Bordetella bronchiseptica dermonecrotizing toxin induces reorganization of actin stress fibers through deamidation of Gln-63 of the GTP-binding protein Rho.
Bordetella dermonecrotizing toxin causes assembly of actin stress fibers and focal adhesions in some cultured cells and induces mobility shifts of the small GTP-binding protein Rho on electrophoresis. We attempted to clarify the molecular basis of the toxin action on Rho. Analysis of the amino acid sequence of toxin-treated RhoA revealed the deamidation of Gln-63 to Glu. The substitution of Glu for Gln-63 of RhoA by site-directed mutagenesis caused a mobility shift on electrophoresis, which was indistinguishable from that of the toxin-treated RhoA. Neither mutant RhoA-bearing Glu-63 nor toxin-treated RhoA significantly differed from untreated wild type RhoA in guanosine 5'-[gamma-thio]triphosphate binding activity but both showed a 10-fold reduction in GTP hydrolysis activity relative to untreated RhoA. C3H10T1/2 cells transfected with cDNA of the mutant RhoA bearing Glu-63 showed extensive formation of actin stress fibers similar to the toxin-treated cells. These results indicate that the toxin catalyzes deamidation of Gln-63 of Rho and renders it constitutively active, leading to formation of actin stress fibers.
Horiguchi
Y
Y
Department of Bacterial Toxicology, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamada-oka, Suita, Osaka 565, Japan. horiguti@biken.osaka-u.ac.jp
Inoue
N
N
Masuda
M
M
Kashimoto
T
T
Katahira
J
J
Sugimoto
N
N
Matsuda
M
M
D000199:Actins / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001427:Bacterial Toxins / Q000633:toxicity*; D016950:Bordetella bronchiseptica; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D018698:Glutamic Acid; D005973:Glutamine; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D011503:Transglutaminases; D010566:Virulence Factors, Bordetella; D020742:rhoA GTP-Binding Protein
D000199:Actins; D001427:Bacterial Toxins; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; C060969:dermonecrotic toxin, Bordetella; D005973:Glutamine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D018698:Glutamic Acid; D006160:Guanosine Triphosphate; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein