Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1


The actin cytoskeleton is regulated by GTP-hydrolysing proteins, the Rho GTPases1,2, which act as molecular switches in diverse signal-transduction processes3. Various bacterial toxins can inactivate Rho GTPases by ADP-ribosylation1 or glucosylation4. Previous research has identified Rho proteins as putative targets for Escherichia coli cytotoxic necrotizing factors 1 and 2 (CNF1 and 2)5,6. These toxins induce actin assembly and multinucleation in culture cells. Here we show that treatment of RhoA with CNF1 inhibits the intrinsic GTPase activity of RhoA and completely blocks GTPase activity stimulated by the Rho-GTPase-activating protein (rhoGAP). Analysis by mass spectrometry and amino-acid sequencing of proteolytic peptides derived from CNF1-treated RhoA indicate that CNF1 induces deamidation of a glutamine residue at position 63 (Gln 63) to give constitutively active Rho protein.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Induction of multinucleation and increase in F-actin by CNF1.
Figure 2: Effects of CNF1 on the migration behaviour of Rho.
Figure 3: a, Nucleotide-binding of CNF1-treated RhoA.
Figure 4: Tandem MS spectrum of the doubly charged tryptic peptide Gln 52–Arg 68 of the CNF I-exposed RhoA.
Figure 5: GTPase activity and nucleotide-binding of Gln63Glu-RhoA.
Figure 6: Induction of stress fibres by microinjection of Gln63Glu-RhoA.

Similar content being viewed by others


  1. Paterson, H. F. Microinjection of recombinant p21rhoinduces rapid changes in cell morphology. J. Cell Biol. 111, 1001–1007 (1990).

    Article  CAS  Google Scholar 

  2. Machesky, L. M. & Hall, A. Rho: A connection between membrane receptor signalling and the cytoskeleton. Trends Cell Biol. 6, 304–310 (1996).

    Article  CAS  Google Scholar 

  3. Lim, L., Manser, E., Leung, T. & Hall, C. Regulation of phosphorylation pathways by p21 GTPases—the p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways. Eur. J. Biochem. 242, 171–185 (1996).

    Article  CAS  Google Scholar 

  4. Just, al. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375, 500–503 (1995).

    Article  ADS  CAS  Google Scholar 

  5. Oswald, al. Cytotoxic necrotizing factor type 2 produced by virulent Escherichia coli modifies the small GTP-binding proteins Rho involved in assembly of actin stress fibers. Proc. Natl Acad. Sci. USA 91, 3814–3818 (1994).

    Article  ADS  CAS  Google Scholar 

  6. Fiorentini, al. Escherichia coli cytotoxic necrotizing factor 1: Evidence for induction of actin assembly by constitutive activation of the p21 Rho GTPase. Infect. Immun. 63, 3936–3944 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Caprioli, A., Falbo, V., Roda, L. G., Ruggeri, F. M. & Zona, C. Partial purification and characterization of an Escherichia coli toxic factor that induces morphological cell alterations. Infect. Immun. 39, 1300–1306 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. de Rycke, al. Evidence for two types of cytotoxic necrotizing factor in human and animal clinical isolates of Escherichia coli. J. Clin. Microbiol. 28, 694–699 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Falzano, al. Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor type 1. Mol. Microbiol. 9, 1247–1254 (1993).

    Article  CAS  Google Scholar 

  10. Leonard, D. A., Evans, T., Hart, M., Cerione, R. A. & Manor, D. Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy. Biochemistry 33, 12323–12328 (1994).

    Article  CAS  Google Scholar 

  11. Ridley, A. J. Microinjection of Rho and Rac into quiescent Swiss 3T3 cells. Methods Enzymol. 256, 313–320 (1995).

    Article  CAS  Google Scholar 

  12. Diekmann, D. & Hall, A. In vitro binding assay for interactions of Rho and Rac with GTPase-activating proteins and effectors. Methods Enzymol. 256, 207–215 (1995).

    Article  CAS  Google Scholar 

  13. Renshaw, M. W., Toksoz, D. & Schwartz, M. A. Involvement of the small GTPase Rho in integrin-mediated activation of mitogen-activated protein kinase. J. Biol. Chem. 271, 21691–21694 (1996).

    Article  CAS  Google Scholar 

  14. Der, C. J., Finkel, T. & Cooper, G. M. Biological and biochemical properties of human rasHgenes mutated at codon 61. Cell 44, 167–176 (1986).

    Article  CAS  Google Scholar 

  15. Schweins, al. Substrate-assisted catalysis as a mechanism for GTP hydrolysis of p21rasand other GTP-binding proteins. Nature Struct. Biol. 2, 36–44 (1995).

    Article  CAS  Google Scholar 

  16. Just, al. Clostridium difficile toxin B acts on the GTP-binding protein Rho. J. Biol. Chem. 269, 10706–10712 (1994).

    CAS  PubMed  Google Scholar 

  17. Hiratsuka, T. New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes. Biochim. Biophys. Acta 742, 496–508 (1983).

    Article  CAS  Google Scholar 

  18. Falbo, V., Pace, T., Picci, L., Pizzi, E. & Caprioli, A. Isolation and nucleotide sequence of the gene encoding cytotoxic necrotizing factor 1 of Escherichia coli. Infect. Immun. 61, 4909–4914 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Aktories, K. & Just, I. In vitro ADP-ribosylation of Rho by bacterial ADP-ribosyltransferases. Methods Enzymol. 256, 184–195 (1995).

    Article  CAS  Google Scholar 

  20. Just, I., Selzer, J., Von Eichel-Streiber, C. & Aktories, K. The low molecular mass GTP-binding protein Rho is affected by toxin A from Clostridium difficile. J. Clin. Invest. 95, 1026–1031 (1995).

    Article  CAS  Google Scholar 

  21. Horiguchi, Y., Senda, T., Sugimoto, N., Katahira, J. & Matsuda, M. Bordetella bronchiseptica dermonecrotizing toxin stimulates assembly of actin stress fibers and focal adhesions by modifying the small GTP-binding protein rho. J. Cell Sci. 108, 3243–3251 (1995).

    CAS  PubMed  Google Scholar 

  22. Wilm, M. & Mann, M. Analytical properties of the nanoelectrospray ion source. Anal. Chem. 68, 1–8 (1996).

    Article  CAS  Google Scholar 

Download references


We thank I. Just for critical reading of the manuscript.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Klaus Aktories.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schmidt, G., Sehr, P., Wilm, M. et al. Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature 387, 725–729 (1997).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing