Ras effector pathway activation by epidermal growth factor is inhibited invivo by exoenzyme S ADP-ribosylation of Ras

We have examined the functional consequences of ADP-ribosyltransferase modi fication of Ras by the exoenzyme S (ExoS) protein of Pseudomonas aeruginosa . ExoS has been shown previously to ADP-ribosylate a number of proteins, in cluding members of the Ras superfamily, which play an essential role in the processes of cell proliferation, differentiation, motility and cell divisi on. HeLa and NIH3T3 cells were infected with ExoS protein, which was delive red via the type III secretion system of the heterologous host Yersinia pse udotuberculosis. Infection of mammalian cells with ExoS results in a change in the ratio of GTP/GDP bound directly to Ras in vivo. This ADP-ribosylati on of Ras in vivo is mediated by the C-terminal domain of ExoS. Further, Ex oS ADP-ribosylation of Ras in vivo inhibits activation of Ras and the abili ty to interact with the Ras binding domain of Raf upon stimulation with epi dermal growth factor (EGF). In the present study, we show that ExoS activit y does not interfere with EGF receptor phosphorylation itself, nor with the formation of a Grb2-activated Shc complex upon EGF stimulation, consistent with ExoS blockage of this mitogenic signalling pathway at the level of Ra s. This is further supported by our observation of a substantial inhibition of extracellular signal-regulated kinase and protein kinase B/Akt kinase a ctivation in response to EGF upon ExoS infection. In conclusion, in the pre sent study, the consequences of ExoS infection on Ras effector pathway in v ivo have been defined.