Identification of residues in the N-terminal domain of the Yersinia tyrosine phosphatase that are critical for substrate recognition

YopH is a 468-amino acid protein-tyrosine phosphatase that is produced by p athogenic Yersinia species. YopH is translocated into host mammalian cells via a type III protein secretion system. Translocation of YopH into human e pithelial cells results in dephosphorylation of p130(Cas) and paxillin, dis ruption of focal adhesions, and inhibition of integrin-mediated bacterial p hagocytosis, Previous studies have shown that the N-terminal 129 amino acid s of YopH comprise a bifunctional domain. This domain binds to the SycH cha perone in Yersinia to orchestrate translocation and to tyrosine-phosphoryla ted target proteins in host cells to mediate substrate recognition. We used random mutagenesis in combination with the yeast two-hybrid system to iden tify residues in the YopH N-terminal domain that are involved in substrate- binding activity. Four single codon changes (Q11R, V31G, A33D, and N34D) we re identified that interfered with binding of the YopH N-terminal domain to tyrosine-phosphorylated p130(Cas) but not to SycH. These mutations did not impair YopH translocation into HeLa cells infected with Yersinia pseudotub erculosis, Introduction of the V31G substitution into catalytically inactiv e (substrate-trapping) forms of YopH interfered with the ability of these p roteins to bind to p130(Cas) and to localize to focal adhesions in HeLa cel ls. In addition, the V31G substitution reduced the ability of catalytically active YopH to dephosphorylate target proteins in HeLa cells. These data i ndicate that the substrate- and SycH-binding activities of the YopH N-termi nal domain can be separated and that the former activity is important for r ecognition and dephosphorylation of substrates by YopH in vivo.