leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitination of Ras leads to Ras activation. Mutation of the primary site of monoubiquitination impairs the ability of activated K-Ras (one of the three mammalian isoforms of Ras) to promote tumor growth. To determine the mechanism of human Ras activation, we chemically ubiquitinated the protein and analyzed its function by NMR, computational modeling and biochemical activity measurements. We established that monoubiquitination has little effect on the binding of Ras to guanine nucleotide, GTP hydrolysis or exchange-factor activation but severely abrogates the response to GTPase-activating proteins in a site-specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation.

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Figure 1: Monoubiquitination of Ras.
Figure 2: mUbRas retains intrinsic stability and activity.
Figure 3: Rosetta model of native and chemical ubiquitination of Ras.
Figure 4: Surfaces of Ras and ubiquitin affected by monoubiquitination.
Figure 5: Monoubiquitination decreases the sensitivity of Ras to downregulation by GAPs.
Figure 6: Modification of Ras with PDZ2 resembles modification with ubiquitin.
Figure 7: The impaired GAP sensitivity of mUbRas is site specific.

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References

  1. Cox, A.D. & Der, C.J. The dark side of Ras: regulation of apoptosis. Oncogene 22, 8999–9006 (2003).

    Article  CAS  Google Scholar 

  2. Kjeldgaard, M., Nyborg, J. & Clark, B.F. The GTP binding motif: variations on a theme. FASEB J. 10, 1347–1368 (1996).

    Article  CAS  Google Scholar 

  3. Herrmann, C. Ras-effector interactions: after one decade. Curr. Opin. Struct. Biol. 13, 122–129 (2003).

    Article  CAS  Google Scholar 

  4. Scheffzek, K. et al. The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277, 333–339 (1997).

    Article  CAS  Google Scholar 

  5. Sprang, S. GEFs: master regulators of G-protein activation. Trends Biochem. Sci. 26, 266–267 (2001).

    Article  CAS  Google Scholar 

  6. Sprang, S.R. G proteins, effectors and GAPs: structure and mechanism. Curr. Opin. Struct. Biol. 7, 849–856 (1997).

    Article  CAS  Google Scholar 

  7. Bos, J.L. Ras oncogenes in human cancer: a review. Cancer Res. 49, 4682–4689 (1989).

    CAS  PubMed  Google Scholar 

  8. Adjei, A.A. Blocking oncogenic Ras signaling for cancer therapy. J. Natl. Cancer Inst. 93, 1062–1074 (2001).

    Article  CAS  Google Scholar 

  9. Karnoub, A.E. & Weinberg, R.A. Ras oncogenes: split personalities. Nat. Rev. Mol. Cell Biol. 9, 517–531 (2008).

    Article  CAS  Google Scholar 

  10. Hicke, L. Protein regulation by monoubiquitin. Nat. Rev. Mol. Cell Biol. 2, 195–201 (2001).

    Article  CAS  Google Scholar 

  11. Jura, N., Scotto-Lavino, E., Sobczyk, A. & Bar-Sagi, D. Differential modification of Ras proteins by ubiquitination. Mol. Cell 21, 679–687 (2006).

    Article  CAS  Google Scholar 

  12. Kawabe, H. et al. Regulation of Rap2A by the ubiquitin ligase Nedd4-1 controls neurite development. Neuron 65, 358–372 (2010).

    Article  CAS  Google Scholar 

  13. Nethe, M. & Hordijk, P.L. The role of ubiquitylation and degradation in RhoGTPase signalling. J. Cell Sci. 123, 4011–4018 (2010).

    Article  CAS  Google Scholar 

  14. Visvikis, O. et al. Activated Rac1, but not the tumorigenic variant Rac1b, is ubiquitinated on Lys 147 through a JNK-regulated process. FEBS J. 275, 386–396 (2008).

    Article  CAS  Google Scholar 

  15. Sasaki, A.T. et al. Ubiquitination of K-Ras enhances activation and facilitates binding to select downstream effectors. Sci. Signal. 4, ra13 (2011).

    Article  Google Scholar 

  16. Merkley, N., Barber, K.R. & Shaw, G.S. Ubiquitin manipulation by an E2 conjugating enzyme using a novel covalent intermediate. J. Biol. Chem. 280, 31732–31738 (2005).

    Article  CAS  Google Scholar 

  17. Chen, J., Ai, Y., Wang, J., Haracska, L. & Zhuang, Z. Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis. Nat. Chem. Biol. 6, 270–272 (2010).

    Article  CAS  Google Scholar 

  18. Virdee, S. et al. Traceless and site-specific ubiquitination of recombinant proteins. J. Am. Chem. Soc. 133, 10708–10711 (2011).

    Article  CAS  Google Scholar 

  19. Kumar, K.S., Spasser, L., Ohayon, S., Erlich, L.A. & Brik, A. Expeditious chemical synthesis of ubiquitinated peptides employing orthogonal protection and native chemical ligation. Bioconjug. Chem. 22, 137–143 (2011).

    Article  CAS  Google Scholar 

  20. Eger, S. et al. Generation of a mono-ubiquitinated PCNA mimic by click chemistry. ChemBioChem 12, 2807–2812 (2011).

    Article  CAS  Google Scholar 

  21. Purbeck, C., Eletr, Z.M. & Kuhlman, B. Kinetics of the transfer of ubiquitin from UbcH7 to E6AP. Biochemistry 49, 1361–1363 (2010).

    Article  CAS  Google Scholar 

  22. Mott, H.R., Carpenter, J.W. & Campbell, S.L. Structural and functional analysis of a mutant Ras protein that is insensitive to nitric oxide activation. Biochemistry 36, 3640–3644 (1997).

    Article  CAS  Google Scholar 

  23. Reuther, G.W. & Der, C.J. The Ras branch of small GTPases: Ras family members don't fall far from the tree. Curr. Opin. Cell Biol. 12, 157–165 (2000).

    Article  CAS  Google Scholar 

  24. Takai, Y., Sasaki, T. & Matozaki, T. Small GTP-binding proteins. Physiol. Rev. 81, 153–208 (2001).

    Article  CAS  Google Scholar 

  25. Wohlgemuth, S. et al. Recognizing and defining true Ras binding domains I: biochemical analysis. J. Mol. Biol. 348, 741–758 (2005).

    Article  CAS  Google Scholar 

  26. Campbell-Burka, S.L., Domaillea, P., Starovasnik, M., Boucher, W. & Laue, E. Sequential assignment of the backbone nuclei of c-H-Ras(1–166) GDP using a novel 4D NMR strategy. J. Biomol. NMR 2, 639–646 (1992).

    Article  Google Scholar 

  27. Hagai, T. & Levy, Y. Ubiquitin not only serves as a tag but also assists degradation by inducing protein unfolding. Proc. Natl. Acad. Sci. USA 107, 2001–2006 (2010).

    Article  CAS  Google Scholar 

  28. Isom, D.G., Marguet, P.R., Oas, T.G. & Hellinga, H.W. A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity. Proteins 79, 1034–1047 (2011).

    Article  CAS  Google Scholar 

  29. Janakiraman, M. et al. Genomic and biological characterization of exon 4 KRAS mutations in human cancer. Cancer Res. 70, 5901–5911 (2010).

    Article  CAS  Google Scholar 

  30. Feig, L.A. & Cooper, G.M. Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated Ras proteins. Mol. Cell. Biol. 8, 2472–2478 (1988).

    Article  CAS  Google Scholar 

  31. Gu, H. et al. A novel analytical method for in vivo phosphate tracking. FEBS Lett. 580, 5885–5893 (2006).

    Article  CAS  Google Scholar 

  32. Saha, A., Lewis, S., Kleiger, G., Kuhlman, B. & Deshaies, R.J. Essential role for ubiquitin-ubiquitin–conjugating enzyme interaction in ubiquitin discharge from Cdc34 to substrate. Mol. Cell 42, 75–83 (2011).

    Article  CAS  Google Scholar 

  33. Leaver-Fay, A. et al. Rosetta3: an object-oriented software suite for the simulation and design of macromolecules. In Methods in Enzymology Vol. 487 (eds. Johnson, M.L. & Brand, L.) Ch. 19, 545–574 (Academic Press, 2011).

  34. Ito, Y. et al. Regional polysterism in the GTP-bound form of the human c-Ha-Ras protein. Biochemistry 36, 9109–9119 (1997).

    Article  CAS  Google Scholar 

  35. Sondermann, H. et al. Structural analysis of autoinhibition in the Ras activator Son of sevenless. Cell 119, 393–405 (2004).

    Article  CAS  Google Scholar 

  36. Scheffzek, K. et al. Structural analysis of the GAP-related domain from neurofibromin and its implications. EMBO J. 17, 4313–4327 (1998).

    Article  CAS  Google Scholar 

  37. Kozlov, G., Gehring, K. & Ekiel, I. Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor. Biochemistry 39, 2572–2580 (2000).

    Article  CAS  Google Scholar 

  38. Wennerberg, K., Rossman, K.L. & Der, C.J. The Ras superfamily at a glance. J. Cell Sci. 118, 843–846 (2005).

    Article  CAS  Google Scholar 

  39. Edkins, S. et al. Recurrent KRAS Codon 146 mutations in human colorectal cancer. Cancer Biol. Ther. 5, 928–932 (2006).

    Article  CAS  Google Scholar 

  40. Weber, P.L., Brown, S.C. & Mueller, L. Sequential proton NMR assignments and secondary structure identification of human ubiquitin. Biochemistry 26, 7282–7290 (1987).

    Article  CAS  Google Scholar 

  41. Lenzen, C., Cool, R.H. & Wittinghofer, A. Analysis of intrinsic and CDC25-stimulated guanine nucleotide exchange of p21ras-nucleotide complexes by fluorescence measurements. Methods Enzymol. 255, 95–109 (1995).

    Article  CAS  Google Scholar 

  42. Lenzen, C., Cool, R.H., Prinz, H., Kuhlmann, J. & Wittinghofer, A. Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide exchange factor Cdc25Mm. Biochemistry 37, 7420–7430 (1998).

    Article  CAS  Google Scholar 

  43. Shutes, A. & Der, C.J. Real-time in vitro measurement of intrinsic and Ras GAP-mediated GTP hydrolysis. Methods Enzymol. 407, 9–22 (2006).

    Article  CAS  Google Scholar 

  44. Ahmadian, M.R., Mittal, R., Hall, A. & Wittinghofer, A. Aluminium fluoride associates with the small guanine nucleotide binding proteins. FEBS Lett. 408, 315–318 (1997).

    Article  CAS  Google Scholar 

  45. Rohl, C.A., Strauss, C.E.M., Misura, K.M.S. & Baker, D. Protein structure prediction using Rosetta. in Methods in Enzymology Vol. 383 (eds. Brand, L. & Johnson, M.L.) 66–93 (Academic Press, 2004).

  46. Dunbrack, R.L. Jr. & Karplus, M. Backbone-dependent rotamer library for proteins application to side-chain prediction. J. Mol. Biol. 230, 543–574 (1993).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. Lee (University of North Carolina at Chapel Hill (UNC-CH)) for supplying the PDZ2 domain. We would also like to thank D. Isom (UNC-CH) for his fQCR expertise and input on data analysis and J. Kuriyan (University of California, Berkeley) for the Soscat plasmid. This work was supported by US National Institutes of Health (NIH) grants R01CA089614 (S.L.C.), R01GM073180-06S1 (H.G.D.), R01GM073960 and R01GM073151 (B.K.) and R01GM41890 and P01CA117969 (L.C.C.). S.M.L. was supported by the Program in Molecular and Cellular Biophysics at UNC-CH and NIH grant T32GM008570. A.T.S. was supported, in part, by the Japanese Society for the Promotion of Science Research Fellowship for Research Abroad, the Kanae Foundation for Research Abroad and a Genentech fellowship.

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Author notes

  1. Atsuo T Sasaki

    Present address: Present address: Department of Internal Medicine, Division of Hematology and Oncology, University of Cincinnati Neuroscience Institute, Brain Tumor Center, Cincinnati, Ohio, USA.,

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Rachael Baker, Steven M Lewis, Emily M Wilkerson, Brian Kuhlman, Henrik G Dohlman & Sharon L Campbell

  2. Division of Signal Transduction, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

    Atsuo T Sasaki, Jason W Locasale & Lewis C Cantley

  3. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    Atsuo T Sasaki, Jason W Locasale & Lewis C Cantley

  4. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Brian Kuhlman, Henrik G Dohlman & Sharon L Campbell

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Contributions

R.B. performed biochemical and NMR studies. S.M.L. and B.K. conducted Rosetta modeling. R.B. and E.M.W. performed site-specific ubiquitination assays. A.T.S., J.W.L. and L.C.C. performed cell-lysate experiments and kinetic modeling. R.B., H.G.D. and S.L.C. designed experiments, analyzed data and wrote the manuscript.

Corresponding authors

Correspondence to Henrik G Dohlman or Sharon L Campbell.

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Baker, R., Lewis, S., Sasaki, A. et al. Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function. Nat Struct Mol Biol 20, 46–52 (2013). https://doi.org/10.1038/nsmb.2430

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