Auto-ADP-ribosylation of Pseudomonas aeruginosa ExoS. J Biol Chem 2002 Apr 05;277(14):12082-8
Date
02/01/2002Pubmed ID
11821389DOI
10.1074/jbc.M109039200Scopus ID
2-s2.0-0037023740 (requires institutional sign-in at Scopus site) 53 CitationsAbstract
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.
Author List
Riese MJ, Goehring UM, Ehrmantraut ME, Moss J, Barbieri JT, Aktories K, Schmidt GAuthor
Joseph T. Barbieri PhD Professor in the Microbiology and Immunology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
ADP Ribose TransferasesADP-Ribosylation Factors
Adenosine Diphosphate Ribose
Animals
Arginine
Bacterial Toxins
Blotting, Western
CHO Cells
Cattle
Cell Membrane
Cells, Cultured
Cricetinae
Dose-Response Relationship, Drug
Electrophoresis, Polyacrylamide Gel
Exons
Lung
Mutagenesis, Site-Directed
Poly(ADP-ribose) Polymerases
Precipitin Tests
Protein Binding
Protein Structure, Tertiary
Pseudomonas aeruginosa
Recombinant Proteins
Substrate Specificity
Tetanus Toxin
Time Factors
ras Proteins