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J. Biol. Chem., Vol. 280, Issue 43, 35953-35960, October 28, 2005

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Uncoupling Crk Signal Transduction by Pseudomonas Exoenzyme T^*

From the Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Exoenzyme T (ExoT) is a bifunctional type III cytotoxin of Pseudomonas aeruginosa that possesses both Rho GTPase-activating protein and ADP-ribosyltransferase activities. The ADP-ribosyltransferase activity of ExoT stimulated depolymerization of the actin cytoskeleton independent of Rho GTPase-activating protein function, and ExoT was subsequently shown to ADP-ribosylate Crk (CT10 regulator of kinase)-I and Crk-II. Crk proteins are eukaryotic adaptor proteins comprising SH2 and SH3 domains that are components of the integrin signaling pathway leading to Rac1 and Rap1 functions. Mass spectroscopic analysis identified Arg²⁰ as the site of ADP-ribosylation by ExoT. Arg²⁰ is a conserved residue located within the SH2 domain that is required for interactions with upstream signaling molecules such as paxillin and p130^cas. Glutathione S-transferase pull-down and far Western assays showed that ADP-ribosylated Crk-I or Crk-I(R20K) failed to bind p130^cas or paxillin. This indicates that ADP-ribosylation inhibited the direct interaction of Crk with these focal adhesion proteins. Overexpression of wild-type Crk-I reduced cell rounding by ExoT, whereas expression of dominant-active Rac1 interfered with the ability of ExoT to round cells. Thus, the ADP-ribosylation of Crk uncouples integrin signaling by direct inhibition of the binding of Crk to focal adhesion proteins.

Received for publication, May 4, 2005 , and in revised form, August 9, 2005.

^* This work was supported by United States Public Health Service Grant RO1 AI30162 from the National Institutes of Health (to J. T. B.). 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.

¹ Present address: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.

² 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: 414-567-6535; E-mail: jtb01{at}mcw.edu.

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