Change in substrate specificity of cytotoxic necrotizing factor unmasks proteasome-independent down-regulation of constitutively active RhoA.
Cytotoxic necrotizing factors CNF1 and CNF2 are produced by pathogenic Escherichia coli strains. They constitutively activate small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. Recently, a novel CNF (CNF(Y)) encompassing 65% identity to CNF1 has been identified in Yersinia pseudotuberculosis. In contrast to the E. coli toxins, which activate several isoforms of Rho family GTPases, CNF(Y) is a strong and selective activator of RhoA in vivo. By constructing chimeras between CNF1 and CNF(Y), we show that this substrate specificity is based on differences in the catalytic domains, whereas the receptor binding and translocation domains have no influence. We further define a loop element (L8) on the surface of the catalytic domains as important for substrate recognition. A single amino acid exchange in L8 is sufficient to shift substrate specificity of CNF1. Moreover, it is shown that RhoA activation by CNF1 is transient, which may be the consequence of the broader substrate specificity of the E. coli toxin, leading to cross-talk between the activated GTPases.
Hoffmann
Claudia
C
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Albert-Strasse 25, 79104 Freiburg, Germany.
Aktories
Klaus
K
Schmidt
Gudula
G
D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity / Q000235:genetics; D015011:Yersinia pseudotuberculosis / Q000378:metabolism / Q000472:pathogenicity; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; C086995:cytotoxic necrotizing factor type 2; C060296:cytotoxic necrotizing factor type 1; D020742:rhoA GTP-Binding Protein