Identification of p130Cas as a substrate of Yersinia YopH (Yop51), a bacterial protein tyrosine phosphatase that translocates into mammalian cells and targets focal adhesions.
A number of pathogenic bacteria utilize type III secretion pathways to translocate virulence proteins into host eukaryotic cells. We identified a host target of YopH, a protein tyrosine phosphatase that is translocated into mammalian cells by Yersiniae. A catalytically inactive 'substrate-trapping' mutant, YopHC403S, was used as a probe to determine where YopH substrates localize in eukaryotic cells. Immunofluorescence microscopy demonstrated that YopHC403S localized to focal adhesions in human epithelial cells infected with Y. pseudotuberculosis. YopHC403S stabilized focal adhesions, as shown by its dominant-negative effect on focal adhesion disassembly mediated by YopE, a translocated protein which disrupts actin stress fibers. Conversely, YopH destabilized focal adhesions, even in the absence of YopE, as shown by loss of phosphotyrosine staining. Immunoprecipitation revealed that YopHC403S was trapped in a complex with a hyperphosphorylated 125-135 kDa protein, identified by immunoblotting as the focal adhesion protein p130Cas. YopHC403S bound directly to p130Cas in a phosphotyrosine-dependent manner in vitro. Translocation of YopH into cells plated on fibronectin resulted in rapid and selective dephosphorylation of p130Cas. These results demonstrate that YopH targets focal adhesions in host cells and that p130Cas, a docking protein for multiple SH2 domains, is a direct substrate of this enzyme in vivo.
Black
D S
DS
Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, 11794-5222, USA.
Bliska
J B
JB
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001692:Biological Transport; D002448:Cell Adhesion; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism*; D050719:Crk-Associated Substrate Protein; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D015011:Yersinia pseudotuberculosis / Q000472:pathogenicity*; D018909:src Homology Domains
C493841:BCAR1 protein, human; D001425:Bacterial Outer Membrane Proteins; D050719:Crk-Associated Substrate Protein; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D017027:Protein Tyrosine Phosphatases; C067571:yopH protein, Yersinia