Uncoupling Crk signal transduction by Pseudomonas exoenzyme T.
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 p130cas. Glutathione S-transferase pull-down and far Western assays showed that ADP-ribosylated Crk-I or Crk-I(R20K) failed to bind p130cas 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.
Deng
Qing
Q
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Sun
Jianjun
J
Barbieri
Joseph T
JT
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001427:Bacterial Toxins / Q000378:metabolism*; D015153:Blotting, Western; D002448:Cell Adhesion; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007525:Isoelectric Focusing; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D050737:Proto-Oncogene Proteins c-crk / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D018909:src Homology Domains
D001427:Bacterial Toxins; C477521:ExoT protein, Pseudomonas aeruginosa; D020690:GTPase-Activating Proteins; D050737:Proto-Oncogene Proteins c-crk; D011994:Recombinant Proteins; C085379:rho GTPase-activating protein; D049452:Green Fluorescent Proteins; D001120:Arginine; D036002:ADP Ribose Transferases; D005982:Glutathione Transferase