ADP-ribosylation of oncogenic Ras proteins by pseudomonas aeruginosa exoenzyme S in vivo.
The exoenzyme S (ExoS)-producing Pseudomonas aeruginosa strain, 388, and corresponding ExoS knock-out strain, 388deltaexoS, were used in a bacterial and mammalian co-culture system as a model for the contact-dependent delivery of ExoS into host cells. Examination of DNA synthesis and Ras ADP-ribosylation in tumour cell lines expressing normal and mutant Ras revealed a decrease in DNA synthesis concomitant with ADP-ribosylation of Ras proteins after exposure to ExoS-producing bacteria, but not after exposure to non-ExoS-producing bacteria. Examination of normal H-Ras, K-Ras and N-Ras by two-dimensional electrophoresis after exposure to bacteria revealed differences in the degree of ADP-ribosylation by ExoS, with H-Ras being modified most extensively. ADP-ribosylation of oncogenic forms of Ras was examined in vivo using cancer lines expressing mutant forms of H-, N- or K-Ras. The mutant Ras proteins were modified in a manner qualitatively similar to their normal counterparts. Using Ras/Raf-1 co-immunoprecipitation after co-culture, it was found that exposure to ExoS-producing bacteria caused a decrease in the amount of Raf-1 associated with EGF-activated Ras and oncogenic Ras. The results from this study indicate that ExoS ADP-ribosylates both normal and mutant Ras proteins in vivo and inhibits signalling through Ras.
Vincent
T S
TS
Departments of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston 29425, USA. vincents@musc.edu
Fraylick
J E
JE
McGuffie
E M
EM
Olson
J C
JC
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D001427:Bacterial Toxins; D015153:Blotting, Western; D002460:Cell Line; D018920:Coculture Techniques; D004247:DNA / Q000096:biosynthesis; D003720:Densitometry; D015180:Electrophoresis, Gel, Two-Dimensional; D011233:Precipitin Tests; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000254:growth & development; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D001427:Bacterial Toxins; D011994:Recombinant Proteins; D000246:Adenosine Diphosphate Ribose; D004247:DNA; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins