The Yersinia tyrosine phosphatase YopH targets a novel adhesion-regulated signalling complex in macrophages

The Yersinia protein tyrosine phosphatase (PTP) YopH is translocated into eukaryotic cells by a type III secretion system that requires bacterial–host cell contact. YopH is composed of two modular effector domains: a substrate-binding domain located in the N-terminal region (residues 1–130) and a PTP catalytic domain located in the C-terminal region (residues 206–468). Previous studies have shown that YopH selectively targets tyrosine-phosphorylated proteins of approximate molecular weight 120 kDa (p120) and 55 kDa (p55) in murine macrophages. It has been demonstrated that p120 actually represents two tyrosine-phosphorylated target proteins, Cas and Fyb. We used the substrate-binding domain of YopH to affinity purify tyrosine-phosphorylated target proteins from lysates of J774A.1 macrophages. Protein microsequencing identified p55 as murine SKAP-HOM. Direct interaction between SKAP-HOM and a catalytically inactive form of YopH was demonstrated in vitro and in macrophages. In addition, we obtained evidence that SKAP-HOM is tyrosine phosphorylated in response to macrophage cell adhesion and that it forms a signalling complex with Fyb. We suggest that dephosphorylation of SKAP-HOM and Fyb by YopH allows yersiniae to interfere with a novel adhesion-regulated signal transduction pathway in macrophages.     

Interaction between the Yersinia protein tyrosine phosphatase YopH and eukaryotic Cas/Fyb is an important virulence mechanism

The tyrosine phosphatase YopH is an essential virulence factor produced by pathogenic Yersinia species. YopH is translocated into host cells via a type III secretion system and its dephosphorylating activity causes disruption of focal complex structures and blockage of the phagocytic process. Among the host cell targets of YopH are the focal adhesion proteins Crk-associated substrate (p130Cas) and focal adhesion kinase (FAK) in epithelial cells, and p130Cas and Fyn-binding protein (Fyb) in macrophages. Previous studies have shown that the N-terminal domain of YopH acts as a substrate-binding domain. In this study, the mechanism and biological importance of the targeting of YopH to focal complexes relative to its interaction with p130Cas/Fyb was elucidated. Mutants of YopH that were defective in p130Cas/Fyb binding but otherwise indistinguishable from wild type were constructed. Mutants unable to bind p130Cas did not localize to focal complex structures in infected cells, indicating that the association with p130Cas is critical for appropriate subcellular localization of YopH. These yopH mutants were also clearly attenuated in virulence, showing that binding to p130Cas and/or Fyb is biologically relevant in Yersinia infections.     

Interaction between the Yersinia tyrosine phosphatase YopH and its macrophage substrate, Fyn-binding protein, Fyb

Pathogenic Yersinia species can evade phagocytosis by injecting virulence effectors that interfere with the phagocytic machinery of host cells. One of these virulence effectors is the protein tyrosine phosphatase YopH. Through its enzymatic activity, YopH interferes with the initial phagocytic process by affecting signalling for cytoskeletal rearrangements. Fyb (Fyn-binding protein), which is an immune cell-specific adaptor protein, has been identified as a substrate of YopH in macrophages. In this study, the interaction between YopH and Fyb is studied. We show that YopH binds to Fyb via different regions in both phosphotyrosine-dependent and phosphotyrosine-independent ways. The phosphotyrosine substrate binding N-terminal part (1-130) of YopH as well as the C-terminal catalytic region binds to Fyb in a phosphotyrosine-dependent manner. We also show that a central part of YopH (130-260) interacts with the Fyb C-terminus (548-783) in a phosphotyrosine-independent manner. Further, we demonstrate that the N-terminal binding region of YopH is important for YopH-mediated functions on macrophages such as dephosphorylation of Fyb, blockage of phagocytosis, and cytotoxic effects.     

A yeast two-hybrid study of human p97/Gab2 interactions with its SH2 domain-containing binding partners

p97/Gab2 is a recently characterized member of a large family of scaffold proteins that play essential roles in signal transduction. Gab2 becomes tyrosine-phosphorylated in response to a variety of growth factors and forms multimolecular complexes with SH2 domain-containing signaling molecules such as the p85-regulatory subunit of the phosphoinositide-3-kinase (p85-PI3K), the tyrosine phosphatase SHP-2 and the adapter protein CrkL. To characterize the interactions between Gab2 and its SH2-containing binding partners, we designed a modified yeast two-hybrid system in which the Lyn tyrosine kinase is expressed in a regulated manner in yeast. Using this assay, we demonstrated that p97/Gab2 specifically interacts with the SH2 domains of PI3K, SHP-2 and CrkL. Interaction with p85-PI3K is mediated by tyrosine residues Y452, Y476 and Y584 of Gab2, while interaction with SHP-2 depends exclusively on tyrosine Y614. CrkL interaction is mediated by its SH2 domain recognizing Y266 and Y293, despite the latter being in a non-consensus (YTFK) environment.     

Fyn binding protein, Fyb, interacts with mammalian actin binding protein, mAbp1

The immune cell specific protein Fyn-T binding protein (Fyb) has been identified as a target of the Yersinia antiphagocytic effector Yersinia outer protein H (YopH), but its role in macrophages is unknown. By using Fyb domains as bait to screen a mouse lymphoma cDNA library, we identified a novel interaction partner, mammalian actin binding protein 1 (mAbp1). We show that mAbp1 binds the Fyb N-terminal via its C-terminally located src homology 3 domain. The interaction between Fyb and mAbp1 is detected in macrophage lysates and the proteins co-localize with F-actin in the leading edge. Hence, mAbp1 is likely to constitute a downstream effector of Fyb involved in F-actin dynamics.     

Inhibition of Yersinia tyrosine phosphatase by furanyl salicylate compounds

To avoid detection and targeting by the immune system, the plague-causing bacterium Yersinia pestis uses a type III secretion system to deliver a set of inhibitory proteins into the cytoplasm of immune cells. One of these proteins is an exceptionally active tyrosine phosphatase termed YopH, which paralyzes lymphocytes and macrophages by dephosphorylating critical tyrosine kinases and signal transduction molecules. Because Y. pestis strains lacking YopH are avirulent, we set out to develop small molecule inhibitors for YopH. We used a novel and cost-effective approach, in which leads from a chemical library screening were analyzed and computationally docked into the crystal structure of YopH. This resulted in the identification of a series of novel YopH inhibitors with nanomolar Ki values, as well as the structural basis for inhibition. Our inhibitors lack the polar phosphate-mimicking moiety of rationally designed tyrosine phosphatase inhibitors, and they readily entered live cells and rescued them from YopH-induced tyrosine dephosphorylation, signaling paralysis, and cell death. These inhibitors may become useful for treating the lethal infection by Y. pestis.     

Lck dephosphorylation at Tyr-394 and inhibition of T cell antigen receptor signaling by Yersinia phosphatase YopH

A key virulence factor for Yersinia pestis, the etiologic agent of plague, is the tyrosine phosphatase YopH, which the bacterium injects into host cells. We report that treatment of human T lymphocytes with a recombinant membrane-permeable YopH resulted in severe reduction in intracellular tyrosine phosphorylation and inhibition of T cell activation. The primary signal transducer for the T cell antigen receptor, the Lck tyrosine kinase, was specifically precipitated by a substrate-trapping YopH mutant, and Lck was dephosphorylated at its positive regulatory site, Tyr-394, in cells containing active YopH. By turning off Lck, YopH blocks T cell antigen receptor signaling at its very first step, effectively preventing the development of a protective immune response against this lethal bacterium.     

Bacterial and viral protein tyrosine phosphatases

Unrestricted protein tyrosine phosphatase (PTPase) activity may play a role in pathogenesis. For instance, the virulence determinant gene, yopH, of Yersinia pseudotuberculosis encodes a PTPase. The phosphatase activity of the YopH protein is essential for the pathogenesis of Y. pseudotuberculosis. Yersinia pestis, the bacterium which causes the bubonic plague, also contains a gene closely related to yopH. The action of YopH on host proteins appears to break down signal transduction mechanisms in many cell types including those of the immune system. This may contribute to the ability of the bacterium to escape effective surveillance by the immune system. The vaccinia virus VH1 gene, like yopH in the Yersinia bacteria, encodes a protein phosphatase. The VH1 PTPase defines a new class of phosphatases capable of dephosphorylating both phosphoserine/threonine and tyrosine containing substrates. Proteins sharing sequence identity to this dual-specificity phosphatase have been identified from other viruses, yeast and man. Although a complete understanding of the function of these dual-specificity phosphatases is not presently available, they clearly play important roles in cell cycle regulation, growth control and mitogenic signaling mechanisms. The unique catalytic properties of the dual specificity phosphatases suggest that these catalysts constitute a distinct subfamily of phosphatases.     

Cell type-specific effects of Yersinia pseudotuberculosis virulence effectors

One important feature of Yersinia pseudotuberculosis that enables resistance against the host immune defence is delivery of the antiphagocytic effectors YopH and YopE into phagocytic cells. The tyrosine phosphatase YopH influences integrin signalling, and YopE impairs cytoskeletal dynamics by inactivating Rho GTPases. Here, we report the impact of these effectors on internalization by dendritic cells (DCs), which internalize antigens to orchestrate host immune responses. We found that this pathogen resists internalization by DCs via YopE. YopH that is important for blocking phagocytosis by macrophages and neutrophils and which is also present inside the DCs does not contribute to the resistance. However, the YopH targets Fyb and p130Cas show higher expression levels in macrophages than in DCs. Furthermore, live cell microscopy revealed that the cells internalize Y. pseudotuberculosis in different ways: the macrophages utilize a locally restricted receptor-mediated zipper mechanism, whereas DCs utilize macropinocytosis involving constitutive ruffling that randomly catches bacteria into membrane folds. We conclude that YopH impacts early phagocytic signalling from the integrin receptor to which the bacterium binds and that this tight receptor-mediated stimulation is absent in DC macropinocytosis. Inactivation of cytoskeletal dynamics by YopE affects ruffling activity and hence also internalization. The different modes of internalization can be coupled to the major functions of these respective cell types: elimination by phagocytosis and antigen sampling.     

Engagement of Gab1 and Gab2 in erythropoietin signaling.

Several signaling cascades are activated during engagement of the erythropoietin receptor to mediate the biological effects of erythropoietin. The members of the insulin receptor substrate (IRS) family of proteins play a central role in signaling for various growth factor receptors and cytokines by acting as docking proteins for the SH2 domains of signaling elements, linking cytokine receptors to diverse downstream pathways. In the present study we provide evidence that the recently cloned IRS-related proteins, Gab1 and Gab2, of the Gab family of proteins, are rapidly phosphorylated on tyrosine during erythropoietin treatment of erythropoietin-responsive cells and provide docking sites for the engagement of the SHP2 phosphatase and the p85 subunit of the phosphatidylinositol 3'-kinase. Furthermore, our data show that Gab1 is the primary IRS-related protein activated by erythropoietin in primary erythroid progenitor cells. In studies to identify the erythropoietin receptor domains required for activation of Gab proteins, we found that tyrosines 425 and 367 in the cytoplasmic domain of the erythropoietin receptor are required for the phosphorylation of Gab2. Taken together, our data demonstrate that Gab proteins are engaged in erythropoietin signaling to mediate downstream activation of the SHP2 and phosphatidylinositol 3'-kinase pathways and possibly participate in the generation of the erythropoietin-induced mitogenic responses.     

Host adaptor proteins Gab1 and CrkII promote InlB-dependent entry of Listeria monocytogenes

The bacterial surface protein InlB mediates internalization of Listeria monocytogenes into mammalian cells through interaction with the host receptor tyrosine kinase, Met. InlB/Met interaction results in activation of the host phosphoinositide (PI) 3-kinase p85-p110, an event required for bacterial entry. p85-p110 activation coincides with tyrosine phosphorylation of the host adaptor Gab1, and formation of complexes between Gab1 and the p85 regulatory subunit of PI 3-kinase. When phosphorylated in response to agonists, Gab1 is known to recruit several Src-homology 2 (SH2) domain-containing proteins including p85, the tyrosine phosphatase Shp2 and the adaptor CrkII. Here, we demonstrate that Gab1.p85 and Gab1.CrkII complexes promote entry of Listeria. Overexpression of wild-type Gab1 stimulated entry, whereas Gab1 alleles unable to recruit all SH2 proteins known to bind wild-type Gab1 inhibited internalization. Further analysis with Gab1 alleles defective in binding individual effectors suggested that recruitment of p85 and CrkII are critical for entry. Consistent with this data, overexpression of wild-type CrkII stimulated bacterial uptake. Experiments with mutant CrkII alleles indicated that both the first and second SH3 domains of this adaptor participate in entry, with the second domain playing the most critical role. Taken together, these findings demonstrate novel roles for Gab1 and CrkII in Listeria internalization.     

Crystal structure of the Cys-NO modified YopH tyrosine phosphatase

Protein tyrosine phosphatases (PTPs) are key virulence factors in pathogenic bacteria, consequently, they have become important targets for new approaches against these pathogens, especially in the fight against antibiotic resistance. Among these targets of interest YopH (Yersinia outer protein H) from virulent species of Yersinia is an example. PTPs can be reversibly inhibited by nitric oxide (NO) since the oxidative modification of cysteine residues may influence the protein structure and catalytic activity. We therefore investigated the effects of NO on the structure and enzymatic activity of Yersinia enterocolitica YopH in vitro. Through phosphatase activity assays, we observe that in the presence of NO YopH activity was inhibited by 50%, and that this oxidative modification is partially reversible in the presence of DTT. Furthermore, YopH S-nitrosylation was clearly confirmed by a biotin switch assay, high resolution mass spectrometry (MS) and X-ray crystallography approaches. The crystal structure confirmed the S-nitrosylation of the catalytic cysteine residue, Cys403, while the MS data provide evidence that Cys221 and Cys234 might also be modified by NO. Interestingly, circular dichroism spectroscopy shows that the S-nitrosylation affects secondary structure of wild type YopH, though to a lesser extent on the catalytic cysteine to serine YopH mutant. The data obtained demonstrate that S-nitrosylation inhibits the catalytic activity of YopH, with effects beyond the catalytic cysteine. These findings are helpful for designing effective YopH inhibitors and potential therapeutic strategies to fight this pathogen or others that use similar mechanisms to interfere in the signal transduction pathways of their hosts.     

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 pathogenic Yersinia species. YopH is translocated into host mammalian cells via a type III protein secretion system. Translocation of YopH into human epithelial cells results in dephosphorylation of p130(Cas) and paxillin, disruption of focal adhesions, and inhibition of integrin-mediated bacterial phagocytosis. Previous studies have shown that the N-terminal 129 amino acids of YopH comprise a bifunctional domain. This domain binds to the SycH chaperone in Yersinia to orchestrate translocation and to tyrosine-phosphorylated target proteins in host cells to mediate substrate recognition. We used random mutagenesis in combination with the yeast two-hybrid system to identify residues in the YopH N-terminal domain that are involved in substrate-binding activity. Four single codon changes (Q11R, V31G, A33D, and N34D) were 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 pseudotuberculosis. Introduction of the V31G substitution into catalytically inactive (substrate-trapping) forms of YopH interfered with the ability of these proteins to bind to p130(Cas) and to localize to focal adhesions in HeLa cells. In addition, the V31G substitution reduced the ability of catalytically active YopH to dephosphorylate target proteins in HeLa cells. These data indicate that the substrate- and SycH-binding activities of the YopH N-terminal domain can be separated and that the former activity is important for recognition and dephosphorylation of substrates by YopH in vivo.     

Two substrate-targeting sites in the Yersinia protein tyrosine phosphatase co-operate to promote bacterial virulence

YopH is a protein tyrosine phosphatase and an essential virulence determinant of the pathogenic bacterium Yersinia. Yersinia delivers YopH into infected host cells using a type III secretion mechanism. YopH dephosphorylates several focal adhesion proteins including p130Cas in human epithelial cells, resulting in disruption of focal adhesions and cell detachment from the extracellular matrix. How the C-terminal protein tyrosine phosphatase domain of YopH targets specific substrates such as p130Cas in the complex milieu of the host cell has not been fully elucidated. An N-terminal non-catalytic domain of YopH binds p130Cas in a phosphotyrosine-dependent manner and functions as a novel substrate-targeting site. The structure of the YopH protein tyrosine phosphatase domain bound to a model phosphopeptide substrate was solved and the resulting structure revealed a second substrate-targeting site ('site 2') within the catalytic domain. Site 2 binds to p130Cas in a phosphotyrosine-dependent manner, and co-operates with the N-terminal domain ('site 1') to promote efficient recognition of p130Cas by YopH in epithelial cells. The identification of two substrate-targeting sites in YopH that co-operate to promote epithelial cell detachment and bacterial virulence reinforces the importance of protein-protein interactions for determining protein tyrosine phosphatase specificity in vivo, and highlights the sophisticated nature of microbial pathogenicity factors.     

Gab1 transduces PI3K-mediated erythropoietin signals to the Erk pathway and regulates erythropoietin-dependent proliferation and survival of erythroid cells

In this study, we examined the biological functions of Gab1 in erythropoietin receptor (EPOR)-mediated signaling in vivo. Knockdown of Gab1 by the introduction of the Gab1 siRNA expression vector into F-36P human erythroleukemia (F-36P-Gab1-siRNA) cells resulted in a reduction of cell proliferation and survival in response to EPO. EPO-induced activation of Erk1/2 but not of Akt was significantly suppressed in F-36P-Gab1-siRNA cells compared with mock-transfected F-36P cells. The co-immunoprecipitation experiments revealed an EPO-enhanced association of Gab1 with the Grb2–SOS1 complex and SHP-2 in F-36P cells. A selective inhibitor of phosphatidylinositol 3-kinase (PI3K) LY294002 and short interfering RNA (siRNA) duplexes targeting the p85 regulatory subunit of PI3K (p85-siRNA) independently suppressed tyrosine phosphorylation of Gab1; its association with Grb2, SHP-2 and p85; and the activation of Erk in EPO-treated F-36P cells. LY294002 inhibited EPO-induced tyrosine phosphorylation of Gab1 and its association with Grb2 in human primary EPO-sensitive erythroid cells. The co-immunoprecipitation experiments using the Jak inhibitor AG490 or siRNA duplexes targeting Jak2 and in vitro binding experiments demonstrated that Jak2 regulated Gab1-mediated Erk activation through tyrosine phosphorylation of Gab1. Taken together, these results suggest that Gab1 couples PI3K-mediated EPO signals with the Ras/Erk pathway and that Gab1 plays an important role in EPOR-mediated signal transduction involved in the proliferation and survival of erythroid cells.     

Identification of an amino-terminal substrate-binding domain in the Yersinia tyrosine phosphatase that is required for efficient recognition of focal adhesion targets

YopH is a protein tyrosine phosphatase (PTP) that is delivered into host mammalian cells via a type III secretion pathway in pathogenic Yersinia species. Although YopH is a highly active PTP, it preferentially targets a subset of tyrosine-phosphorylated proteins in host cells, including p130^Cas. Previous in vitro studies have indicated that the carboxy-terminal PTP domain contributes specificity to the interaction of YopH with substrates. However, it is not known if the PTP domain is sufficient for substrate recognition by YopH. Here, we have identified paxillin as an additional substrate of YopH in HeLa cells. In addition, we have identified a domain in the amino-terminal region of YopH that binds to both p130^Cas and paxillin and is required for the efficient recognition of substrates by the wild-type enzyme. This 'substrate-binding' domain exhibits a ligand specificity that is similar to that of the Crk Src homology 2 (SH2) domain, and it binds substrates directly in a phosphotyrosine-dependent manner. The substrate-binding domain of YopH may represent a novel type of protein–protein interaction module, as it lacks significant sequence similarity with any known SH2 or phosphotyrosine-binding (PTB) domain.     

Dissociation of FAK/p130CAS/c-Src Complex during Mitosis: Role of Mitosis-specific Serine Phosphorylation of FAK

At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. We have found that components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130^CAS (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading.We have found two significant alterations in FAK-mediated signal transduction during mitosis. First, the association of FAK with CAS or c-Src is greatly inhibited, with levels decreasing to 16 and 13% of the interphase levels, respectively. Second, mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells. Mitosis-specific phosphorylation is responsible for the disruption of FAK/CAS binding because dephosphorylation of mitotic FAK in vitro by protein serine/threonine phosphatase 1 restores the ability of FAK to associate with CAS, though not with c-Src. These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading.     

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.     

Gab1 contributes to cytoskeletal reorganization and chemotaxis in response to platelet-derived growth factor

Gab1 is a scaffolding/docking protein that has been suggested to play a role in signal transduction downstream of certain plasma membrane receptors, including platelet-derived growth factor (PDGF) receptors. We found that PDGF induced a rapid Gab1 phosphorylation, which depended on the recruitment of Grb2, indicating that Grb2 acts as a bridge between Gab1 and the PDGF beta-receptor. PDGF also enhanced the binding of Gab1 to the phosphatase SHP-2, but not to p85. To further study the role of Gab1 in PDGF signaling, we transfected porcine aortic endothelial cells with a doxycycline-inducible Gab1 construct. Increased Gab1 expression enhanced the recruitment and activation of SHP-2, as well as the phosphorylation of the mitogen-activated protein kinases Erk and p38 by PDGF. Gab1 expression also enhanced the formation of lamellipodia and cellular protrusions. In Gab1-deficient mouse embryonic fibroblasts, the same phenotype was induced by restoring the expression of wild-type Gab1, but not a mutant Gab1 that was unable to associate with SHP-2. These effects of PDGF on the actin cytoskeleton were not altered by the inhibition of p38 or Erk, but could be blocked by a dominant-negative form of Rac (Asn(17)). Finally, Gab1-deficient fibroblasts showed a decreased chemotactic response toward gradients of PDGF as compared with wild-type cells. In conclusion, Gab1 plays a selective role in the regulation of the mitogen-activated protein kinases Erk and p38 downstream of the PDGF beta-receptor, and contributes to cytoskeletal reorganization and chemotaxis in response to PDGF.