Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras.
We have examined the functional consequences of ADP-ribosyltransferase modification of Ras by the exoenzyme S (ExoS) protein of Pseudomonas aeruginosa. ExoS has been shown previously to ADP-ribosylate a number of proteins, including members of the Ras superfamily, which play an essential role in the processes of cell proliferation, differentiation, motility and cell division. HeLa and NIH3T3 cells were infected with ExoS protein, which was delivered via the type III secretion system of the heterologous host Yersinia pseudotuberculosis. Infection of mammalian cells with ExoS results in a change in the ratio of GTP/GDP bound directly to Ras in vivo. This ADP-ribosylation of Ras in vivo is mediated by the C-terminal domain of ExoS. Further, ExoS ADP-ribosylation of Ras in vivo inhibits activation of Ras and the ability to interact with the Ras binding domain of Raf upon stimulation with epidermal growth factor (EGF). In the present study, we show that ExoS activity 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 Ras. This is further supported by our observation of a substantial inhibition of extracellular signal-regulated kinase and protein kinase B/Akt kinase activation in response to EGF upon ExoS infection. In conclusion, in the present study, the consequences of ExoS infection on Ras effector pathway in vivo have been defined.
Henriksson
M L
ML
Department of Cell and Molecular Biology, University of Umeâ, S-901 87 Umeâ, Sweden.
Rosqvist
R
R
Telepnev
M
M
Wolf-Watz
H
H
Hallberg
B
B
D016475:3T3 Cells; D036002:ADP Ribose Transferases / Q000737:chemistry / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins; D001665:Binding Sites; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011506:Proteins / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D015011:Yersinia pseudotuberculosis / Q000502:physiology; D018631:ras Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D001427:Bacterial Toxins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C495949:Grb2 protein, mouse; D011506:Proteins; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D004815:Epidermal Growth Factor; D006160:Guanosine Triphosphate; D036002:ADP Ribose Transferases; C444820:exoenzyme S; D066246:ErbB Receptors; D018631:ras Proteins