Among the various CagA-interacting molecules reported to date, only SHP-2 and Csk bind specifically to the tyrosine-phosphorylated form of CagA (2
). Upon complex formation, CagA stimulates their catalytic activities. Thus, SHP-2 and/or Csk may mediate some if not all of the phosphorylation-dependent CagA activities. Indeed, we have already shown that activation of SHP-2 by CagA is both essential and sufficient for induction of the hummingbird phenotype (19
). In this study, we demonstrated that CagA reduces the tyrosine phosphorylation level of FAK, a tyrosine kinase that plays a critical role in focal adhesion turnover, in a manner dependent on CagA phosphorylation. This decrease in FAK tyrosine phosphorylation could be explained by either CagA-Csk or CagA-SHP-2 interaction. In the former case, CagA-activated Csk inhibits SFK activity and thereby prevents SFK-dependent FAK phosphorylation. In the latter case, CagA-activated SHP-2 directly or indirectly dephosphorylates FAK. To investigate these two possibilities, we made use of the EPIYA sites of CagA. The present work revealed that in gastric epithelial cells Csk specifically binds to the EPIYA-A or EPIYA-B site, whereas SHP-2 has been shown to bind to the EPIYA-C site (20
). In this regard, we previously reported that Csk is capable of binding CagA through either the EPIYA-A/B sites or EPIYA-C site when they are overexpressed in COS-7 cells (51
). The differences between the previous and present results may be due to different levels of CagA expression. Indeed, the level of transfected CagA in COS-7 cells was more than 15-fold greater than that of transfected CagA in AGS cells, which is comparable to the level of CagA transduced by infection with cagA
-positive H. pylori
). Given that SHP-2 and Csk bind to CagA in a mutually exclusive manner (data not shown), the interaction between Csk and the EPIYA-C site may be competitively inhibited by the high-affinity interaction between SHP-2 and the EPIYA-C site in AGS cells, where endogenous SHP-2 is in relative excess to CagA (21
). On the other hand, in COS-7 cells, overexpression of CagA results in the accumulation of CagA proteins, which are phosphorylated at the EPIYA-C site but not bound to SHP-2 because of their relative excess to endogenous SHP-2 proteins. Such SHP-2-unbound CagA molecules may then bind to Csk via the EPIYA-A/B sites or EPIYA-C site in COS-7 cells. Accordingly, we consider that results obtained using AGS cells are more reflective of the pathophysiologically relevant situation.
The finding of requirement of the EPIYA-C site, but not the EPIYA-A/B sites, for the CagA activity to reduce FAK tyrosine phosphorylation raised the possibility that CagA-activated SHP-2 is responsible for the biochemical event. It has been reported that SHP-2 can directly activate SFKs by dephosphorylating the C-terminal inhibitory tyrosine residue (40
). More recently, Zhang et al. demonstrated that SHP-2-deficient fibroblasts exhibit reduced SFK activity and suggested that SHP-2 positively regulates SFK activity by controlling the ability of PAG/Cbp to recruit Csk to the membrane through PAG/Cbp dephosphorylation (58
). Since both of the reported SHP-2 activities on SFKs result in the activation, they cannot explain the current observation that CagA-stimulated SHP-2 reduces the level of FAK tyrosine phosphorylation (8
). Indeed, our present work shows that CagA expression, while activating SHP-2, inhibits rather than activates SFKs in gastric epithelial cells. This inhibition of SFK kinase activity by CagA was attributed to CagA-Csk interaction, but not to CagA-SHP-2 interaction, since the abCCC CagA mutant, which binds SHP-2 but not Csk, still retained the ability to reduce FAK tyrosine phosphorylation (Fig. ) but did not modify SFK activity (Fig. ). Accordingly, while SHP-2 is capable of activating SFKs either directly or through PAG/Cbp dephosphorylation (40
), this SHP-2 activity is counteracted by CagA-Csk interaction, which stimulates Csk and thereby inhibits SFKs independent of PAG/Cbp. It should also be noted that expression of the abCCC CagA mutant, which binds SHP-2 but not Csk, or siRNA-mediated knockdown of SHP-2 did not alter the SFK kinase activity in gastric epithelial cells. Thus, the degree of involvement of SHP-2 in the regulation of SFK activity may be cell context dependent. In this regard, the possibility also exists that CagA sequesters SHP-2 away from its normal targets, leading to a paradoxical inactivation of SFKs, which results in the reduced level of FAK tyrosine phosphorylation. However, the results of our experiment using a general tyrosine kinase inhibitor indicate that inhibition of tyrosine kinase activities including those of SFKs in cells cannot mimic the CagA activity to reduce the level of FAK tyrosine phosphorylation. Furthermore, ABccc CagA, which binds Csk but not SHP-2, inhibits SFK activity, whereas abCCC CagA, which binds SHP-2 but not Csk, fails to do so. The results indicate that inhibition of SFK is mediated by CagA-activated Csk but not by sequestration of SHP-2 by CagA from its normal substrates. Given that ABccc CagA cannot reduce the level of FAK tyrosine phosphorylation, the results further suggest that SFK inhibition by CagA is independent of FAK dephosphorylation. In addition, SHP-2 knockdown, which may mimic abnormal sequestration of SHP-2 by CagA from its normal targets, does not inhibit SFK activity. Together with the observation that SFK activity is efficiently inhibited by CagA even in SHP-2-knockdown cells, these results collectively rule out the possibility that CagA-SHP-2 interaction causes SFK inactivation, which results in reduction in the level of FAK tyrosine phosphorylation.
The above-described observations indicate that CagA-activated SHP-2 is directly involved in the reduction in the level of FAK tyrosine phosphorylation. Indeed, the results of a series of present works support an enzyme-substrate relationship between SHP-2 and FAK. First, enhanced tyrosine phosphorylation of FAK is observed in SHP-2-knockdown cells. Second, overexpression of constitutively active SHP-2 reduces the level of FAK tyrosine phosphorylation. Third, FAK is dephosphorylated by SHP-2 in vitro. Fourth, FAK specifically binds to the substrate-trapping mutant of SHP-2. From these observations, we concluded that FAK is an in vivo substrate of SHP-2. FAK is activated via autophosphorylation at Tyr-397, which is initiated by integrin activation. Upon phosphorylation, Tyr-397 becomes a binding site for SFKs, which phosphorylate FAK at Tyr-576 and Tyr-577 to further activate FAK kinase activity. FAK is also reportedly phosphorylated at Y407, Y861, and Y925 (8
). Among these FAK tyrosine residues, Tyr-397, Tyr-576, and Tyr-577 are selectively and constitutively phosphorylated in gastric epithelial cells and CagA-activated SHP-2 dephosphorylates these tyrosine residues. Accordingly, CagA binds and activates SHP-2, which in turn dephosphorylates the activating phosphotyrosine residues and thereby inhibits FAK kinase activity.
It has been reported that tyrosine phosphorylation of FAK in response to integrin signaling was impaired in mouse embryonic fibroblasts rendered acutely deficient in SHP-2 (58
). It has also been reported that the levels of FAK tyrosine phosphorylation in embryonic fibroblasts prepared from WT and SHP-2-
knockout mice were comparable (57
). The differences between those results and our results may be due to different cell types (fibroblasts versus epithelial cells) and/or different experimental systems (SHP-2
knockout versus SHP-2 knockdown) employed. It should also be noted that SHP-2 is recruited to the membrane by receptor tyrosine kinase or a scaffolding/adapter protein such as Gab in response to a growth factor, whereas it is translocated to the membrane by SHPS-1/SIRP-1α in integrin signaling (35
). Accordingly, the effect of SHP-2 on FAK might differ depending on upstream molecules that recruit SHP-2 to the membrane.
Cells with the hummingbird phenotype show increased motility and exhibit a tendency to detach from the culture plate. Thus, CagA has been suspected to perturb intracellular signaling that regulates cell adhesion and cell movement in a tyrosine phosphorylation-dependent manner (19
). In this respect, FAK is a legitimate downstream target of CagA because it plays pivotal roles in cell adhesion and cell morphology as well as cell motility (39
). Two lines of evidence support the idea that reduced FAK activity plays a role in the morphogenetic activity of CagA. First, a constitutively active mutant of FAK (K578E/K581E), which has phosphorylation-independent enhanced kinase activity, inhibited induction of the hummingbird phenotype by CagA. Second, ectopic expression of kinase-dead FAK (K454R) or a dephosphorylated form of FAK (Y576A/Y577A) was capable of inducing cell elongation that resembles the hummingbird phenotype. In this regard, many studies have implicated FAK as a positive regulator of cell motility in response to integrin signaling (24
). However, recent studies have also shown that downregulation of FAK activity plays an important role in growth factor-induced changes in cell morphology and cell movement. Lu et al. demonstrated that treatment of human A431 epidermal carcinoma cells with epidermal growth factor elicits rapid tyrosine dephosphorylation and inhibition of FAK, which is associated with elongated cell shape and increased cell motility (27
). Vadlamudi et al. reported that heregulin induces FAK dephosphorylation, which is also associated with increased migratory potential, in breast cancer cells (53
). Both studies suggested that tyrosine phosphatases such as SHP-2 may be involved in dephosphorylation and inactivation of FAK in growth factor-stimulated epithelial cells. Yano et al. also reported that downregulation of FAK by siRNA resulted in increased cell migration, in association with the induction of aberrant large protrusions, in HeLa cells (56
). These observations are consistent with results of the present study showing that inhibition of FAK by CagA-activated SHP-2 is involved in induction of hummingbird cells with elevated cell motility.
In the present study, approximately 20% of the AGS cells transfected with the CagA expression vector exhibited the hummingbird phenotype at 36 h after transfection. The low frequency of the hummingbird phenotype compared to the high transfection efficiency (~85%) and significant reduction in the level of FAK tyrosine phosphorylation (~65%) can be explained as follows. First, the hummingbird phenotype is a rapid and dynamic cellular process that is associated with multiple rounds of extension and retraction of the protrusions (19
). Thus, a single CagA-expressing AGS cell never stays in its elongated state. Second, the hummingbird phenotype may be induced only in a fraction of CagA-expressing cells whose FAK kinase activity is decreased to a level within certain ranges. More specifically, only CagA-expressing cells in which FAK kinase activity is inhibited but not totally lost might develop the hummingbird phenotype. This idea is supported by the finding that a small amount of active FAK is present in cells with the hummingbird phenotype (see later discussion). Third, there may be other signaling pathways that participate to achieve maximal CagA response in inducing the hummingbird phenotype in addition to FAK inhibition.
Focal adhesions are sites where integrin-mediated adhesion links the actin cytoskeleton. FAK localizes to focal adhesions via its C-terminal focal adhesion-targeting (FAT) domain. This FAT region contains binding sites for integrin-associated proteins such as paxillin and talin (39
). Cell migration is not able to take place in the absence of focal adhesion turnover. Although FAK per se is not essential for the formation of focal adhesion complexes (24
), studies in many laboratories have shown that FAK activation plays a crucial role in focal contact formation (13
). Recent studies have shown that FAK phosphorylates and activates the type I phosphatidylinositol phosphate kinase isoform-γ661 (PIPKIγ661), which is involved in the formation of focal adhesion sites (26
). FAK also functions to promote the disassembly of focal contacts, in part by activating intracellular proteases such as calpain, promoting turnover of focal adhesions (10
). Thus, the kinase enhances both assembly and disassembly of the complexes, and the two seemingly opposite functions may underlie the ability of FAK to regulate focal adhesion turnover. Accordingly, downregulation of FAK by CagA impairs the focal adhesion system, resulting in altered amounts and intracellular distribution of active focal adhesion sites. The decrease in the focal adhesion sites promotes detachment of CagA-expressing cells from the plate. Intriguingly, there still remains a small amount of active FAK molecules, which are specifically enriched at the tips of the membrane protrusions, in CagA-expressing cells with the hummingbird phenotype. This observation indicates that a specific compartmentalization of active FAK, which has escaped from CagA-stimulated SHP-2, may promote assembly of new focal adhesion complexes that generate precursor sites for membrane protrusions. Such a polarized localization of active FAK should also be important for a single cell to move from one place to another with a small number of focal adhesions. As a result, cells with the hummingbird phenotype may exhibit high motility while showing a net decrease in FAK tyrosine phosphorylation. Obviously, cytoskeletal molecules that are regulated by FAK, SHP-2, and/or SFKs should be involved in the morphogenetic activities of CagA in gastric epithelial cells. In fact, it has been suggested that dephosphorylation of cortactin plays a role in the development of elongated cell shape induced by CagA (47
). We have also observed that expression of CagA in AGS cells results in decreased tyrosine phosphorylation of paxillin (data not shown), which is phosphorylated and dephosphorylated by FAK and SHP-2, respectively (6
). Accordingly, molecules such as paxillin may play crucial roles in induction of the hummingbird phenotype by acting as downstream effectors of the CagA-SHP-2-FAK pathway.
Morphological transformation as well as increased motility of gastric epithelial cells induced by CagA may disrupt the normal architecture of gastric mucosa and enhance local inflammation by H. pylori infection in the stomach. Continuous mucosal damage caused by cagA-positive H. pylori would obviously stimulate epithelial cell turnover, increasing the chances for accumulation of genetic mutations that promote multistep gastric carcinogenesis.