title: The Yersinia pseudotuberculosis cytotoxic necrotizing factor (CNFY) selectively activates RhoA.
abstract: The cytotoxic necrotizing factors (CNF)1 and CNF2 from pathogenic Escherichia coli strains activate RhoA, Rac1, and Cdc42 by deamidation of Gln63 (RhoA) or Gln61 (Rac and Cdc42). Recently, a novel cytotoxic necrotizing factor termed CNFY was identified in Yersinia pseudotuberculosis strains (Lockman, H. A., Gillespie, R. A., Baker, B. D., and Shakhnovich, E. (2002) Infect. Immun. 70, 2708-2714). We amplified the cnfy gene from genomic DNA of Y. pseudotuberculosis, cloned and expressed the recombinant protein, and studied its activity. Recombinant GST-CNFY induced morphological changes in HeLa cells and caused an upward shift of RhoA in SDS-PAGE, as is known for GST-CNF1 and GST-CNF2. Mass spectrometric analysis of GST-CNFY-treated RhoA confirmed deamidation at Glu63. Treatment of RhoA, Rac1, and Cdc42 with GST-CNFY decreased their GTPase activities, indicating that all of these Rho proteins could serve as substrates for GST-CNFY in vitro. In contrast, RhoA, but not Rac or Cdc42, was the substrate of GST-CNFY in culture cells. GST-CNFY caused marked stress fiber formation in HeLa cells after 2 h. In contrast to GST-CNF1, formation of filopodia or lamellipodia was not induced with GST-CNFY. Accordingly, effector pull-down experiments with lysates of toxin-treated cells revealed strong activation of RhoA but no activation of Rac1 or Cdc42 after 6 h of GST-CNFY-treatment. Moreover, in rat hippocampal neurons, GST-CNFY results in the retraction of neurites, indicating RhoA activation. In contrast, no activation of Rac or Cdc42 was found. Altogether, our data suggest that CNFY from Y. pseudotuberculosis is a strong, selective activator of RhoA, which can be used as a powerful tool for constitutive RhoA activation without concomitant activation of Rac1 or Cdc42.
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Hoffmann
authors.forename: Marius
authors.initials: M
authors.lastname: Pop
authors.forename: Jost
authors.initials: J
authors.lastname: Leemhuis
authors.forename: Jörg
authors.initials: J
authors.lastname: Schirmer
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
authors.forename: Gudula
authors.initials: G
authors.lastname: Schmidt
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003603:Cytotoxins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006367:HeLa Cells; D006801:Humans; D016296:Mutagenesis; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D015011:Yersinia pseudotuberculosis / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D003603:Cytotoxins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C086995:cytotoxic necrotizing factor type 2; C060296:cytotoxic necrotizing factor type 1; D020742:rhoA GTP-Binding Protein