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) and Arg(128). Since Arg(41) is adjacent to the switch 1 region of Ras, ADP-ribosylation could interfere with Ras-mediated signal transduction via several mechanisms, including interaction with Raf, or guanine nucleotide exchange factor-stimulated or intrinsic nucleotide exchange. Initial experiments showed that ADP-ribosylated Ras (ADP-r-Ras) and unmodified Ras (Ras) interacted 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 Ras was not a dominant negative phenotype. Ras-R41K and ADP-r-Ras R41K possessed similar exchange rates as Ras, indicating that ADP-ribosylation at Arg(128) did not inhibit Cdc25-stimulated nucleotide exchange. Consistent with the slower nucleotide exchange rate of ADP-r-Ras as compared with Ras, ADP-r-Ras bound its guanine nucleotide 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 step in Ras signal transduction, the activation of Ras by its guanine nucleotide exchange factor.
Ganesan
A K
AK
Medical College of Wisconsin, Microbiology and Molecular Genetics, Milwaukee, Wisconsin 53226, USA.
Vincent
T S
TS
Olson
J C
JC
Barbieri
J T
JT
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001120:Arginine; D001427:Bacterial Toxins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D018920:Coculture Techniques; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008322:Mammals; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism*; D020666:ras-GRF1
D001427:Bacterial Toxins; D018797:Cell Cycle Proteins; D011993:Recombinant Fusion Proteins; D020666:ras-GRF1; D000246:Adenosine Diphosphate Ribose; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D001120:Arginine; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D018631:ras Proteins