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J. Biol. Chem., Vol. 277, Issue 14, 12082-12088, April 5, 2002
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From the 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 [32P]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 [32P]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.
Auto-ADP-ribosylation of Pseudomonas aeruginosa
ExoS*
**,,
, and Microbiology and Molecular Genetics, Medical
College of Wisconsin, Milwaukee, Wisconsin 53226, the
¶ Pulmonary-Critical Care Medicine Branch, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892, and the
§ Institut für Experimentelle und Klinische
Pharmakologie und Toxikologie der Albert-Ludwigs-Universität
Freiburg, Otto-Krayer-Haus, Albertstrasse 25, Freiburg D-79104,
Germany
*
This work was supported by the Deutsche
Forschungsgemeinschaft DFG-SCHM 1385/1-1.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by Grant A1-30162 from the National Institutes of Health.
To whom correspondence should be addressed. Tel.:
49-761-203-5301; Fax: 49-761-203-5311; E-mail:
aktories@uni-freiburg.de.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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