The Yersinia pseudotuberculosis cytotoxic necrotizing factor (CNFY) selectively activates RhoA.
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.
Hoffmann
Claudia
C
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
Pop
Marius
M
Leemhuis
Jost
J
Schirmer
Jörg
J
Aktories
Klaus
K
Schmidt
Gudula
G
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*
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