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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
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Pseudomonas aeruginosa ExoT ADP-ribosylates CT10 Regulator of Kinase (Crk) Proteins*

Jianjun Sun and Joseph T. Barbieri {ddagger}

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 2–3 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.

{ddagger} 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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