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Originally published In Press as doi:10.1074/jbc.M304290200 on June 13, 2003
J. Biol. Chem., Vol. 278, Issue 35, 32794-32800, August 29, 2003
Pseudomonas aeruginosa ExoT ADP-ribosylates CT10 Regulator of Kinase (Crk) Proteins*
Jianjun Sun and
Joseph T. Barbieri
From the
Department of Microbiology and Molecular Genetics, Medical College of
Wisconsin, Milwaukee, Wisconsin 53226
Pseudomonas aeruginosa ExoT is a type III cytotoxin that functions
as an anti-internalization factor with an N-terminal RhoGAP domain and a
C-terminal ADP-ribosyltransferase domain. Although ExoT RhoGAP stimulates
actin reorganization through the inactivation of Rho, Rac, and Cdc42, the
function of the ADP-ribosylation domain is unknown. The present study
characterized the mammalian proteins that are ADP-ribosylated by ExoT, using
two-dimensional SDS-PAGE and matrix-assisted laser desorption ionization/time
of flight (MALDI-TOF) analysis. ExoT ADP-ribosylated two cytosolic proteins in
cell lysates upon type III delivery into cultured HeLa cells. MALDI-TOF mass
spectrometry analysis identified the two proteins as Crk-I and Crk-II that are
Src homology 23 domains containing adaptor proteins, which mediate
signal pathways involving focal adhesion and phagocytosis. ExoT
ADP-ribosylated recombinant Crk-I at a rate similar to the ADP-ribosylation of
soybean trypsin inhibitor by ExoS. ExoS did not ADP-ribosylate Crk-I.
ADP-ribosylation of Crk-I may be responsible for the anti-phagocytosis
phenotype elicited by ExoT in mammalian cells.
Received for publication, April 24, 2003
, and in revised form, May 29, 2003.
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Supported by National Institutes of Health Grants HL68912 and AI30165 and by
the Cystic Fibrosis Foundation. To whom correspondence should be addressed:
Dept. of Microbiology and Molecular Genetics, Medical College of Wisconsin,
8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8412; Fax:
414-456-6535; E-mail:
jtb01{at}mcw.edu.

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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
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