PSEUDOMONAS-AERUGINOSA EXOENZYME-S ADP-RIBOSYLATES RAS AT MULTIPLE SITES

Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras to a sto ichiometry of similar to 2 molecules of ADP-ribose incorporated per mo lecule of Ras, which suggested that ExoS could ADP-ribosylate Ras at m ore than one arginine residue, SDS-polyacrylamide gel electrophoresis analysis showed that ADP-ribosylated Ras possessed a slower mobility t han non-ADP-ribosylated Ras, Analysis of the ADP-ribosylation of in vi tro transcribed/translated Ras by ExoS identified two electrophoretica lly shifted forms of Ras, which was consistent with the ADP-ribosylati on of Ras at two distinct arginine residues, Analysis of ADP-ribosylat ed in vitro transcribed/translated Ras mutants possessing individual A rg-to-Ala substitutions showed that Arg-41 was the preferred site of A DP-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 Ras Delta CAAX and Ras Delta CAAXR41K supported the conclusion t hat Arg-41 was the preferred site of ADP-ribosylation. Arg-41 is locat ed adjacent to the switch 1 region of Ras, which is involved in effect or interactions. Introduction of ExoS into eukaryotic cells inhibited Ras-mediated eukaryotic signal transduction since infection of PC-12 c ells with an ExoS-producing strain of P. aeruginosa inhibited nerve gr owth factor-stimulated neurite formation, This is the first demonstrat ion that ExoS disrupts a Ras-mediated signal transduction pathway.