IDENTIFICATION OF P130(CAS) 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 identif ied a host target of YopH, a protein tyrosine phosphatase that is tran slocated into mammalian cells by Yersiniae. A catalytically inactive ' substrate-trapping' mutant, YopHC403S, was used as a probe to determin e where YopH substrates localize in eukaryotic cells, Immunofluorescen ce microscopy demonstrated that YopHC403S localized to focal adhesions in human epithelial cells infected with Y.pseudotuberculosis. YopHC40 3S stabilized focal adhesions, as shown by its dominant-negative effec t on focal adhesion disassembly mediated by YopE, a translocated prote in which disrupts actin stress fibers, Conversely, YopH destabilized f ocal adhesions, even in the absence of YopE, as shown by loss of phosp hotyrosine staining, Immunoprecipitation revealed that YopHC403S was t rapped in a complex with a hyperphosphorylated 125-135 kDa protein, id entified by immunoblotting as the focal adhesion protein p130(Cas)., Y opHC403S bound directly to p130(Cas) in a phosphotyrosine-dependent ma nner in vitro. Translocation of YopH into cells plated on fibronectin resulted in rapid and selective dephosphorylation of p130(Cas). These results demonstrate that YopH targets focal adhesions in host cells an d that p130(Cas), a docking protein for multiple SH2 domains, is a dir ect substrate of this enzyme in vivo.