Regulation of Cell Adhesion by Protein-tyrosine Phosphatases

Cell-cell adhesion is critical to the development and maintenance of multicellular organisms. The stability of many adhesions is regulated by protein tyrosine phosphorylation of cell adhesion molecules and their associated components, with high levels of phosphorylation promoting disassembly. The level of tyrosine phosphorylation reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. Many protein-tyrosine phosphatases associate with the cadherin-catenin complex, directly regulating the phosphorylation of these proteins, thereby affecting their interactions and the integrity of cell-cell junctions. Tyrosine phosphatases can also affect cell-cell adhesions indirectly by regulating the signaling pathways that control the activities of Rho family G proteins. In addition, receptor-type tyrosine phosphatases can mediate outside-in signaling through both ligand binding and dimerization of their extracellular domains. This review will discuss the role of protein-tyrosine phosphatases in cell-cell interactions, with an emphasis on cadherin-mediated adhesions.


PTPs and Cell-Cell Junctions
Epithelial tissues typically display stable cell-cell adhesion that is accompanied by prominent cell junctions between interacting cells. Tight junctions (TJs) are responsible for the barrier function of many epithelia, whereas adherens junctions (AJs) and desmosomes mediate strong intercellular adhesion. The assembly and function of TJs is typically dependent on the state of AJs, so that modulating AJ function often affects TJ barrier properties. The major adhesion molecules in both AJs and desmosomes belong to the cadherin family; however, cadherins can also contribute to adhesions between cells where distinct junctions do not develop and where adhesion is more dynamic. Additional adhesion molecules, such as nectins (3), are also present in AJs, but we will focus here on cadherins. Whereas the extracellular domain of cadherins participates in calcium-dependent homophilic adhesion, the cytoplasmic domain binds p120 ctn and ␤-catenin (4 -6). The former regulates the stability of cadherins on the cell surface (7), and ␤-catenin provides a link to ␣-catenin and the actin cytoskeleton, although the details of the bridge remain controversial (8). Tyrosine phosphorylation of cadherins and their associated proteins has major effects on the stability of adherens junctions (9,10). In early work, it was shown that inhibiting PTPs with pervanadate-elevated tyrosine phosphorylation in adherens junctions and promoted the disassembly of these structures (11). However, the same group later observed that in some situations elevation of tyrosine phosphorylation first transiently stimulated AJ assembly before resulting in the eventual disassembly of the same structures (12). Activation of PTKs or inhibition of PTPs can lead to increased tyrosine phosphorylation of members of the cadherin-catenin complex, dissociation of the AJ from the cytoskeleton, and disruption of cell-cell adhesion (13)(14)(15). For example, phosphorylation of tyrosine residues 755 and 756 on E-cadherin leads to its ubiquitination and subsequent endocytosis, resulting in loss of junctional integrity (16). Similarly, phosphorylation of Tyr 658 and Tyr 731 in the cytoplasmic domain of VE-cadherin prevents binding of p120 ctn and ␤-catenin, respectively, and causes a decrease in barrier function (17). Therefore, maintenance of junctional integrity is regulated in part by reversible tyrosine phosphorylation that results from a competing balance of PTK and PTP activity. Receptor-PTPs such as PTP, DEP-1, and VE-PTP, as well as the cytosolic PTPs, PTP1B, and Shp-2, have been shown to bind to members of the cadherin-catenin complex (18 -23) (Table 1) and to regulate cell-cell adhesion by regulating phosphorylation of the cadherin-catenin complex.

Direct Regulation of the Cadherin-Catenin Complex
The extracellular domains of PTP can mediate cell-cell adhesion via homophilic interactions (24,25). Together with its binding to p120 ctn and the cytoplasmic domain of cadherins, the homophilic interactions of PTP combine to localize it to cell-cell junctions (18,19,26,27). Localization at AJs orients the catalytic domain of PTP in close proximity with substrates VEcadherin and p120 ctn (19,27). In addition, knockdown of PTP by siRNA increases permeability of endothelial monolayers demonstrating its role in the regulation of junctional integrity (27). Although many of the effects of PTP at AJs are undoubtedly due to its phosphatase activity, other domains of the protein may also contribute to junctional stability. Evidence for this came from a study of prostate carcinoma cells lacking endogenous PTP, which were unable to form AJs even though E-cadherin and the catenin proteins were present. Expression of PTP restored cadherin-mediated cell-cell adhesion, but strikingly this could also be achieved by expression of catalytically dead PTP (28). This finding suggests that PTP can act as a scaffold and recruit additional regulatory proteins to sites of cell-cell adhesion.
Another PTP important for junction formation and maintenance is high cell density enhanced PTP-1 (DEP-1). Expression of DEP-1 is increased more than 10-fold in many cell types as they approach confluence, suggesting it contributes to cell-cell adhesion and contact inhibition of growth (29). DEP-1 is present at the apical surface of endothelial cells but also co-localizes with VEcadherin at intercellular junctions (30,31). DEP-1 indirectly associates with VE-cadherin by binding to p120 ctn , ␥-catenin (plakoglobin), and ␤-catenin (Table 1); DEP-1 regulates their phosphorylation state, preserving their interactions with the cadherins and promoting cell-cell adhesion (20,30). The role of DEP-1 in organizing cell-cell junctions is supported by the observation that in cells lacking strong cell-cell adhesion, such as fibroblasts, overexpression of DEP-1 results in a change in localization of cadherins from discrete areas of cell-cell contact to large areas reminiscent of continuous AJ found in epithelial cells (32).
A PTP in the same family as DEP-1 is vascular endothelial PTP (VE-PTP), which is selectively expressed in endothelial cells and binds to VE-cadherin but not to ␤-catenin (33). VE-PTP associates with VE-cadherin via its membraneproximal extracellular domain, and its recruitment results in the dephosphorylation of VE-cadherin (21). Expression of wild type VE-PTP decreases paracellular permeability in endothelial monolayers, while the catalytically inactive mutant has no effect, indicating that the phosphatase activity of VE-PTP acting on VE-cadherin is necessary for maintaining the integrity of endothelial junctions (21).
Cytosolic PTPs such as PTP1B and Shp-2 have also been implicated in regulating cell-cell adhesion by controlling phosphorylation of cadherin-catenin proteins (22,23,34). PTP1B binds directly to the cytoplasmic domain of N-cadherin, an interaction that promotes ␤-catenin binding and targeting of the cadherin-catenin complex to the cell membrane (35). Expression of a catalytically inactive mutant of PTP1B disrupts cadherin-mediated adhesion, with concomitant increases in tyrosine phosphorylation of ␤-catenin and reduction in the association of N-cadherin with the actin cytoskeleton, suggesting that the catalytic activity of PTP1B is important for junctional maintenance (23,34). Shp-2 is another cytosolic PTP associated with the cadherin-catenin complex in confluent endothelial cell monolayers, specifically interacting with ␤-catenin (22). Thrombin treatment of endothelial cells induces Shp-2 tyrosine phosphorylation and dissociation from ␤-catenin followed by junctional breakdown and the formation of large intercellular gaps, thus supporting the hypothesis that Shp-2 localization to junctions also helps maintain endothelial junctional strength and integrity (22). The loss of Shp-2 binding is accompanied by an increase in phosphorylation of ␤-catenin, plakoglobin, and p120 ctn . AJs are not the only adhesive complexes where PTPs play a role in regulating signal transduction. Shp-2 binds to platelet endothelial cell adhesion molecule-1 (PECAM-1) (36, 37) and intercellular adhesion molecule 1 (ICAM-1) (38,39). In response to ICAM-1 engagement, phosphorylated ICAM-1 binds to Shp-2 and this interaction is necessary for Src activation as well as p38 MAPK activation (39).
The concentration of PTPs, both cytosolic and transmembrane, at AJs indicates the importance of maintaining low levels of tyrosine phosphorylation at these sites, except when the junctions need to be remodeled or disassembled. In general, the suppression of bulk PTP activity by inhibitors or the suppression of individual PTPs by siRNA elevates the tyrosine phosphorylation of cadherins and their associated proteins. In turn, this results in destabilization of the junctions and diminished epithelial or endothelial barrier functions.

Rho GTPases and Cell-Cell Adhesion
Just as with cell-matrix adhesions (2), there is bidirectional interplay between Rho family GTPases and cell-cell adhesions. Not only is the assembly of AJs affected by the activities of Rho proteins, but cell-cell adhesion can stimulate or depress the activities of these G proteins, suggesting complex feedback loops. Several studies have shown that Rac1 activation promotes assembly of epithelial AJs and inhibition of either Rac1 or RhoA activity results in disassembly (40 -42). Conversely, other studies show that high levels of active Rac1 or RhoA can actually disrupt both TJs (43) and AJs (44,45); this might reflect different cellular contexts or the need for tight regulation of GTPase activity. The formation of AJs can activate Rac1 and Cdc42 (46, 47) but inhibit RhoA (47). PTPs may play critical roles in these upstream and downstream pathways by regulating the phosphorylation and activity of GTPaseactivating proteins (GAPs) and guanine nucleotide exchange factors (2). For example, p190 RhoGAP tyrosine phosphorylation was implicated in the depression of RhoA activity downstream from cadherin engagement (48).
With endothelial monolayers, several agents that promote increased permeability also stimulate Rac1 activity. PTPs are also likely to be important in this response to Rac1 because in these cells Rac1 has been shown to generate reactive oxygen species (ROS) (49). In turn, ROS are potent inhibitors of PTPs (50) and are responsible for the elevation in junctional protein tyrosine phosphorylation that results from Rac1 activation (45,49,51).

Regulation of PTP Activity
The resemblance of the extracellular domains of RPTPs to cell adhesion molecules has stimulated a search for potential binding partners and by extension, for modes of regulation via such interactions. Several mechanisms for modulation of specific PTP activity exist (Fig. 1). In some cases, homophilic interactions have been detected, as first shown with PTP (24,25). Two closely related PTPs, PTP and PTP, have similarly been shown to interact in a homophilic manner (52,53). The extracellular domains of several RPTPs participate in heterophilic interactions with extracellular matrix or other components. For example, LAR binds the laminin-nidogen complex, which is prominent in many basement membranes (54). Similarly, PTP binds to heparin sulfate proteoglycans (55), and PTP␣ binds to contactin (56). Although several interactions involving the extracellular domains of RPTPs have been identified, in most cases no effect on the activity of the PTP has been demonstrated. One exception is the interaction of PTP␤/ with pleiotrophin, which inhibits PTP activity and results in an increase in ␤-catenin phosphorylation (57). Pleiotro-  In some instances, such as homophilic interaction, it is not clear whether the interaction affects activity (shown with a yellow cytoplasmic domain). However, heterophilic interactions can be either activating (shown in green) or inhibitory (shown in red) and appear to be ligand/PTP-specific. B, RPTPs also interact in cis on the cell surface. In most cases dimerization is associated with inactive PTPs. However, the known conformational change by which dimerization inhibits activity is not consistent for the interactions of all RPTPs. Therefore, in some cases dimerization may increase PTP activity. Further investigation into this is needed. C, in several cell types ROS are generated in signaling pathways, such as downstream from Rac1 activation. The reversible oxidation of the catalytic cysteine by ROS inhibits PTP activity.
phin is a heparin binding growth-associated molecule, and its interaction with PTP␤/ promotes cortical neuron migration (57). In contrast, DEP-1 phosphatase activity is up-regulated by the binding of its extracellular domain to an unknown component of Matrigel (58). The observation that the activities of RPTPs can be regulated by the interactions of their extracellular domains will be broadly important if further work can establish that this is a general characteristic of RPTPs. It is easy to imagine many scenarios in which tyrosine phosphorylation levels could be regulated by interactions of the extracellular domains of RPTPs with other cells, with matrix molecules or soluble ligands (Fig. 1A).
Dimerization of RPTPs provides another potential mode of regulating their activity. RPTPs have traditionally been considered to be inactive when dimerized and active when monomers (59). This idea was first proposed when a chimera of the extracellular domain of EGFR fused to the intracellular domain of CD45 was inhibited by EGF-induced dimerization (60). Further support was generated when studies revealed RPTP␣ exists as dimers on the cell surface and that this dimerization inhibits the activity of the phosphatase via an interaction of the tandem catalytic domains, preventing the binding of substrates (61)(62)(63). This suggests a model where RPTPs exist as inactive dimers on the cell surface in the absence of a ligand and binding of the ligand dissociates the dimers and activates the PTP (Fig. 1B). However, dimerization-induced inactivation of RPTPs through blockage of the active site by an opposing PTP domain may not be a universal mechanism. Structural analysis of the membrane-proximal catalytic domain of PTP and the entire cytoplasmic domain of PTP-LAR suggests that these PTPs are not inhibited by dimerization (64,65). In addition, RPTPs such as DEP-1 and VE-PTP contain a single catalytic domain and therefore may also not be inhibited by dimerization.
The regulation of PTP activity by ROS is an area developing very rapidly and too large to review in detail here. The cysteine residue in the catalytic site of classical PTPs has long been known to be sensitive to oxidation and this has been the basis for broad specificity inhibitors, such as H 2 0 2 and pervanadate. Subsequent work has shown that reversible oxidation of the catalytic cysteine can provide a physiological mechanism for regulating PTPs (50) (Fig. 1C). The generation of ROS was originally identified as a defense mechanism in the phagocytic killing of bacteria by leukocytes, but more recently the generation of low levels of ROS by other cells has been recognized as a widespread occurrence with important physiological consequences (67). The formation of ROS is downstream of active Rac1 (49,68). For example, in endothelial cells, active Rac1 promotes endothelial permeability and is associated with increased tyrosine phosphorylation of junctional proteins, such as VE-cadherin and ␤-catenin (49,51). These effects are blocked by inhibiting the generation of ROS and are mimicked by pervanadate (49). Whether these effects are the result of one or a few PTPs being inhibited or reflect broad inhibition of all PTPs within these cells has not been established. It will be interesting to determine the degree to which ROS can act locally on one or a few PTPs rather than globally by inhibiting all PTPs within a cell. Techniques have been developed for analyzing the reversible oxidation of PTPs (69), and so this area should advance rapidly. There are many physiological situations in which Rac1 is activated, and it will be important to determine whether the generation of ROS and the consequent inactivation of PTPs is a general signaling pathway downstream from Rac1 or whether this only occurs in particular situations.

Concluding Remarks
It is clear that PTPs play a central role regulating tyrosine phosphorylation in cell-cell adhesions. With respect to RPTPs, the interactions of their extracellular domains command considerable interest due to their similarity with cell adhesion molecules. Identifying their ligands is a high priority, as is determining whether these interactions regulate PTP activity. The large number of PTPs complexing with, and acting on, cadherins and their associated proteins is striking. Inhibiting PTP activity and elevating tyrosine phosphorylation at AJs promotes junctional disassembly and affects TJ permeability. Together these observations demonstrate the importance of maintaining low tyrosine phosphorylation of junctional components to maintain normal epithelial and endothelial barrier functions. An exciting corollary of this is that agents that increase permeability or cells that need to cross these junctions, such as leukocytes or tumor cells, may modulate cell junctions by manipulating PTPs. Exploring this possibility is one of the challenges facing this field.