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Sun, J and Barbieri, JT (2003) Pseudomonas aeruginosa ExoT ADP-ribosylates CT10 regulator of kinase (Crk) proteins. J. Biol. Chem. 278:32794-800


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.


PubMed Online version:10.1074/jbc.M304290200


ADP Ribose Transferases/metabolism; Adenosine Diphosphate/metabolism; Animals; CHO Cells; Cell Adhesion; Cricetinae; Cytosol/metabolism; DNA, Complementary/metabolism; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; GTPase-Activating Proteins; Genetic Vectors; Glutathione Transferase/metabolism; HeLa Cells; Humans; Mutagenesis, Site-Directed; Mutation; Phagocytosis; Phenotype; Protein Kinases/metabolism; Protein Structure, Tertiary; Proto-Oncogene Proteins/metabolism; Proto-Oncogene Proteins c-crk; Pseudomonas aeruginosa/metabolism; Recombinant Proteins/metabolism; Ribose/metabolism; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Subcellular Fractions; Trypsin Inhibitors/metabolism



Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

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