Article

• The EMBO Journal (1997) 16, 2307 - 2318
• doi:10.1093/emboj/16.9.2307

The PTPase YopH inhibits uptake of Yersinia, tyrosine phosphorylation of p130^Cas and FAK, and the associated accumulation of these proteins in peripheral focal adhesions

Cathrine Persson¹, Nivia Carballeira¹, Hans Wolf-Watz¹ and Maria Fällman¹

1. Department of Cell and Molecular Biology, Umeå University, S-901 87 Umeå, Sweden

Received 16 December 1996; Revised 21 January 1997

• Keywords:

  • FAK,
  • focal adhesions,
  • p130^Cas,
  • Yersinia,
  • YopH

Introduction

Pathogenic Yersiniae infect both animals and humans and cause diseases ranging from mild gastroenteritis (Yersinia pseudotuberculosis and Y.enterocolitica) to bubonic plague (Y.pestis). The bacteria resist the non-specific host defence and proliferate extracellularly in lymphatic tissues (Hanski et al., 1989; Simonet et al., 1990). This ability is linked to the presence of a common virulence plasmid that encodes a number of secreted proteins denoted Yersinia outer proteins (Yops), seven of which have been shown to be essential for virulence (Cornelis, 1992; Straley et al., 1993; Forsberg et al., 1994). Upon intimate contact with a eukaryotic target cell, the extracellularly located bacteria secrete Yops by the type III secretion machinery that is encoded by the virulence plasmid (Forsberg et al., 1994). The Yop effectors are then translocated through the plasma membrane into the interior of the target cell. Secretion and translocation of the Yops are mediated by a polarized mechanism that prevents secretion of Yops to the surrounding medium (Sory and Cornelis, 1994; Rosqvist et al., 1994; Persson et al., 1995; Sory et al., 1995). In contrast to extracellularly located bacteria, bacteria within a target cell do not express Yops (Rosqvist and Wolf-Watz, 1986; Rosqvist et al., 1994; Persson et al., 1995).

YopH has the same signature sequence that is typical of eukaryotic protein tyrosine phosphatases (PTPases) EC 3.1.3.48 (Guan and Dixon, 1990). Moreover, the specific activity of YopH is the highest of all the PTPases described thus far (Zhang et al., 1992), and this activity is essential for the virulence of pathogenic Yersiniae (Bliska et al., 1991; Andersson et al., 1996). YopH has been shown to cause dephosphorylation of target cell proteins (Bliska et al., 1991, 1992; Hartland et al., 1994; Green et al., 1995) and to interrupt early phosphotyrosine signalling associated with the bacterial uptake process (Andersson et al., 1996). The ability of a cell surface attached bacterium to avoid being ingested implies that the mechanism involved is rapid and specific, which in turn suggests that the effect of YopH is exerted close to the site of interaction between the bacteria and the target cell (Forsberg et al., 1994). Y.pseudotuberculosis bacteria lacking the virulence plasmid are taken up by eukaryotic cells. This uptake is dependent on interactions between the bacterial protein invasin (chromosomally encoded) and 1-integrins on the surface of the eukaryotic cell, and in the absence of invasin, the bacteria are not taken up at all (Rosenshine et al., 1992; Isberg and Tran van Nhieu, 1994; Fällman et al., 1995). Invasin stimulated uptake is associated with increased tyrosine kinase activity in the ingesting cell, and it has also been shown that the uptake is obstructed by the presence of tyrosine kinase inhibitors (Rosenshine et al., 1992; Fällman et al., 1995). Furthermore, upon 1-integrin mediated interactions with Y.pseudotuberculosis strains that express Yops this uptake process is blocked (Persson et al., 1995), and it has been proposed that YopH acts by dephosphorylating proteins that are involved in regulation of the cellular cytoskeleton and thereby inhibiting integrin mediated uptake (Andersson et al., 1996). However, the specific role of YopH in this process has been difficult to evaluate, since other Yop effectors concomitantly expressed by this pathogen also influence the uptake process (Cornelis, 1992; Straley et al., 1993; Forsberg et al., 1994).

Integrins take part in adhesive interactions of eukaryotic cells by serving as a link between the extracellular matrix and the actin cytoskeleton at sites of close cell substratum contact (Lo and Chen, 1994). These sites, termed focal adhesions (FAs), are multi-molecular complexes in which clustered integrins are associated with a large number of cytoplasmic derived molecules that exhibit structural (-actinin, vinculin, talin) and/or regulatory (focal adhesion kinase, Src family tyrosine kinases, paxillin and tensin) functions (Lo and Chen, 1994; Miyamoto et al., 1995b). The FA components participate in a variety of signalling events generated by ligation and clustering of integrins, for instance those mediating cytoskeletal rearrangement (Lo and Chen, 1994; Clark and Brugge, 1995). The precise mechanism underlying assembly of FAs is not known, although it has been shown to involve occupancy and clustering of integrins in association with tyrosine kinase activity (Burridge et al., 1992; Miyamoto et al., 1995a, 1995b). Rho family GTPases appear to play a regulatory role in the very early events of integrin clustering and subsequent FA assembly (Ridley and Hall, 1992; Hotchin and Hall, 1995; Nobes and Hall, 1995). Disassembly of FAs involves PTPase activity and occurs during processes that require cells to detach from the substratum and to reorganize their cytoskeleton (Dunlevy and Couchman, 1993; Nakamura et al., 1995).

One FA protein involved in the integrin mediated signalling cascade is the focal adhesion kinase (FAK). This kinase interacts with and phosphorylates several proteins and is therefore believed to function as a signal amplifier/transmitter. The mechanism for its activity, however, is not clear (Richardson and Parsons, 1995). The localization of FAK to FAs does not depend on integrin binding and tyrosine phosphorylation (Hildebrand et al., 1993), and the activity of the kinase is stimulated by a mechanism involving interactions with integrins (Richardson and Parsons, 1995). Polte and Hanks (1995) used the two hybrid screen technique to gain insight into the FAK signalling pathway and found that the Crk associated tyrosine kinase substrate p130^Cas interacts with FAK and that a proline-rich sequence of FAK serves as a binding site for the SH3 domain of p130^Cas. Besides the SH3 domain, p130^Cas contains several putative tyrosine phosphorylation sites that can serve as binding sites for SH2-containing molecules (Sakai et al., 1994; Burnham et al., 1996). The role of p130^Cas in FAK-related signal transduction is not clear, although it was recently found that this protein is phosphorylated upon cellular adhesion to fibronectin, and has been found localized to focal adhesion, which suggests that p130^Cas, like FAK, is involved in integrin mediated adhesion (Nojima et al., 1995; Petch et al., 1995; Vuori and Rouslahti, 1995; Harte et al., 1996; Vouri et al., 1996).

In the present study, we have examined the molecular mechanism by which YopH blocks 1-integrin mediated uptake of bacteria by HeLa cells. We found that YopH, in infected cells, caused p130^Cas and FAK to become dephosphorylated, which suggests that these proteins constitute native targets of YopH. We also observed that bacterial infection stimulated phosphorylation of p130^Cas and FAK, and this was associated with recruitment of the former and accumulation of the latter in focal complexes located at the cell periphery.

YopH inhibits the uptake of bacteria by HeLa cells

Yersiniae pseudotuberculosis blocks its own uptake into eukaryotic cells and one bacterial key protein in this process is the PTPase YopH. Since the uptake process starts immediately upon contact between the pathogen and the target cell, YopH must instantly affect the uptake machinery of the cell. In accordance with this, it has been shown that YopH affects early phosphotyrosine signalling (Andersson et al., 1996). In general, PTPases exhibit low substrate specificity when studied in vitro but are strictly regulated in vivo, and it has been suggested that the targeting to specific intracellular locations is an important mechanism that regulates PTPase activity (Mauro and Dixon, 1994). Therefore, we have in this study chosen an in vivo infection model. We used a specifically engineered multiple Yop mutant (MYM) strain of Y.pseudotuberculosis (Håkansson et al., 1996), to examine the molecular mechanism of the PTPase YopH. This strain does not express YadA, YopH, YopE, YopM, YopK or YpkA, but it is still able to regulate, secrete and translocate Yops as the wild-type Y.pseudotuberculosis strain does. A multicopy plasmid that encodes a given Yop can be introduced into MYM to study the behaviour of that particular Yop in the intracellular compartment of the target cell without interference from other Yop effectors (Håkansson et al., 1996). We examined the effects of the PTPase YopH by using the MYM strain and an isogenic pair of plasmid-bearing MYM strains, i.e. MYM expressing the wild-type YopH protein (MYMpyopH) and MYM expressing a catalytically inactive form of YopH (MYMpyopHC403A) (all strains used are described in Table I). The YopHC403A protein has a single amino acid substitution (Cys403Ala) which totally abolishes the PTPase activity (Guan and Dixon, 1990). These three strains as well as the wild-type strain were used to infect cultured HeLa cells, and the level of bacterial uptake into these cells was determined (Heesemann and Laufs, 1985; Rosqvist et al., 1988). When MYM or MYMpyopHC403A infected HeLa cells were analyzed, 84 or 75% of the cell associated bacteria were intracellularly located, while only 27% were intracellularly located when the MYMpyopH strain was studied (Figure 1A). The efficiency of the latter strain to resist being ingested by the HeLa cells mimic that of the wild-type strain (Figure 1A). The uptake of the pathogen into eukaryotic cells is dependent on binding of the bacterial protein invasin to 1-integrins present on the surface of the target cell (Isberg and Tran van Nhieu, 1994); consequently, an invasin mutant that lacks interactions with 1-integrins was not ingested (data not shown). These results indicated that high expression of YopH alone was sufficient to impair 1-integrin mediated bacterial uptake.

Figure 1.

YopH abrogates the phosphotyrosine signal associated with uptake of Yersinia by HeLa cells. (A) HeLa cells were infected with MYM, MYMpyopH, MYMpyopHC403A or with wild-type Y.pseudotuberculosis. The infections were performed at a calculated bacteria:cell ratio of 20:1 at 37°C, for 30 min and the intra- and extracellular location of bacteria associated with target cells were distinguished by a double fluorescent antibody test. The values represent the mean SEM of four separate experiments and are expressed as percentages of HeLa cell associated bacteria that were located intracellularly. (B and C) Immunoblots (anti-phosphotyrosine) from lysates of HeLa cells infected with a calculated bacteria:cell ratio of 100:1 of different strains of Y.pseudotuberculosis; the infection was done at 37°C for the periods of time indicated. (B) Infection with either the MYM strain, YP100 (invA) or YP100(pIRR1) (invA/pinvA⁺). (C) Infection with either MYMpyopHC403A or MYMpyopH. In the right blot, HeLa cells were infected with the MYMpyopHC403A strain for 60 min and thereafter infected again with MYMpyopH for the periods of time indicated. Positions of the molecular weight markers and of the 120–130 kDa phosphotyrosine proteins (arrow) are indicated to the left of the blots.

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Table 1: Bacterial strains and plasmids used in this study

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Uptake of bacteria by HeLa cells is associated with increased tyrosine phosphorylation

To evaluate the involvement of tyrosine kinase activity in bacterial uptake by HeLa cells, phosphotyrosine proteins were analyzed at different time points after infection of the cells with the MYM strain. The uptake of the bacteria was associated with a time dependent increase in the tyrosine phosphorylated state of, in particular, one protein class with a molecular weight of 120 kDa (Figure 1B). To explore the importance of invasin for stimulating the increased phosphorylation seen with the MYM strain, an isogenic pair of plasmid cured Y.pseudotuberculosis strains YP100 (invA) and YP100(pIRR1) (invA/pinvA⁺) was used. The invasin expressing strain caused an increased tyrosine phosphorylation of proteins in the 120 kDa range, similar to that seen with the MYM strain (Figure 1B). The invasin mutant, which was not taken up by the HeLa cells, did not induce any phosphotyrosine signal (Figure 1B). Thus, the presence of invasin was a prerequisite for the observed increase of tyrosine phosphorylation in the infected cells. Infection with the MYMpyopHC403A strain led to increased tyrosine phosphorylation of several proteins, with the predominant protein species being in the 120–130 kDa range (Figure 1C), and the increase in the tyrosine phosphorylated state of the 120–130 kDa proteins was much more pronounced than that caused by the MYM strain. One possible explanation for this could be that the presence of YopHC403A protected against dephosphorylation by endogenous PTPases, and this in turn suggested that the mutated form of YopH interacted with the proteins indicated.

Earlier studies have shown that YopH caused an overall dephosphorylation of cellular proteins (Bliska et al., 1991, 1992; Hartland et al., 1994). In contrast to this, we found that constitutively tyrosine phosphorylated proteins were only slightly affected after prolonged exposure to YopH, but the infection-induced increase in tyrosine phosphorylation of the 120–130 kDa proteins was not detected (Figure 1C). This demonstrated that although YopH is an extremely potent PTPase, it was not unrestrained within the HeLa cells and the absence of the increased phosphorylation of the 120–130 kDa proteins suggested that YopH specifically interrupted the signal transduction involved in the uptake of the bacteria.

To determine if YopH could dephosphorylate the proteins showing increased tyrosine phosphorylation induced by the MYMpyopHC403A strain, HeLa cells were infected with the MYMpyopHC403A strain for 60 min (after exposure for that time, the 120–130 kDa proteins were highly phosphorylated) and then infected again but now with MYMpyopH. At different times after the second infection, the cellular content of phosphotyrosine proteins was determined. The presence of active YopH resulted in a rapid (detected within 5 min) dephosphorylation of the 120–130 kDa proteins (Figure 1C), suggesting that this protein class was a substrate of YopH.

YopHC403A associates with FAK and a highly phosphorylated form of p130^Cas

To determine whether YopHC403A interacted with the 120–130 kDa phosphotyrosine proteins immunoprecipitations using anti-YopH antisera were performed, employing the method described by Bliska et al. (1992). Immunoprecipitation of YopHC403A from infected cells resulted in a co-precipitation of 120–130 kDa phosphotyrosine proteins (Figure 2A). Interestingly, the association between YopHC403A and these proteins could be detected already after 2 min of infection and no phosphotyrosine proteins of other molecular weights were found in the precipitate. Furthermore, immunoprecipitation of YopHC403A depleted the 120–130 kDa phosphotyrosine proteins from the lysate (Figure 2B), demonstrating that the majority of this protein class was associated with the catalytically inactive YopH.

Figure 2.

YopHC403A interacts with FAK and a highly phosphorylated form of p130^Cas. (A) Immunoprecipitation of YopH from lysates of HeLa cells infected with MYMpyopHC403A; infection was done at a calculated bacteria:cell ratio of 100:1; at 37°C for the periods of time indicated. The precipitates were subjected to immunoblotting with anti-phosphotyrosine antibodies. (B) Immunoblot (anti-phosphotyrosine) from lysates of HeLa cells infected with MYMpyopHC403A before (-) and after (+) immunoprecipitation of the YopHC403A protein. The cells were infected for 60 min. (C) Immunoprecipitation of YopH from lysates of HeLa cells infected with MYM, MYMpyopH or MYMpyopHC403A for 60 min. The precipitates were subjected to immunoblotting with anti-phosphotyrosine antibodies, anti-p130^Cas antibodies or with anti-FAK antibodies. (D) Immunoblot (anti-p130^Cas) of immunoprecipitates of YopH or p130^Cas from lysates of HeLa cells infected with MYMpyopHC403A for 60 min.

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1-integrin mediated adhesion stimulates tyrosine phosphorylation of two proteins, p130^Cas and FAK, with molecular weights of 120–135 kDa (Nojima et al., 1995; Petch et al., 1995; Vuori and Rouslahti, 1995). This finding, and the fact that the uptake of Yersinia by HeLa cells is mediated by 1-integrins (Isberg and Tran van Nhieu, 1994) suggested that the phosphotyrosine proteins associated with YopHC403A were p130^Cas and/or FAK. Therefore, the YopH immunocomplexes were immunoblotted with anti-p130^Cas and anti-FAK antibodies (Figure 2C). Both p130^Cas and FAK were present in the YopH immunocomplex obtained from cells infected with the MYMpyopHC403A strain (Figure 2C). On the other hand, no tyrosine phosphorylated proteins were detected in YopH precipitates from cells infected with MYM or MYMpyopH (Figure 2C). The absence of FAK and p130^Cas in the immunocomplex of PTPase-active YopH indicated that the interaction of YopH with these proteins required them to be tyrosine phosphorylated.

To test the specificity of the association between YopHC403A, p130^Cas and FAK, we examined whether other phosphotyrosine proteins of similar molecular weight (Cbl, vinculin, 1-integrin and pp120) or proteins possibly involved in the regulation of p130^Cas and FAK (Crk, Src, Abl and paxillin) were present in the precipitate. The tested proteins were found in the lysates before and after precipitation of YopHC403A, but none of these were detected in the precipitate (data not shown). The anti-FAK antibody recognized a protein corresponding to the lower part of the 120–130 kDa YopHC403A immunocomplex, whereas the anti-p130^Cas antibody displayed a broad band with a migration correlating to the upper part of the 120–130 kDa protein class (Figure 2C). There are three forms of p130^Cas: CasA, CasB and CasC. The former two arise from alternative splicing and CasC is a highly phosphorylated form of the protein, which can be discriminated from the other two forms by PAGE (Sakai et al., 1994; Polte and Hanks, 1995). Most of the total p130^Cas protein (immunoprecipitated with anti-p130^Cas antibodies) had a lower molecular weight than the fraction of p130^Cas associated with YopHC403A (Figure 2D), implying that YopHC403A interacted specifically with the highly phosphorylated form of the protein i.e. CasC.

The above findings indicated that the 120–130 kDa proteins that were phosphorylated during infection with MYM and MYMpyopHC403A (see Figure 1) were p130^Cas and FAK. Immunoprecipitation of p130^Cas or FAK revealed that these proteins were tyrosine phosphorylated upon infection of HeLa cells with the strains indicated above, and as expected, the MYMpyopHC403A strain caused a more pronounced tyrosine phosphorylation of the proteins (Figure 3). The opposite was found when the wild-type YopH protein was studied: in this case, both p130^Cas and FAK were totally dephosphorylated (Figure 3), showing that p130^Cas and FAK were substrates of YopH. Moreover, these results did also suggest that these two proteins were involved in the 1-integrin associated signalling that mediates uptake of bacteria, and that dephosphorylation of these proteins by YopH impaired the uptake process.

Figure 3.

The p130^Cas protein and FAK are tyrosine phosphorylated upon bacterial infection and are dephosphorylated in the presence of YopH. Immunoprecipitation of p130^Cas or FAK from lysates of HeLa cells infected with MYM, MYMpyopH or MYMpyopHC403A at a calculated bacteria:cell ratio of 100:1 at 37°C for 60 min. The precipitates were subjected to immunoblotting with the corresponding antibodies or with the anti-phosphotyrosine antibody.

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FAK and p130^Cas are recruited to peripheral FAs upon infection

To analyse the subcellular localization of p130^Cas after bacterial infection, HeLa cells were infected with either MYMpyopH or MYMpyopHC403A; samples were taken at different time points and further processed for visualization by confocal microscopy. The p130^Cas protein was found distributed in the cytosol and enriched in the perinuclear region of uninfected cells (Figure 4A). After infection with MYMpyopHC403A, p130^Cas was detected in distinct regions lining the edges of the HeLa cell; after 30 min of infection 60% of the peripheral HeLa cells exhibited this type of staining pattern, and after 60 min this was seen in all peripheral cells and no morphological changes of the infected cells were detected (Figure 4A). When the MYMpyopH strain was used to infect HeLa cells, p130^Cas was not recruited to the distinct regions at the edges of the cells; instead, the cells became round in shape and were cytotoxically affected (Figure 4A). To ascertain whether the dotted staining pattern of p130^Cas at the edges of the infected HeLa cell was due to enrichment of this protein in peripheral FAs, double immunofluorescence staining of p130^Cas and FA component FAK was performed. The p130^Cas protein was found to consistently co-localize with FAK in distinct regions lining the edges of the MYMyopHC403A infected HeLa cells (Figure 4B). Although FAK was present in peripheral FAs before the infection (Figure 4B), it was evident that this protein also accumulated at the same sites as p130^Cas in response to MYMyopHC403A exposure. These results suggested that p130^Cas and FAK were recruited to peripheral FAs as a response to the MYMyopHC403A infection, and interestingly, this relocation correlated with the infection induced phosphorylation of FAK and p130^Cas.

Figure 4.

FAK and p130^Cas are recruited to peripheral focal adhesions upon infection with MYMpyopHC403A. (A) Confocal fluorescence images of HeLa cells infected with MYMpyopHC403A or MYMpyopH at 37°C for the indicated periods of time. Samples were analyzed for the presence of p130^Cas by laser scanning confocal microscopy using indirect immunofluorescence techniques. Localization of p130^Cas at the edges of the cells are indicated with arrowheads. All sections were scanned under identical conditions and show the basolateral side of the cells. Scale bar: 10 m. (B) Double staining of p130^Cas and FAK in HeLa cells infected with MYMpyopHC403A for 60 min. The leftmost and the middle picture shows the same section of one infected HeLa cell and the arrowheads indicate some of the overlapping staining pattern between the two proteins. The rightmost picture shows the localization of FAK in non-infected HeLa cells. All pictures show the basolateral side of the cells and no background staining of the bacteria are detected. Scale bar: 5 m.

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YopH disrupts FAs

The immunoprecipitation experiments showed that YopHC403A was associated with tyrosine phosphorylated forms of p130^Cas and FAK, and that these two FA components were dephosphorylated by active YopH. Therefore, we explored the possibility that YopH could have an effect on the integrity of the FAs and the associated cytoskeleton in the target cell. HeLa cells, either not infected or infected for different periods of time with MYM, MYMyopHC403A or MYMyopH, were fixed and permeabilized, thereafter cellular F-actin was stained with FITC conjugated phalloidin, and the FAs were visualized by indirect immunofluorescence using an antibody recognizing vinculin. Infection with the MYM or MYMpyopHC403A strains had no destructive effects on these structures and gave the same type of staining pattern as in the non-infected control cells. The microfilament bundles were evenly distributed and the FA marker vinculin decorated the ends of the actin fibres facing the basal lateral side of the cell, even after 50 min of infection (Figure 5). Notably, a 4-fold increase in the number of FAs was observed after 30 min of infection with these strains (data not shown). When the MYMpyopH strain was used to infect HeLa cells, a different staining pattern was observed. In this case, the F-actin stress fibres were progressively disrupted with increasing time of infection, and after 50 min these structures were completely absent (Figure 5). The cortical actin, however, could still be seen as ring-shaped structures lining the margin of the cytotoxically affected cells. Interestingly, the presence of active YopH resulted in a rapid disappearance of FA structures (Figure 5), and it is therefore likely that the YopH mediated change in the morphology of the infected HeLa cells was a consequence of a direct effect of YopH on the integrity of FAs.

Figure 5.

The PTPase activity of YopH affects the integrity of focal adhesions. HeLa cells non-infected or infected with MYM, MYMpyopHC403A or MYMpyopH. This was done at a calculated bacteria:cell ratio of 20:1 at 37°C for the periods of time indicated. Thereafter, the cells were fixed, the microfilaments were visualized by staining with fluorescein conjugated phalloidin (green), and vinculin containing focal adhesions were visualized by indirect immunofluorescence (red). The yellow colour represents co-localization of microfilaments and vinculin. Vinculin-containing focal adhesions (arrowheads) and vinculin-containing retraction fibres (arrows) are shown. All sections were scanned under identical conditions and show the basolateral side of the cells. Scale bar: 10 m.

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The spatial localization of YopH and YopHC403A was investigated after infection of HeLa cells using either MYMpyopH or MYMpyopHC403A strains. YopHC403A was enriched at focal adhesion sites lining the edges of the cells and co-localized with the FA proteins paxillin and vinculin at these sites (Figure 6). Consistent with the biochemical data, immunofluorescence staining revealed that YopHC403A co-localized with p130^Cas at focal adhesions (Figure 6). The YopHC403A protein was also seen in the cytosol of the infected cells, this phenomenon increased with time and seemed to be a result of 'overloading' (Figure 6). By contrast, when the strain expressing active YopH was used to infect HeLa cells, YopH was evenly distributed in the cytosol together with the main pool of FA marker proteins, and no obvious co-localization between YopH and the FA proteins could be detected (Figure 6). The p130^Cas protein was only detected in the cytosol whereas paxillin and vinculin were also found in retraction fibres, distinct from native FA structures (Figure 6).

Figure 6.

YopHC403A localizes to focal adhesions. The localization of YopHC403A/YopH (green) and proteins localized at FAs (paxillin, vinculin; red) or p130^Cas (red) was determined by confocal microscopy of HeLa cells infected with either MYMpyopHC403A or MYMpyopH at 37°C, for 30 min. Co-localization between YopHC403A and FA proteins are seen as yellow colour (arrowheads). The presence of the PTPase YopH results in cytotoxically affected HeLa cells and therefore they appear smaller compared with the flat unaffected MYMpyopHC403A infected cells. All sections were scanned under identical conditions and show the basolateral side of the cells. Scale bar, 10 m.

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The observed enrichment of YopHC403A at FAs indicated the target site of this enzyme. Thus, the active form of YopH present in a high local concentration at this intracellular location should be able to compete with and displace YopHC403A. With that in mind, a reinfection experiment was performed in which the HeLa cells were infected with the MYMpyopHC403A strain for 60 min and then infected again but now with the MYMpyopH strain. The active PTPase YopH released YopHC403A from FAs in a time dependent fashion (compare Figure 7 with 1C), after 60 min FAs were disrupted; and consequently, no YopH antigen was found associated with either FAs or with the vinculin and paxillin containing retraction fibres.

Figure 7.

Displacement of YopHC403A from focal adhesions by active YopH. HeLa cells were infected with MYMpyopHC403A at a calculated bacteria:cell ratio of 20:1 at 37°C, for 60 min. Thereafter, the cells were incubated for an additional 60 min or were infected again, but now with MYMpyopH for 30 or 60 min. The localization of YopH/YopHC403A (green) and the FA-component vinculin (red) was determined by confocal microscopy. Co-localization between YopHC430A and vinculin is seen as yellow colour (arrow heads) and vinculin containing retraction fibres are indicated with an arrow. All sections were scanned under identical conditions and show the basolateral side of the cells. Scale bar: 10 m.

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Our study focused on one specific interaction between the bacteria and the eukaryotic target cell: the invasin stimulated, 1-integrin mediated uptake of Y.pseudotuberculosis by HeLa cells. This model system limits the number of potential signalling pathways involved in bacterial uptake, to encompass possibly only one. To explore the virulence determinant YopH without any interference of other Yops, we used a specifically engineered bacterial strain to introduce this PTPase alone into the interior milieu of the target cell. By using this minimalistic approach, we were able to identify two eukaryotic proteins, p130^Cas and FAK, that were phosphorylated on tyrosine in response to interaction with the bacteria and that were recognized and dephosphorylated by YopH. Moreover, this activity of YopH resulted in disruption of FA structures and this was associated with impaired ability of the target cell to ingest the pathogen. This conclusion is based on results obtained by using three different experimental approaches: bacterial genetics, biochemistry and confocal microscopy studies. Infection of HeLa cells with a YopH mutant strain resulted in a rapid increase in tyrosine phosphorylation of FAK and p130^Cas. This phosphorylation was not observed in the presence of PTPase-active YopH. In addition, active YopH rapidly dephosphorylated infection induced phosphorylated forms of FAK and p130^Cas, and this was observed already 5 min after addition of the YopH expressing strain. Although YopH has a broad substrate specificity upon prolonged incubations (Bliska et al., 1991, 1992; Hartland et al., 1994), these rapid events were only observed for FAK and p130^Cas. In accordance, phosphorylated forms of FAK and p130^Cas were found to bind to the catalytically inactive mutant form of YopH (YopHC403A). This interaction was observed within 2 min after infection, and no other phosphotyrosine proteins were detected in the immunocomplex. Interestingly, at this very early time point, FAK and p130^Cas constituted a minor class of tyrosine phosphorylated proteins in the HeLa cells, indicating a high specificity of YopH for these proteins. Prolonged incubation resulted in elevated amounts of FAK and p130^Cas in the immunocomplex, and this paralleled the infection induced phosphorylation of these proteins (see Figures 1 and 2). Moreover, the precipitation of YopH depleted the HeLa cell lysate of the majority of tyrosine phosphorylated FAK and p130^Cas, further strengthening the hypothesis that phosphorylated FAK and p130^Cas constitute targets for YopH. These biochemical results were confirmed by confocal microscopy, where we could demonstrate that the inactive form of YopH co-localized with FAK and p130^Cas in peripheral FAs in situ. We could also show that PTPase active YopH disrupted FAK and p130^Cas containing focal adhesion, paralleling the results obtained from the biochemical results (see Figures 1C and 7).

The uptake of the pathogen into eukaryotic cells is dependent on binding of the bacterial protein invasin to 1-integrins present on the surface of the target cell (Isberg and Tran van Nhieu, 1994). The interaction between the bacteria and the target cell results in occupancy and clustering of the 1-integrin receptors, a condition that has been shown to generate a phosphotyrosine signal that is essential for linkage of 1-integrins to F-actin via FAs (Miyamoto et al., 1995b). It is likely that bacterial uptake stimulated by invasin binding to 1-integrins generates an assembly of FA components, similar to the assembly seen in experiments with 1-integrin ligand coated beads (Plopper and Ingber, 1993; Miyamoto et al., 1995a,b; Plopper et al., 1995). In line with this, the invasin stimulated uptake requires tyrosine kinase activity and is blocked by tyrosine kinase inhibitors (Rosenshine et al., 1992; Andersson et al., 1996). Therefore, it can be assumed that YopH counteracts the phosphotyrosine signal that is involved in the uptake of Yersinia mediated by 1-integrin receptors.

FAK is located in FAs and it has been implicated in the regulation of these multi-component structures (Richardson and Parsons, 1995, 1996). We found that infection with strains lacking YopH activity caused an increased tyrosine phosphorylation of FAK and an accumulation of this protein in peripheral FAs. A similar observation was recently presented by Sasakawa and co-workers who showed that increased tyrosine phosphorylation of FAK was involved in 1-integrin mediated uptake of Shigella flexneri by CHO cells (Watarai et al., 1996). These two findings suggest a role of FAK in 1-integrin mediated bacterial uptake. It is known that FAK is tyrosine phosphorylated upon 1-integrin clustering, and interestingly, in a recent study it was suggested that tyrosine phosphorylation of FAK is important in regulating the formation of new FAs (Richardson and Parsons, 1996). The role of FAK in FA assembly has been questioned, since FAK deficient cells were shown to contain FAs (Ilic et al., 1995). These kinds of results are, however, difficult to interpret due to functional redundancy and the FAK^-/- cells did in fact exhibit defects in migration, suggesting a role of FAK in FA remodelling during cell motility (Ilic et al., 1996). Gilmore and Romer (1996) used a GST–C-terminal fusion of FAK that were loaded into cells and found similar effects on cellular migration. Consequently, although the exact role of FAK in FA turnover is unclear, it is, based on the data presented herein, reasonable to suggest that the observed dephosphorylation of FAK by YopH could interfere with remodelling and/or assembly of FA complexes involved in 1-integrin mediated internalization of the bacteria.

Phosphorylation of p130^Cas causes a relocalization of this protein from the cytosolic fraction to the membrane fraction (Sakai et al., 1994). It is likely that the membrane association reflects the presence of phosphorylated p130^Cas at FAs, an assumption supported by the recent findings that p130^Cas interacts with the FA component FAK (Polte and Hanks, 1995; Harte et al., 1996). This is further strengthened by our finding that upon infection both FAK and p130^Cas were recruited to peripheral FAs that contained YopHC403A. Interestingly, this relocation correlated with the infection induced phosphorylation of both FAK and p130^Cas. The pathogen binds to the 1-integrin exposed at the edges of a cell (Rosqvist et al., 1991; Young et al., 1992), i.e. in the region of peripheral FAs in which p130^Cas and FAK were accumulated. Importantly, at early time points the bacteria are in close contact with FAs at the edges of the infected HeLa cells. Enrichment of FA protein-containing structures in the cell periphery represents FAs under formation (Nobes and Hall, 1995; Richardson and Parsons, 1996), and inhibition of FAK tyrosine phosphorylation results in an impaired formation of such structures (Richardson and Parsons, 1996). Consequently, the observed co-localization of YopHC403A and the p130^Cas and FAK proteins at this site supports our hypothesis that the YopH PTPase exerts its activity in the vicinity of the bacterium and that the site of action is FAs under formation. Moreover, no p130^Cas was present in FAs facing the basal lateral side of the cell, and, in contrast to the FA component vinculin, p130^Cas did not associate with FAs in uninfected cells. This, and the fact that p130^Cas was recruited to this site of action, suggests that this protein plays a specific role in FA formation that is important for the uptake process.

In contrast to YopHC403A, which was enriched in target cell FAs, active YopH was found evenly distributed in the cytosol, HeLa cells exposed to active YopH detached peripherally from the underlying substratum and became round in shape and the F-actin stress fibres and FAs in these cells were disrupted. Due to the observed interactions of YopHC403A with FA proteins, it is reasonable to believe that FAs are the primary site for the YopH PTPase activity. Active YopH could release YopHC403A from FAs which indicates that active YopH dephosphorylated the molecular targets. The involvement of endogenous PTPases in the regulation of FAs and associated stress fibres has been suggested in several studies. Treatment of cells with PTPase inhibitors has been found to increase the number of FAs and to protect FAs and stress fibres from cytochalasin D mediated disruption (Nobes et al., 1994; Defilippi et al., 1995). In addition, the transmembranous PTPase LAR (leukocyte common antigen related), which is a homologue of YopH, is present in FAs, where it is associated with the proximal (disassembled) ends of the adhesion complex (Serra-Pagès et al., 1995). This further indicates the involvement of such enzyme activity in regulation of these structures (Serra-Pagès et al., 1995). Together, all of these findings strongly support a direct effect of YopH on FAs. This is reinforced by our observation that YopH specifically dephosphorylated the FA associated proteins p130^Cas and FAK, and it is possible that dephosphorylation of these two proteins leads to disassembly of FAs.

Since the presence of YopH causes inhibition of phagocytic uptake of Yersinia by cells of the non-specific immune defence (Rosqvist et al., 1988; Fällman et al., 1995; Andersson et al., 1996) it is tempting to speculate about the molecular mechanism that mediates inhibition in this system. The uptake of non-opsonized Yersinia by phagocytes resembles that in HeLa cells with respect to the requirement of the presence of invasin on the bacterial surface (Fällman et al., 1995). It is therefore likely that YopH mediated dephosphorylation of FAK, p130^Cas or related proteins thereof is of importance for phagocytic inhibition. Interestingly, upon exposure to macrophages YopH causes a very rapid dephosphorylation of phosphotyrosine proteins in the 120 kDa range. The majority of the tyrosine phosphorylated proteins are located in the detergent insoluble fraction, suggesting it to be cytoskeletally associated. In accordance with this, a 120 kDa phosphotyrosine protein co-precipitates with an inactive form of YopH (Bliska et al., 1992) (unpublished data). In addition, YopH also inhibits other kinds of phagocytic uptake like those mediated by complement receptors (2-integrin receptors; Ruckdeschel et al., 1996) and Fc receptors (Fällman et al., 1995). Noteworthy in this context is that also these types of receptors mediate phosphorylation of FAK or p130^Cas upon stimulation (Haimovich et al., 1996; Petruzelli et al., 1996). It will be of particular importance to further elucidate the role of FAK, p130^Cas or related proteins in phagocytic cells.

Antibodies

The following antibodies were used: anti-Crk mAb (1:2000), anti-pp120 mAb (1:2000), anti-paxillin mAb (1:50), anti-p130^Cas mAb (1:20; Transduction Laboratories, Lexington, KY), anti-Src mAb (1:2000), anti-FAK mAb 2A7 (Upstate Biotechnology, Lake Placid, NY), anti-1-integrin K20 (1:2000; Immunotech, Marseille-Cedex, France), anti-Abl mAb 24-11(1:2000), anti-Cbl mAb C15 (1:1000), rabbit polyclonal anti-FAK antibodies C-20 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-vinculin mAb VIN-11-5 (1:50; Sigma Chemical Company, St Louis, MO), rabbit polyclonal anti-p130^Cas antibodies (1:2000; H. Hirai) and goat polyclonal anti-YopH antibodies that recognized YopH and YopHC403A equally well. The YopH antisera were affinity purified as described previously (Persson et al., 1995) and the rabbit polyclonal anti-FAK antibodies were affinity purified against the GST–FAK fusion protein (amino acids 903–1052; 902; Santa Cruz Biotechnology). The following secondary antibodies were used: FITC conjugated donkey anti-goat antibodies (1:100), FITC conjugated donkey anti-rabbit antibodies (1:100), Lissamine conjugated donkey anti-mouse antibodies (1:100) and Lissamine conjugated donkey anti-rabbit antibodies (1:100; Jackson Immuno Research Laboratories Inc., West Grove, PA). Horseradish peroxidase conjugated antibodies: recombinant anti-phosphotyrosine antibody RC20 (1:2500; Transduction Laboratories, Lexington, KY), sheep anti-mouse or donkey anti-rabbit antibodies (1:4000; Amersham Sweden AB, Solna, Sweden). The dilutions given were used for Western blotting or immunostaining. To increase the sensitivity of the secondary antibodies used in immunofluorescence staining, the antiseras were run through six cycles of subtractive immunoabsorption to HeLa cells, as described in detail elsewhere (Persson et al., 1995).

Bacterial strains

The Y.pseudotuberculosis strains and plasmids used in this study are listed in Table I. The plasmids were isolated using standard methods (Sambrook et al., 1989) and introduced into the multiple mutant strain by electroporation (Conchas and Carniel, 1990) using a Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, CA).

Cultivation and infection of HeLa cells

HeLa cells were cultured in Leibovitz L-15 medium containing 10% heat inactivated fetal calf serum (FCS) and 100 IU/ml penicillin at 37°C in a humidified atmosphere. For detection of bacterial uptake or immunofluorescent staining, HeLa cells were seeded on sterile cover slips (12 mm; 110⁵ cells/cm²) placed in 24-well tissue culture plates 2 days prior to infection. On day 2, the HeLa cultures were washed free from penicillin, and 1 ml Leibovitz L-15 medium containing 10% heat-inactivated FCS was added. The Y.pseudotuberculosis strains used to infect HeLa cells were grown overnight at 26°C in Luria broth (LB) medium. The suspension was diluted 200 times in Leibovitz L–15 medium containing 10% heat inactivated FCS without antibiotics and grown for 1 h at 37°C. Thereafter, 0.1 ml bacteria suspension was added to the HeLa cells (210⁶ bacteria/well). To facilitate contact between bacteria and cells, the infected cell cultures were centrifuged for 3 min at 400 g and then incubated at 37°C.

For immunoprecipitation and Western blot experiments, HeLa cells grown to semi-confluence in 280 cm² cell culture bottles (1–310⁷/cells) were washed twice with 37°C PBS and incubated in penicillin-free culture medium for 2 h. The cells were infected by replacing the medium with 4 ml bacterial suspension that gave a calculated bacteria:HeLa cell ratio of 100:1.

Detection of bacterial uptake by HeLa cells

HeLa cells grown on cover slips were infected with different strains of Y.pseudotuberculosis, and the intra- and extracellular locations of bacteria associated with target cells were distinguished by a double fluorescent antibody test described elsewhere (Heesemann and Laufs, 1985; Rosqvist et al., 1988). The specimens were examined in a fluorescent microscope (Zeiss Axioskop 50) with epifluorescence illumination and a 100/1.4 plan-apochromate oil immersion lens (final magnification 1000). In each experiment, 80 HeLa cells were counted in randomly selected fields without previewing. Extracellularly located bacteria were detected with a rhodamine filter system, and the total number of cell associated (intracellular and extracellular) bacteria was determined with a fluorescein filter system.

Cell lysis and immunoprecipitation

Infections were terminated by removing the bacteria, washing the HeLa cells with 37°C phosphate buffered saline (PBS) pH 7.4 and then adding 4 ml ice-cold immunoprecipitation buffer pH 7.4 (50 mM Tris–HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM Na₃VO₄, 1 mM NaF and Complete™ protease inhibitor cocktail (Boehringer Mannheim Scandinavia AB, Bromma, Sweden). The cells were lysed for 30 min at 4°C and the lysates were clarified by centrifugation at 14 000 g for 10 min. For detection of total cellular phosphotyrosine proteins, aliquots of the clarified lysates were heated in an equal volume of 2 sample buffer for 5 min at 90°C and subjected to Western blot. For immunoprecipitations, the clarified lysates were precleared by incubation for 1 h at 4°C with protein G–Sepharose beads (Pharmacia Biotech Norden, Sollentuna, Sweden) precoupled to preimmune goat IgG or normal mouse IgG. The beads were then pelleted by a brief centrifugation, and the supernatants were incubated for 3 h at 4°C with protein G–Sepharose beads (20 l beads/ml lysate) precoupled to goat anti-YopH IgG or anti-p130^Cas mAb or anti-FAK (mAb 2A7). The immunocomplexes were washed three times in immunoprecipitation buffer, heated in 2 sample buffer for 5 min at 90°C, and subjected to Western blot analysis.

Western blot

Proteins were separated by 7.5% SDS–PAGE according to Laemmli (1970), and then electrotransferred to Immobilon-P (Millipore Corp., Bedford, MA). For detection of phosphotyrosine proteins, the membranes were incubated with the horse radish peroxidase conjugated recombinant anti-phosphotyrosine antibody RC20 as recommended by the manufacturer (Transduction Laboratories). For detection of specific proteins the membranes were blocked in Tris buffered saline containing 5% milk powder and 0.1% Tween 20 and then incubated with primary antibodies. Thereafter the membranes were washed and incubated with horseradish peroxidase conjugated secondary antibodies. The proteins were detected using a ECL chemoluminescence kit (Amersham Sweden AB, Solna, Sweden).

Immunofluorescence staining

Bacterial infections were terminated by washing the cell monolayers twice in PBS. The cells were fixed in 2% paraformaldehyde, permeabilized with 0.5% Triton X-100 and further processed for indirect immunofluorescence labelling. To visualize microfilaments and the integrity of FAs in the target cell, the fixed and permeabilized cells were incubated with FITC conjugated Phalloidin (0.5 ng/ml; Sigma Chemical Company, St Louis, MO) for 30 min and then overlaid with the anti-vinculin mAb for 1 h, followed by 1 h with purified Lissamine conjugated donkey anti-mouse secondary antibodies. In double labelling experiments, the cells were incubated with primary mAbs against FA proteins and then with the Lissamine conjugated secondary antibodies. The specimens were then incubated with goat anti-YopH antibodies or anti-FAK antibodies, and then with the FITC conjugated secondary antibodies. All incubations were performed at 37°C, and the specimens were mounted in a medium containing Cityflour (Cityflour Limited, London, UK).

Fluorescence confocal microscopy and image processing

The fluorescence images of infected HeLa cells were obtained with a confocal laser scanning microscope equipped with dual detectors and an argon-krypton (Ar/Kr) laser for simultaneous scanning of two fluorochromes (Multiprobe 2001, Molecular Dynamics, Sunnyvale, CA). The same section was scanned simultaneously for Lissamine (excitation 568 nm) and FITC (excitation 488 nm) tagged markers. In each experiment, laser power and gain were set by using cells labelled with either fluorochrome alone, so that there was no crossover of signals from green to red or red to green channels. The sections with the image size of 10241024 were scanned with 0.07 m pixel size, 0.3 m step size and the pinhole setting was 100 m. To determine possible co-localization between FA proteins and YopH/YopHC403A in the infected cell, the images obtained were scrutinized with the ImageSpace™ version 3.10 software (Molecular Dynamics). Pixels with intensity values >100 in both the green and red channels were considered to co-localize. The co-localizing pixels were visualized as a light yellow colour on top of the companion sections (red and green channel); the companion sections were overlaid in some of the pictures.

We thank Drs H.Semb, C.Sentman and S.Tuck for helpful discussions and critical reading and P.Ödman for linguistic revision of the manuscript. This work was supported by the Swedish Medical Research Council, the Swedish Natural Science Research Council, the King Gustaf Vth 80 year Foundation, the Swedish Rheumatism Association, the Magnus Bergvalls Foundation and the Medical Faculty Research Foundation at Umeå University. C.P. and N.C. were recipients of grants from the Kempe foundation of the University of Umeå. We thank Dr Hisamaru Hirai for anti-p130^Cas antibodies.

Andersson K, Carballeira N, Magnusson K-E, Persson C, Stendahl O, Wolf-Watz H and Fällman M (1996) YopH of Yersinia pseudotuberculosis interrupts early phosphotyrosine signalling associated with phagocytosis. Mol Microbiol, 20, 1057–1069. | Article | PubMed | ISI | ChemPort |

Bliska JB, Guan K, Dixon JE and Falkow S (1991) Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. Proc Natl Acad Sci USA, 88, 1187–1191. | Article | PubMed | ChemPort |

Bliska JB, Clemens JC, Dixon JE and Falkow S (1992) The Yersinia tyrosine phosphatase: specificity of a bacterial virulence determinant for phosphoproteins in the J774A.1 macrophage. J Exp Med, 176, 1625–1630. | Article | PubMed | ISI | ChemPort |

Burnham MR, Harte MT, Richardson A, Parsons JT and Bouton AH (1996) The identification of p130^cas-binding proteins and their role in cellular transformation. Oncogene, 12, 2467–2472. | PubMed | ISI | ChemPort |

Burridge K, Turner CE and Romer LH (1992) Tyrosine phosphorylation of paxillin and pp125^FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol, 119, 893–903. | Article | PubMed | ISI | ChemPort |

Bölin I and Wolf-Watz H (1984) Molecular cloning of temperature-inducible outer membrane protein 1 of Yersinia pseudotuberculosis. Infect Immun, 43, 72–78. | PubMed | ISI |

Chang AYC and Cohen SNJ (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derivied from the P15A cryptic miniplasmid. J Bacteriol, 134, 1141–1156. | PubMed | ISI | ChemPort |

Clark EA and Brugge JS (1995) Integrins and signal transduction pathways: the road taken. Science, 268, 233–239. | Article | PubMed | ISI | ChemPort |

Conchas R and Carniel E (1990) A highly efficient electroporation system for transformation of Yersinia. Gene, 87, 133–137. | Article | PubMed | ISI | ChemPort |

Cornelis GR (1992) Yersiniae, finely tuned pathogens. In Hormache,C.E., Penn,C.W. and Smythe,C.J. (eds), Molecular Biology of Bacterial Infection. Society for General Microbiology Symposium 49, Cambridge University Press, Cambridge, UK, pp. 231–265.

Defilippi P, Retta SF, Olivo C, Palmieri M, Venturino M, Silengo L and Tarone G (1995) p125^FAK tyrosine phosphorylation and focal adhesion assembly: studies with phosphotyrosine phosphatase inhibitors. Exp Cell Res, 221, 141–152. | Article | PubMed | ISI | ChemPort |

Dunlevy JR and Couchman JR (1993) Controlled induction of focal adhesion disassembly and migration in primary fibroblasts. J Cell Sci, 105, 489–500. | PubMed | ISI | ChemPort |

Forsberg Å, Rosqvist R and Wolf-Watz H (1994) Regulation and polarized transfer of the Yersinia outer proteins (Yops) involved in antiphagocytosis. Trends Microbiol, 2, 14–19. | Article | PubMed | ChemPort |

Fällman M, Andersson K, Håkansson S, Magnusson K-E, Stendahl O and Wolf-Watz H (1995) Yersinia pseudotuberculosis inhibits Fc receptor-mediated phagocytosis in J774 cells. Infect Immun, 63, 3117–3124. | PubMed | ISI | ChemPort |

Gilmore AP and Romer LH (1996) Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decrease cell motility and proliferation. Mol Biol Cell, 7, 1209–1224. | PubMed | ISI | ChemPort |

Green SP, Hartland EL, Robins-Browne RM and Philips WA (1995) Role of YopH in the supression of tyrosine phosphorylation and respiratory burst activity in murine macrophages infected with Yersinia enterocolitica. J Leukoc Biol, 57, 972–977. | PubMed | ISI | ChemPort |

Guan K and Dixon JE (1990) Protein tyrosine phosphatase activity of an essential virulence determinant in Yersinia. Science, 249, 553–556. | Article | PubMed | ISI | ChemPort |

Haimovich B, Regan C, DiFazio L, Ginalis E, Ji P, Purohit U, Rowley RB, Bolen J and Greco R (1996) The FcRII receptor triggers pp125^FAK phosphorylation in platelets. J Biol Chem, 271, 16332–16337. | Article | PubMed | ISI | ChemPort |

Hanski C, Kutschka U and Schmoranzer HP (1989) Immunohistochemical and electron microscopic study of interaction of Yersinia enterocolitica serotype O:8 with intestinal mucosa during experimental enteritis. Infect Immun, 57, 673–678. | PubMed | ISI | ChemPort |

Harte MT, Hildebrand JD, Burnham MR, Bouton AH and Parsons JT (1996) p130^Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase. J Biol Chem, 271, 13649–13655. | Article | PubMed | ISI | ChemPort |

Hartland EL, Green SP, Phillips WA and Robins-Browne RM (1994) Essential role of YopD in inhibition of respiratory burst of macrophages by Yersinia enterocolitica. Infect Immun, 62, 4445–4453. | PubMed | ISI | ChemPort |

Heesemann J and Laufs R (1985) Double immunofluorescence microscopic technique for accurate differentiation of extracellular and intracellularly located bacteria in cell culture. J Clin Microbiol, 22, 168–175. | PubMed | ISI | ChemPort |

Hildebrand JD, Schaller MD and Parsons JT (1993) Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125^FAK, to cellular focal adhesions. J Cell Biol, 123, 993–1005. | Article | PubMed | ISI | ChemPort |

Hotchin NA and Hall A (1995) The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. J Cell Biol, 131, 1857–1865. | Article | PubMed | ISI | ChemPort |

Håkansson S, Galyov EE, Rosqvist R and Wolf-Watz H (1996) The Yersinia YpkA Ser/Thr kinase is translocated and subsequently targetted to the inner surface of the HeLa cell plasma membrane. Mol Microbiol, 20, 593–603. | Article | PubMed | ISI | ChemPort |

Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto J, Okada M, Yamamoto T and Aizawa S (1995) Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature, 377, 539–544. | Article | PubMed | ChemPort |

Ilic D, Kanazawa S, Furuta Y, Yamamoto T and Aizawa S (1996) Impairment of mobility in endodermal cells by FAK deficiency. Exp Cell Res, 222, 298–303. | Article | PubMed | ISI | ChemPort |

Isberg RR and Tran van Nhieu G (1994) Binding and internalization of microorganisms by integrin receptors. Trends Microbiol, 2, 10–14. | Article | PubMed | ChemPort |

Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature, 227, 680–685. | Article | PubMed | ISI | ChemPort |

Lo SH and Chen LB (1994) Focal adhesion as a signal transduction organelle. Cancer Metastasis Rev, 13, 9–24. | PubMed | ISI | ChemPort |

Mauro LJ and Dixon JE (1994) 'Zip codes' direct intracellular protein tyrosine phosphatases to the correct cellular 'address'. Trends Biol Sci, 19, 151–155. | Article | ChemPort |

Miyamoto S, Akiyama SK and Yamada KM (1995a) Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science, 267, 883–885. | Article | PubMed | ISI | ChemPort |

Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo PD, Akiyama SK and Yamada KM (1995b) Integrin function: Molecular hierarchies of cytoskeletal and signaling molecules. J Cell Biol, 131, 791–805. | Article | PubMed | ISI | ChemPort |

Nakamura TY, Yamamoto I, Nishitani H, Matozaki T, Suzuki T, Wakabayashi S, Shigekawa M and Goshima K (1995) Detachment of cultured cells from the substratum induced by the neutrophil-derived oxidant NH₂Cl: Synergistic role of phosphotyrosine and intracellular Ca²⁺ concentration. J Cell Biol, 131, 509–524. | PubMed | ISI | ChemPort |

Nobes CD and Hall A (1995) Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lammellipodia, and filopodia. Cell, 81, 53–62. | Article | PubMed | ISI | ChemPort |

Nobes CD, Hawkins P, Stephens L and Hall A (1994) Activation of the small GTP-binding proteins rho and rac by growth factor receptors. J Cell Sci, 108, 225–233. | ISI |

Nojima Y, Morino N, Mimura T, Hamasaki K, Furuya H, Sakai R, Sato T, Tachibana K, Morimoto C and Yazaki Y (1995) Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130^Cas, a Src homology 3-containing molecule having multiple Scr homology 2-binding motifs. J Biol Chem, 270, 15398–15402. | Article | PubMed | ISI | ChemPort |

Persson C, Nordfelth R, Holmström A, Håkansson S, Rosqvist R and Wolf-Watz H (1995) Cell-surface-bound Yersinia translocate the protein tyrosine phosphatase YopH by a polarized mechanism into the target cell. Mol Microbiol, 18, 135–150. | Article | PubMed | ISI | ChemPort |

Petch LA, Bockholt SM, Bouton A, Parsons JT and Burridge K (1995) Adhesion-induced tyrosine phosphorylation of the p130 Src substrate. J Cell Sci, 108, 1371–1379. | PubMed | ChemPort |

Petruzelli L, Takami M and Herrera R (1996) Adhesion through the interaction of lymphocyte function-associated antigen-1 with intracellular adhesion molecule-1 induces tyrosine phosphorylation of p130^cas and its association with c-CrkII. J Biol Chem, 271, 7796–7801. | Article | PubMed | ChemPort |

Plopper G and Ingber DE (1993) Rapid induction and isolation of focal adhesion complexes. Biochem Biophys Res Commun, 193, 571–578. | Article | PubMed | ISI | ChemPort |

Plopper GE, McNamee HP, Dike LE, Bojanowski K and Ingber DE (1995) Converge of integrin and growth factor receptor signaling pathways within the focal adhesion complex. Mol Biol Cell, 6, 1349–1365. | PubMed | ISI | ChemPort |

Polte TR and Hanks SK (1995) Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130^Cas. Proc Natl Acad Sci USA, 92, 10678–10682. | Article | PubMed | ChemPort |

Richardson A and Parsons JT (1995) Signal transduction through integrins: a central role for focal adhesion kinase? Bioessays, 17, 229–236. | Article | PubMed | ISI | ChemPort |

Richardson A and Parsons T (1996) A mechanism for regulation of the adhesion-associated protein tyrosine kinase pp125^FAK. Nature, 380, 538–540. | Article | PubMed | ISI | ChemPort |

Ridley AJ and Hall A (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell, 70, 389–399. | Article | PubMed | ISI | ChemPort |

Rosenshine I, Duronio V and Finlay BB (1992) Tyrosine protein kinase inhibitors block invasin-promoted bacterial uptake by epithelial cells. Infect Immun, 60, 2211–2217. | PubMed | ISI | ChemPort |

Rosqvist R, Bölin I and Wolf-Watz H (1988) Inhibition of phagocytosis in Yersinia pseudotuberculosis: a virulence plasmid-encoded ability involving the Yop2b protein. Infect Immun, 56, 2139–2143. | PubMed | ISI | ChemPort |

Rosqvist R and Wolf-Watz H (1986) Virulence plasmid-associated HeLa cell induced cytotoxicity of Yersinia pseudotuberculosis. Microb Pathog, 1, 229–240. | Article | PubMed | ISI | ChemPort |

Rosqvist R, Skurnik M and Wolf-Watz H (1988) Increased virulence of Yersinia pseudotuberculosis by two independent mutations. Nature, 334, 522–525. | Article | PubMed | ISI | ChemPort |

Rosqvist R, Forsberg Å and Wolf-Watz H (1991) Intracellular targeting of the Yersinia YopE cytotoxin in mammalian cells induces actin microfilament disruption. Infect Immun, 59, 4562–4569. | PubMed | ISI | ChemPort |

Rosqvist R, Magnusson K-E and Wolf-Watz H (1994) Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. EMBO J, 13, 964–972. | PubMed | ISI | ChemPort |

Ruckdeschel K, Roggenkamp A, Schubert S and Heesemann J (1996) Differential contribution of Yersinia enterocolitica virulence factors to evasion of microbiocidal action of neutrophils. Infect Immun, 64, 724–733. | PubMed | ISI | ChemPort |

Sakai R, Iwamatsu A, Hirano N, Ogawa S, Tanaka T, Mano H, Yazaki Y and Hirai H (1994) A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner. EMBO J, 13, 3748–3756. | PubMed | ISI | ChemPort |

Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Sping Harbor Laboratory Press, Cold Spring Harbor, NY.

Serra-Pagès C, Kedersha NL, Fazikas L, Medley Q, Debant A and Streuli M (1995) The LAR transmembrane protein tyrosine phosphatase and a coiled-coil LAR-interacting protein co-localize at focal adhesions. EMBO J, 14, 2827–2838. | PubMed | ISI | ChemPort |

Simonet M, Richard S and Berche P (1990) Electron microscopic evidence for in vivo extracellular localization of Yersinia pseudotuberculosis harboring the pYV plasmid. Infect Immun, 58, 841–845. | PubMed | ISI | ChemPort |

Sory M-P and Cornelis GR (1994) Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol Microbiol, 14, 583–594. | Article | PubMed | ISI | ChemPort |

Sory M-P, Boland A, Lambermont I and Cornelis GR (1995) Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of the macrophages, using the cyaA gene fusion approach. Proc Natl Acad Sci USA, 92, 11998–12002. | Article | PubMed | ChemPort |

Straley SC, Skrzypek E, Plano GV and Bliska JB (1993) Yops of Yersinia spp. pathogenic for humans. Infect Immun, 61, 3105–3110. | PubMed | ISI | ChemPort |

Vuori K and Rouslahti E (1995) Tyrosine phosphorylation of p130^Cas and cortactin accompanies integrin-mediated cell adhesion to extracellular matrix. J Biol Chem, 270, 22259–22262. | Article | PubMed | ISI | ChemPort |

Vuori K, Hirai H, Aizawa S and Rouslahti E (1996) Induction of p130^cas signaling complex formation upon integrin-mediated cell adhesion: a role for Scr family kinases. Mol Cell Biol, 16, 2606–2613. | PubMed | ISI | ChemPort |

Watarai M, Funato S and Sasakawa C (1996) Interaction of Ipa proteins of Shigella flexneri with 51 integrin promotes entry of the bacteria into mammalian cells. J Exp Med, 183, 991–999. | Article | PubMed | ISI | ChemPort |

Young VB, Falkow S and Schoolnik GK (1992) The invasin protein of Yersinia enterocolitica: Internalization of Invasin-bearing bacteria by eukaryotic cells is associated with reorganization of the cytoskeleton. J Cell Biol, 116, 197–207. | Article | PubMed | ISI | ChemPort |

Zhang Z-Y, Clemens JC, Schubert HL, Stuckey JA, Fischer MWF, Hume DM, Saper MA and Dixon JE (1992) Expression, purification, and physicochemical characterization of a recombinant Yersinia protein tyrosine phosphatase. J Biol Chem, 267, 23759–23766. | PubMed | ISI | ChemPort |

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