title: Pseudomonas aeruginosa exoenzyme S ADP-ribosylates Ras at multiple sites.
abstract: Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras to a stoichiometry of approximately 2 molecules of ADP-ribose incorporated per molecule of Ras, which suggested that ExoS could ADP-ribosylate Ras at more than one arginine residue. SDS-polyacrylamide gel electrophoresis analysis showed that ADP-ribosylated Ras possessed a slower mobility than non-ADP-ribosylated Ras. Analysis of the ADP-ribosylation of in vitro transcribed/translated Ras by ExoS identified two electrophoretically shifted forms of Ras, which was consistent with the ADP-ribosylation of Ras at two distinct arginine residues. Analysis of ADP-ribosylated in vitro transcribed/translated Ras mutants possessing individual Arg-to-Ala substitutions showed that Arg-41 was the preferred site of ADP-ribosylation and that the second ADP-ribosylation event occurred at a slower rate than the ADP-ribosylation at Arg-41, but did not occur at a specific arginine residue. Analysis of bacterially expressed wild-type RasDeltaCAAX and RasDeltaCAAXR41K supported the conclusion that Arg-41 was the preferred site of ADP-ribosylation. Arg-41 is located adjacent to the switch 1 region of Ras, which is involved in effector interactions. Introduction of ExoS into eukaryotic cells inhibited Ras-mediated eukaryotic signal transduction since infection of PC-12 cells with an ExoS-producing strain of P. aeruginosa inhibited nerve growth factor-stimulated neurite formation. This is the first demonstration that ExoS disrupts a Ras-mediated signal transduction pathway.
authors.affiliation: Department of Microbiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: A K
authors.initials: AK
authors.lastname: Ganesan
authors.forename: D W
authors.initials: DW
authors.lastname: Frank
authors.forename: R P
authors.initials: RP
authors.lastname: Misra
authors.forename: G
authors.initials: G
authors.lastname: Schmidt
authors.forename: J T
authors.initials: JT
authors.lastname: Barbieri
mesh_terms: D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D051381:Rats; D016415:Sequence Alignment; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D000246:Adenosine Diphosphate Ribose; D001120:Arginine; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C444820:exoenzyme S; D018631:ras Proteins