Pseudomonas aeruginosa exoenzyme S disrupts Ras-mediated signal transduction by inhibiting guanine nucleotide exchange factor-catalyzed nucleotide exchange

Pseudomonas aeruginosa exoenzyme S double ADP-ribosylates Ras at Arg(41) an d Arg(128). Since Arg(41) is adjacent to the switch 1 region of Ras, ADP-ri bosylation could interfere with Ras-mediated signal transduction via severa l mechanisms, including interaction with Raf, or guanine nucleotide exchang e factor-stimulated or intrinsic nucleotide exchange. Initial experiments s howed that ADP-ribosylated Ras (ADP-r-Ras) and unmodified Ras (Ras) interac ted with Raf with equal efficiencies, indicating that ADP-ribosylation did not interfere with Ras-Raf interactions, While ADP-r-Ras and Ras possessed equivalent intrinsic nucleotide exchange rates, guanine nucleotide exchange factor (Cdc25) stimulated the nucleotide exchange of ADP-r-Ras at a 3-fold slower rate than Ras. ADP-r-Ras did not affect the nucleotide exchange of Ras, indicating that the ADP-ribosylation of pas was not a dominant negativ e phenotype. Ras-R41K and ADP-r-Ras R41K possessed similar exchange rates a s Ras, indicating that ADP-ribosylation at Arg(128) did not inhibit Cdc25-s timulated nucleotide exchange, Consistent with the slower nucleotide exchan ge rate of ADP-r-Ras as compared with Ras, ADP-r-Ras bound its guanine nucl eotide exchange factor (Cdc25) less efficiently than Ras in direct binding experiments. Together, these data indicate that ADP-ribosylation of Ras at Arg(41) disrupts Ras-Cdc25 interactions, which inhibits the rate-limiting s tep in Ras signal transduction, the activation of Ras by its guanine nucleo tide exchange factor.