Pseudomonas aeruginosa ExoT ADP-ribosylates CT10 regulator of kinase (Crk) proteins.
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.
Sun
Jianjun
J
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Barbieri
Joseph T
JT
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D000071677:Histidine Kinase; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010587:Phagocytosis; D010641:Phenotype; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012266:Ribose / Q000378:metabolism; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions; D014361:Trypsin Inhibitors / Q000378:metabolism
D018076:DNA, Complementary; C477521:ExoT protein, Pseudomonas aeruginosa; D020690:GTPase-Activating Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D000244:Adenosine Diphosphate; D012266:Ribose; D036002:ADP Ribose Transferases; D005982:Glutathione Transferase; D011494:Protein Kinases; D000071677:Histidine Kinase