Identification of p130Cas as a substrate of Yersinia YopH (Yop51), a bacterial protein tyrosine phosphatase that translocates into mammalian cells and targets focal adhesions
1997; Springer Nature; Volume: 16; Issue: 10 Linguagem: Inglês
10.1093/emboj/16.10.2730
ISSN1460-2075
Autores Tópico(s)Bacterial Genetics and Biotechnology
ResumoArticle15 May 1997free access Identification of p130Cas as a substrate of Yersinia YopH (Yop51), a bacterial protein tyrosine phosphatase that translocates into mammalian cells and targets focal adhesions Deborah S. Black Deborah S. Black Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY, 11794-5222 USA Search for more papers by this author James B. Bliska Corresponding Author James B. Bliska Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY, 11794-5222 USA Search for more papers by this author Deborah S. Black Deborah S. Black Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY, 11794-5222 USA Search for more papers by this author James B. Bliska Corresponding Author James B. Bliska Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY, 11794-5222 USA Search for more papers by this author Author Information Deborah S. Black1 and James B. Bliska 1 1Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY, 11794-5222 USA The EMBO Journal (1997)16:2730-2744https://doi.org/10.1093/emboj/16.10.2730 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Signal transduction pathways in eukaryotic cells are subject to modulation by a large number of pathogenic bacteria. For example, Salmonella, Shigella, enteropathogenic Escherichia coli and Yersinia target various components of eukaryotic signal transduction pathways in order to attach to or enter into host cells (for reviews, see Bliska et al., 1993b; Rosenshine and Finlay, 1993; Galan, 1994; Galan and Bliska, 1996). These bacteria are capable of translocating effector proteins directly into host cells in order to modulate eukaryotic signal transduction pathways (Rosqvist et al., 1994, 1995; Sory and Cornelis, 1994; Persson et al., 1995; Sory et al., 1995). Translocation of these effectors is mediated by a highly conserved type III protein secretion pathway (reviewed in Galan and Bliska, 1996). Although many of the components of the secretory pathways have been characterized, very little is known about the effector proteins themselves or their host targets. Yersinia pseudotuberculosis is an opportunistic enteric pathogen of humans and a close relative of Y.pestis, the agent of bubonic plague (reviewed in Brubaker, 1991). Y.pseudotuberculosis invades and replicates in Peyer's patches, mesenteric lymph nodes and deeper tissues such as the spleen. The infection appears to involve interactions with several different types of host cells, including M (microfold) cells, epithelial cells, platelets and professional phagocytes (Fujimura et al., 1992; Simonet et al., 1990). At late stages of infection, the bacteria preferentially replicate in extracellular niches and suppress cell-mediated immunity in a localized fashion (Brubaker, 1991). Y.pseudotuberculosis infects a variety of cultured mammalian cell lines in vitro, which has allowed for a dissection of this bacterial–host cell interaction at the molecular level (for reviews, see Bliska et al., 1993b; Forsberg et al., 1994; Isberg and Tran Van Nhieu, 1994). The bacteria initially respond to a physiological temperature of 37°C by activating the transcription of factors important for infection (reviewed in Cornelis et al., 1989; Falkow et al., 1992). The initial interaction with host cells occurs when at least one of several different bacterial surface proteins binds to a host cell receptor (Isberg and Tran Van Nhieu, 1994). The best characterized of these proteins is invasin, which binds with high affinity to a subclass of β1 integrin extracellular matrix receptors (Isberg and Tran Van Nhieu, 1994). Once the bacterium is tightly attached, a type III secretion pathway encoded by the Yersinia virulence plasmid is activated, and a set of effectors known as Yops are translocated into the cytoplasm of the host cell at the point of contact (for reviews, see Straley et al., 1993; Forsberg et al., 1994; Galan and Bliska, 1996). Within minutes, cultured mammalian cells lose actin filaments, round up and begin to detach from the extracellular matrix (Portnoy et al., 1981; Rosqvist et al., 1991). In the absence of the virulence plasmid, invasin mediates efficient uptake of the bacteria into host cells by a β1 integrin-mediated phagocytic-like process (Isberg, 1989; Isberg and Tran Van Nhieu, 1994). In contrast, when the bacteria harbor the virulence plasmid, phagocytosis appears to be antagonized by the action of the translocated Yops, a phenomenon referred to as anti-phagocytosis (Forsberg et al., 1994). Cellular disruption and anti-phagocytosis are mediated primarily by two Yops that are translocated into mammalian cells, YopE and YopH (Yop51) (Rosqvist et al., 1988, 1990; Bliska et al., 1993a; Sory and Cornelis, 1994; Persson et al., 1995; Sory et al., 1995; Ruckdeschel et al., 1996). YopE (25 kDa) and YopH (51 kDa) both contain N-terminal domains important for bacterial secretion and translocation into mammalian cells (Persson et al., 1995; Sory et al., 1995). YopE is homologous to the N-terminal domain of ExoS, a toxin which ADP-ribosylates a number of low molecular weight GTP-binding proteins, including Ras (Coburn et al., 1989; Kulich et al., 1994). YopE has a potent cytotoxic effect on host cells and is associated with disruption of actin stress fibers (Rosqvist et al., 1990, 1991). YopH has a weaker cytotoxic effect on host cells that can only be detected in the absence of YopE (Rosqvist et al., 1990; Bliska et al., 1993a). YopH contains a C–terminal protein tyrosine phosphatase (PTPase) domain (Guan and Dixon, 1990) and a central proline-rich region that appears to bind host cell Src homology 3 (SH3) domains (Bliska, 1996). The activity of the PTPase domain has been shown to be essential for the pathogenicity of Y.pseudotuberculosis (Bliska et al., 1991). Production of YopH during bacterial infection of cultured mammalian cells is associated with dephosphorylation of proteins in human epithelial cells and murine macrophages (Bliska et al., 1991, 1992; Hartland et al., 1994; Andersson et al., 1996). Although the primary host cell target(s) of this PTPase has not been identified, it may be involved in integrin-mediated signaling, since wild-type YopH promotes detachment of epithelial cells from the extracellular matrix, while a catalytically inactive form of the protein, in which the essential Cys403 in the PTPase domain is changed to Ser (YopHC403S) or Ala (YopHC403A), is defective for cellular detachment (Bliska et al., 1993a). Thus, although significant emphasis has been placed on the role of YopH as an inhibitor of integrin-mediated bacterial internalization, the PTPase could play an equally or more important role as an inhibitor of integrin-mediated cell adhesion. The first suggestion that a catalytically inactive PTPase could be used to trap substrates in vivo was derived from the observation that tyrosine-phosphorylated host proteins co-precipitated with YopHC403A (Bliska et al., 1992). Subsequently, a number of other inactive PTPases including CSW (C583S) (Herbst et al., 1996), SHP-1 (C453S) (Plas et al., 1996) and PTP-PEST (C231S) (Garton et al., 1996) have been used to trap substrates in vivo. In some cases, these substrate-trapping mutants also promote substrate hyperphosphorylation in vivo, apparently by binding to phosphotyrosyl residues and sequestering these sites from the action of endogenous PTPases (Herbst et al., 1996; Plas et al., 1996). Here we demonstrate that a catalytically inactive PTPase can also be used as a cytological probe to investigate the cellular localization of substrates. Using this approach, we identified the focal adhesion protein p130Cas (Cas) as a direct substrate of YopH in human epithelial cells. Results YopHC403S localizes to focal adhesions The first step of our approach to identify an in vivo substrate of YopH was to determine if the substrate localized to a particular subcellular compartment or structure in host cells. We used confocal immunofluorescence microscopy to determine where YopH localizes in host cells, under the assumption that YopH, and in particular its catalytically inactive derivative YopHC403S, could act as substrate-specific probes (Bliska et al., 1992). Human epithelial (HeLa) cells were used as host cells in these studies because their well defined cellular architecture facilitated our microscopic analysis. HeLa cells were infected with Y.pseudotuberculosis at a multiplicity of infection of 50:1. Two hours after infection, the cells were fixed, permeabilized and processed for confocal immunofluorescence microscopy using a polyclonal rabbit anti-Yop51 antibody (RAY51), either alone, or in combination with a monoclonal anti-phosphotyrosine antibody (4G10). Samples were treated gently during processing to avoid detachment of infected HeLa cells. As a control for the specificity of the RAY51 antibody, we examined HeLa cells that were infected with IP15/pVector, a yopH deletion mutant carrying an empty expression vector, and observed only a low level of background fluorescence (Figure 1B). When the wild-type yopH+ gene was introduced in trans into the yopH mutant (IP15/pYOPH), a strong fluorescent signal was detected in the cytoplasmic compartments of HeLa cells (Figure 1D), demonstrating that RAY51 was specifically recognizing YopH. As shown by the phase images in Figure 1A and C, the HeLa cells infected with either IP15/pVector or IP15/pYOPH were rounded up and partially detached due to the cytotoxic activity of YopE. Therefore, we also carried out infections with a yopEyopH double mutant complemented with the yopH+ gene (IP17/pYOPH) to examine the localization of YopH in the absence of YopE-mediated actin stress fiber disruption. In HeLa cells infected with IP17/pYOPH, some cytotoxicity was still evident, although the cells were significantly less rounded (Figure 2A). YopH also localized to the cytoplasmic compartment, but the RAY51 labeling was distributed more diffusely (Figure 2A). The yopEyopH mutant was used in certain experiments described below in order to prevent the extreme disruption of cell morphology that was associated with the activity of YopE. Figure 1.YopH localizes to the cytoplasmic compartment of HeLa cells infected with Y.pseudotuberculosis. HeLa cells were infected for 2 h with IP15/pVector, a Y.pseudotuberculosis yopH mutant carrying an empty expression plasmid (A and B) or IP15/pYOPH, a yopH mutant complemented in trans with a wild-type yopH+ gene (C and D). The infected cells were processed for confocal immunofluorescence microscopy using an affinity-purified rabbit polyclonal anti-Yop51 antibody (RAY51) (see Materials and methods). Corresponding phase (A and C) and rhodamine (B and D) images are shown. Download figure Download PowerPoint Figure 2.YopHC403S co-localizes with phosphotyrosine in focal adhesions in infected HeLa cells. HeLa cells infected for 2 h with IP17/pYOPH (A and B), IP17/pYOPHC403S (C, D, G and H) or left uninfected (E and F) were processed for immunofluorescence microscopy using RAY51 and mouse monoclonal anti-phosphotyrosine antibody (4G10) or DAPI (see Materials and methods). Corresponding rhodamine (A, C and E) or DAPI (G) and fluorescein (B, D, F and H) images are shown. (A–F) show confocal microscopic images. Images in (G) and (H) were obtained by epifluorescence microscopy. Download figure Download PowerPoint When the yopHC403S gene was introduced in trans into the yopEyopH mutant (IP17/pYOPHC403S), a very different localization pattern was seen in infected HeLa cells. In this case, YopHC403S was found in punctate structures that co-localized with phosphotyrosine (Figure 2C and D, arrows). A similar localization pattern was evident after a 1 h infection, although the signal was weaker (data not shown). Two hour infections were used in subsequent experiments in order to maximize the detection of YopHC403S in these intracellular structures. Similar punctate structures were seen in uninfected HeLa cells labeled with 4G10 (Figure 2F). Analysis of optical sections demonstrated that these punctate structures were located at the basal surface of HeLa cells (data not shown). These structures were reminiscent of focal adhesions, which are multiprotein complexes that form at the interface between integrin receptors and actin filaments and are enriched in tyrosine-phosphorylated proteins (reviewed in Clark and Brugge, 1995). Parallel samples of IP17/pYOPHC403S-infected cells were processed for epifluorescence microscopy using 4′,6′-diamidino-2-phenylindole (DAPI; a probe for DNA) and RAY51 to determine if the infecting bacteria co-localized with YopHC403S. As shown in Figure 2, most of the punctate labeling of YopHC403S occurred in regions that were devoid of bacteria, although on occasion the signals did overlap (compare Figure 2G and H). Thus, the majority of YopHC403S was localizing in structures that were distant from the site of bacterial–host cell interaction. These data suggested that following translocation, YopHC403S localized to focal adhesions because these structures contained a major substrate of this enzyme. Consistent with the idea that focal adhesions contained a substrate of YopH was the observation that the labeling of these structures with 4G10 decreased in HeLa cells infected with IP17/pYOPH (compare Figure 2B and F). Interestingly, the labeling of cells infected with IP17/pYOPHC403S with 4G10 was 2- to 4-fold brighter than that of uninfected cells (compare Figure 2D and F), indicating that YopHC403S not only localized to focal adhesions but actually increased the level of phosphotyrosine in the cells. At this point, we were unable to determine if this resulted from an increase in the number of focal adhesions or an increase in phosphotyrosine in existing focal adhesions. One explanation for the latter possibility was that YopHC403S was binding to its substrate and protecting phosphotyrosyl residues in the substrate from the action of an endogenous PTPase. After fixation and permeabilization of the HeLa cells, these protected phosphotyrosyl residues were nevertheless accessible to the 4G10 antibody (see Discussion for a possible explanation). To determine if YopHC403S was localizing to characteristic focal adhesions, we compared the localization pattern of YopHC403S with that of two well characterized focal adhesion proteins, paxillin and vinculin. Paxillin and vinculin concentrated in focal adhesions in uninfected cells as expected (data not shown). In HeLa cells infected with IP17/pYOPHC403S, YopHC403S co-localized with paxillin (Figure 3A and B) and vinculin (Figure 3C and D). These data confirmed that YopHC403S localized to characteristic focal adhesions. Using paxillin and vinculin as indicators of focal adhesions, we found that there were similar numbers of focal adhesions in uninfected cells and cells infected with IP17/pYOPHC403S (data not shown), which suggested that YopHC403S was not actually increasing the number of these structures in the cell. Figure 3.Co-localization of YopHC403S with paxillin or vinculin in focal adhesions in infected HeLa cells. HeLa cells infected with IP17/pYOPHC403S for 2 h were processed for confocal immuno- fluorescence microscopy using RAY51 and mouse monoclonal anti-paxillin (A and B) or mouse monoclonal anti-vinculin (C and D) antibodies (see Materials and methods). Corresponding rhodamine (A and C) and fluorescein (B and D) images are shown. Download figure Download PowerPoint YopHC403S has a dominant-negative effect on focal adhesion disruption mediated by YopE When HeLa cells were infected with the yopE+yopH strain IP15/pVector and examined by immunofluorescence microscopy using 4G10, anti-vinculin or anti-paxillin antibodies, no focal adhesions were observed, while focal adhesions were observed in IP17/pVector-infected cells (Figure 4A and E and data not shown). This result suggested that YopE had sufficient activity to disrupt focal adhesions, most likely through its ability to destabilize stress fibers (Rosqvist et al., 1991). The loss of actin stress fibers in IP15/pVector-infected cells was confirmed by microscopic examination of the cells after staining with phalloidin (data not shown). Interestingly, when HeLa cells were infected with IP15/pYOPHC403S and examined by immunofluorescence microscopy using 4G10 and anti-vinculin antibodies, significant numbers of focal adhesion-like structures were detected (Figure 4C and G). The focal adhesions detected in IP15/pYOPHC403S-infected cells contained YopHC403S, as shown by the co-localization of RAY51 and 4G10 antibodies (Figure 4C and D) or RAY51 and anti-vinculin antibodies (Figure 4G and H). This result suggested that the interaction of YopHC403S with a substrate in focal adhesions had a dominant-negative effect on disassembly of these structures in response to destabilization of stress fibers. One interpretation of this finding was that dephosphorylation of the protein bound to YopHC403S was necessary for focal adhesion disassembly. Figure 4.YopHC403S has a dominant-negative effect on focal adhesion disruption by YopE. HeLa cells infected for 2 h with IP15/pVector or IP15/pYOPHC403S were processed for confocal immunofluorescence microscopy using 4G10 and RAY51 antibodies (A–D) or mouse monoclonal anti-vinculin and RAY51 antibodies (E–H) (see Materials and methods). Corresponding fluorescein (A, C, E and G) and rhodamine (B, D, F and H) images are shown. Download figure Download PowerPoint Translocation of YopHC403S into HeLa cells is associated with the hyperphosphorylation of a 125–135 kDa protein As a first step toward identification of the focal adhesion protein interacting with YopHC403S, HeLa cells infected with IP17-derived strains were lysed in a relatively harsh detergent mixture (RIPA), separated into soluble and insoluble fractions, and samples of the resulting fractions (equivalent to 5×104 and 1×106 HeLa cells, respectively) were examined by immunoblotting with 4G10. In the RIPA-soluble fraction of HeLa cells, a broad band of hyperphosphorylated protein that migrated between 125 and 135 kDa was detected in cells infected with IP17/pYOPHC4O3S (Figure 5, lane 4), but not in uninfected cells or in cells infected with IP17/pVector or IP17/pYOPH (lanes 1–3, respectively). In addition, a broad band of hyperphosphorylated protein of similar mobility was present in the RIPA-insoluble fraction of HeLa cells infected with IP17/pYOPHC403S (Figure 5, lane 8, right side arrow). Similar results were obtained when the cells were lysed in the less solubilizing detergent Triton X-100 (data not shown). These results demonstrated that a protein (or proteins), hereafter designated p125-135, was hyperphosphorylated in the presence of YopHC403S. Taken together with the preferential localization of YopHC403S to focal adhesions, p125-135 was likely to be associated with these structures. The fact that p125-135 partitioned into a detergent-insoluble fraction of HeLa cells was consistent with this hypothesis, since this cell fraction is known to be enriched in cytoskeleton-associated proteins that are phosphorylated on tyrosine. Figure 5.A 125–135 kDa protein is hyperphosphorylated in response to translocation of YopHC403S into HeLa cells. Uninfected HeLa cells (lanes 1 and 5), or HeLa cells infected with IP17/pVector (lanes 2 and 6), IP17/pYOPH (lanes 3 and 7) or IP17/pYOPHC403S (lanes 4 and 8) were lysed in RIPA and separated into soluble (lanes 1–4) and insoluble (lanes 5–8) protein fractions (see Materials and methods). Soluble and insoluble protein fractions (equivalent to 5×104 and 1×106 HeLa cells, respectively) were separated on a 10% SDS–polyacrylamide gel and analyzed by immunoblotting with 4G10 antibody. The positions of pre-stained molecular weight standards and p125-135 (arrow) are shown on the right. The arrows on the left indicate the positions of major phosphoproteins (p125 and p68) that are dephosphorylated by YopH. Download figure Download PowerPoint Infection of HeLa cells with IP17/pYOPH was associated with decreased phosphorylation of several proteins (Figure 5, lane 3), including a 125 kDa protein (p125) detected as a faint band in lysates of uninfected and IP17/pVector-infected cells (lanes 1 and 2, upper left arrow). Since this protein was dephosphorylated in a YopH-specific manner, it represented a potential candidate for the protein that became hyperphosphorylated in response to expression of YopHC403S. Many proteins, including two more prominent molecules in the 125–135 kDa range, were not dephosphorylated in IP17/pYOPH-infected cells (Figure 5, lane 3), demonstrating that YopH PTPase activity was directed to a subset of tyrosine-phosphorylated proteins in the cell. In addition to the 125 kDa substrate, a prominent protein that migrated as a broad 68 kDa band (p68) (Figure 5, lane 1, lower left arrow) was dephosphorylated in cells infected with IP17/pYOPH (lane 3). However, this protein was not hyperphosphorylated reproducibly in cells infected with IP17/pYOPHC403S (compare lanes 1 and 4). In addition, as shown in the next experiment, this 68 kDa protein did not co-precipitate with YopHC403S (Figure 6). Although it was likely that the 68 kDa protein was dephosphorylated by YopH, it was also possible that it was dephosphorylated in an indirect fashion, for example by stimulation of endogenous PTPase activity during infection with IP17/pYOPH. Because of this uncertainty, we focused our attention on the protein that was hyperphosphorylated in the presence of YopHC403S since, based on previous studies (Herbst et al., 1996; Plas et al., 1996), this molecule was more likely to be a direct substrate of the PTPase. Figure 6.A hyperphosphorylated protein that co-precipitates with YopHC403S and partitions to the insoluble fraction is recognized by a monoclonal anti-Cas antibody. (A–C) Uninfected HeLa cells (lanes 1, 5 and 12), or HeLa cells infected with IP17/pVector (lanes 2, 6 and 11), IP17/pYOPH (lanes 3, 7 and 10) or IP17/pYOPHC403S (lanes 4, 8 and 9) were lysed in Triton X-100, and the lysates were subjected to immunoprecipitation with RAY51 (see Materials and methods). Samples of lysates pre- and post-immunoprecipitation (lanes 1–4 and 5–8, respectively) and the immunoprecipitates (lanes 9–12) were separated on a 7.5% SDS–polyacrylamide gel and analyzed by immunoblotting with 4G10 (A), RAY51 (B) or monoclonal anti-Cas (C) antibodies. In (A), H corresponds to heavy chain of immunoprecipitation antibody (also visible in B and C). (D) Uninfected HeLa cells (lanes 1 and 6), or HeLa cells infected with IP17/pVector (lanes 2 and 7), IP17/pYOPH (lanes 3 and 8) or IP17/pYOPHC403S (lanes 4, 5, 9 and 10) were lysed in Triton X-100 (lanes 1–4 and 6–9) or RIPA (lanes 5 and 10) and separated into soluble and insoluble protein fractions (see Materials and methods). Soluble and insoluble protein fractions (equivalent to 2×104 and 1×106 HeLa cells, respectively) were separated on a 7.5% SDS–polyacrylamide gel and analyzed by immunoblotting with monoclonal anti-Cas antibody. Download figure Download PowerPoint A hyperphosphorylated 125–135 kDa protein that co-precipitates with YopHC403S is recognized by anti-Cas antibodies To determine if p125-135 was physically associated with YopHC403S, lysates of infected HeLa cells prepared in Triton X-100 were subjected to immunoprecipitation with RAY51. The immunoprecipitates were analyzed by immunoblotting with 4G10. The results indicated that p125-135 was bound to YopHC403S, since p125-135 was largely depleted from cell lysates following immunodepletion of >94% of total cellular YopHC403S (Figure 6A, compare lanes 4 and 8), and a broad band of phosphorylated protein which migrated in the 125–135 kDa range co-precipitated with YopHC403S (lane 9, arrow). p125-135 specifically co-precipitated with YopHC403S, since it was not precipitated from lysates of uninfected cells (Figure 6A, lane 12), IP17/pVector-infected cells (lane 11) or IP17/pYOPH-infected cells (lane 10). Immunoblotting with RAY51 demonstrated that equivalent amounts of PTPase protein were recovered from IP17/pYOPHC403S- and IP17/pYOPH-infected cells (Figure 6B, lanes 9 and 10). In an attempt to identify p125-135, immunoblots containing immunoprecipitated YopHC403S were probed with antibodies specific for the proteins focal adhesion kinase (Fak; Schaller et al., 1992), vinculin (Sefton et al., 1981), HEF-1 (Law et al., 1996) and Cas (Sakai et al., 1994), all of which are in the 125–135 kDa range and are known to be tyrosine phosphorylated. A monoclonal anti-Cas antibody specifically recognized a broad 125–135 kDa protein band that co-precipitated with YopHC403S (Figure 6C lane 9), while the other antibodies gave negative results (data not shown). A rabbit polyclonal antibody specific for Cas (Sakai et al., 1994) also specifically recognized p125-135. These results suggested that the p125-135 protein associated with YopHC403S was Cas. Significantly, Cas recently has been shown to be phosphorylated on tyrosine in response to integrin engagement and to localize to focal adhesions (Nojima et al., 1995; Petch et al., 1995; Vuori and Ruoslahti, 1995; Harte et al., 1996). Cas is detected as multiple species on immunoblots, with two distinct bands migrating at 115 and 125 kDa (CasA and B, respectively) (Figure 6C, lane 1) and a broad, diffuse band spreading between 125 and 135 kDa (CasC) (Sakai et al., 1994). CasC is converted into CasA and B by phosphatase treatment and appears to be heavily phosphorylated on tyrosine, which may account for its diffuse gel mobility and poor reactivity with antibodies (Sakai et al., 1994). The phosphorylated protein that co-precipitated with YopHC403S appeared to correspond to the slower migrating CasC (Figure 6C, lane 9), suggesting that YopHC403S was binding specifically to the tyrosine-phosphorylated form of Cas. Translocation of YopHC403S into HeLa cells is associated with increased detergent insolubility of Cas Infection of HeLa cells with IP17/pYOPHC403S was associated with the appearance of a 125–135 kDa tyrosine-phosphorylated protein in the RIPA-insoluble cell fraction (Figure 5, lane 8). We wanted to determine if this protein was Cas and whether its appearance in the RIPA-insoluble fraction was due to increased tyrosine phosphorylation of insoluble protein or a net increase in the amount of insoluble protein. For this purpose, infected HeLa cells were lysed in Triton X-100, separated into soluble and insoluble fractions, and samples of the resulting fractions (equivalent to 2×104 and 1×106 HeLa cells, respectively) were analyzed by immunoblotting with the monoclonal anti-Cas antibody. RIPA-soluble and RIPA-insoluble fractions of IP17/pYOPHC403S-infected cells were also analyzed to allow for a comparison to the data shown in Figure 5. As shown in Figure 6D, there was significantly increased detection of Cas in the insoluble fractions of IP17/pYOPHC403S-infected cells relative to uninfected cells (Figure 6D, compare lanes 6, 9 and 10). Quantitation by densitometry indicated that the insoluble Cas in IP17/pYOPHC403S-infected cells represented only a small fraction (∼1%) of total cellular Cas, although much of the insoluble Cas appeared to correspond to CasC. There was also a small increase in insoluble Cas in IP17/pVector-infected cells that was not seen in IP17/pYOPH-infected cells (Figure 6D, compare lanes 7 and 8), indicating that YopH activity was causing solubilization of Cas. Taken together, these results were consistent with previous data showing increased partitioning of Cas into a particulate cell fraction in response to tyrosine phosphorylation (Sakai et al., 1994). Cas is hyperphosphorylated in the presence of YopHC403S and dephosphorylated in the presence of YopH To confirm the identit
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