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  • Title: Auto-ADP-ribosylation of Pseudomonas aeruginosa ExoS.
    Author: Riese MJ, Goehring UM, Ehrmantraut ME, Moss J, Barbieri JT, Aktories K, Schmidt G.
    Journal: J Biol Chem; 2002 Apr 05; 277(14):12082-8. PubMed ID: 11821389.
    Abstract:
    Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin. The N terminus possesses a Rho GTPase-activating protein (GAP) activity, whereas the C terminus comprises an ADP-ribosyltransferase domain. We investigated whether the ADP-ribosyltransferase activity of ExoS influences its GAP activity. Although the ADP-ribosyltransferase activity of ExoS is dependent upon FAS, a 14-3-3 family protein, factor-activating ExoS (FAS) had no influence on the activity of the GAP domain of ExoS (ExoS-GAP). In the presence of NAD and FAS, the GAP activity of full-length ExoS was reduced about 10-fold, whereas NAD and FAS did not affect the activity of the ExoS-GAP fragment. Using [(32)P]NAD, ExoS-GAP was identified as a substrate of the ADP-ribosyltransferase activity of ExoS. Site-directed mutagenesis revealed that auto-ADP-ribosylation of Arg-146 of ExoS was crucial for inhibition of GAP activity in vitro. To reveal the auto-ADP-ribosylation of ExoS in intact cells, tetanolysin was used to produce pores in the plasma membrane of Chinese hamster ovary (CHO) cells to allow the intracellular entry of [(32)P]NAD, the substrate for ADP-ribosylation. After a 3-h infection of CHO cells with Pseudomonas aeruginosa, proteins of 50 and 25 kDa were preferentially ADP-ribosylated. The 50-kDa protein was determined to be auto-ADP-ribosylated ExoS, whereas the 25-kDa protein appeared to represent a group of proteins that included Ras.
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