Components of adherens junctions are subjected to rapid, reversible tyrosine phosphorylation in a cellular context (Volberg et al., 1991
). Tyrosine phosphorylation of the cadherin–catenin complex has been observed under a variety of conditions, including in response to oncoprotein PTKs, such as Src (Matsuyoshi et al., 1992
; Behrens et al., 1993
), or to oncogenic forms of Ras (Kinch et al., 1995
) and following stimulation of receptor PTKs, such as EGF receptor and Met (Shibamoto et al., 1994
). In addition, PTKs such as EGF receptor and c-erbB2 have been observed to associate with the cadherin–catenin complex in vivo (Hoschuetzky et al., 1994
; Ochiai et al., 1994
). The reversibility of tyrosine phosphorylation in vivo depends upon the coordinated action of both PTKs and PTPs. Therefore, to understand fully the regulation of cadherin function by reversible tyrosine phosphorylation, it will be necessary to identify and characterize the phosphatases that act upon adhesion complexes in vivo. Our observation of association between a receptor PTP, PTPμ, and cadherins in various tissues and cells is consistent with a role for this phosphatase in regulating cadherin function and lends further support to the regulatory importance of tyrosine phosphorylation in cell adhesion.
In this study, we have demonstrated that PTPμ interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. Although PTPμ can interact with several cadherins, it displays a restricted tissue distribution. Therefore, one would anticipate that, if regulation of cadherin function by reversible tyrosine phosphorylation was a general phenomenon, there would be additional PTPs that function in a manner analogous to PTPμ in other cell types. Subsequent to our original demonstration of association between PTPμ and the cadherin– catenin complex (Brady-Kalnay et al., 1995
), several reports have appeared that substantiate the general principle that members of the PTP family may be important regulators of cadherin-mediated adhesion. Association of a number of receptor and nontransmembrane PTPs with different members of the cadherin family in a variety of cell systems has now been reported. PTPκ, a receptor PTP that is closely related in structure to PTPμ (~75% sequence identity with the same overall arrangement of structural motifs), has been shown to associate directly with β-catenin and plakoglobin (Fuchs et al., 1996
). Interestingly, PTPκ displays a much broader expression pattern than PTPμ (Jiang et al., 1993
) and therefore may interact with cadherin–catenin complexes in many tissues. To date, four other PTPs have been shown to interact with cadherin– catenin complexes. Most recently, LAR (Aicher et al., 1997
) and a novel receptor PTP, termed PTPλ (a close relative of PTPs μ and κ; Cheng et al., 1997
), were also shown to associate with β-catenin. These authors observed that the association with β-catenin, like that involving PTPκ (Fuchs et al., 1996
), required the intracellular segment of the phosphatase (Aicher et al., 1997
; Cheng et al., 1997
). A LAR-like receptor PTP was found to associate with the cadherin–catenin complex in PC12 cells, and this association appears to be regulated by nerve growth factor–induced tyrosine phosphorylation of the PTP itself (Kypta et al., 1996
). In addition, a PTP1B-like cytoplasmic phosphatase has been shown to interact with N-cadherin (Balsamo et al., 1996
). The authors suggest that the association of the PTP with N-cadherin facilitates dephosphorylation of β-catenin, which is required for N-cadherin–mediated adhesion and its association with the actin cytoskeleton.
In contrast to this consensus view of the potential importance of PTPs in regulating the tyrosine phosphorylation of cadherin–catenin complexes in vivo, one report (Zondag et al., 1996
) has questioned the validity of our original observation. Therefore, we will respond in detail to the various issues raised in the paper by Zondag et al., in an attempt both to resolve this controversy and clarify the various issues. Zondag et al. report the generation of antibodies to an undefined epitope(s) in the ectodomain of PTPμ that fail to coimmunoprecipitate cadherin–catenin complexes. After successive rounds of immunoprecipitation with one of these antibodies, 3D7, to “clear” PTPμ from the cell lysate, the authors subjected the cleared lysate to immunoprecipitation with our antibody BK2. Even though they were unable to detect PTPμ in the cleared lysate by immunoblotting with their antibodies, they still observed cadherin in BK2 immunprecipitates. From this, the authors concluded that the interaction we observed was due to nonspecific cross-reactivity between BK2 and cadherin. There are two problems with this experiment and the conclusion drawn from it. Firstly, the authors did not blot the cleared lysate with BK2 to check whether there was a pool of PTPμ that was not recognized by their antibodies but was detected by BK2. Secondly, although the authors made the strong assertion of nonspecific cross- reactivity between BK2 and cadherin, they failed to demonstrate such cross-reactivity in a direct binding assay.
The following observations refute their argument. First, we used Sf9 cells, which do not contain detectable levels of endogenous PTPμ or E-cadherin, to express these proteins and reconstitute the complex. Through this approach we demonstrated that E-cadherin was only recovered in immunoprecipitates of PTPμ from lysates of cells in which both proteins had been coexpressed. Furthermore, the complex was detected in the reciprocal experiment, in which PTPμ was recovered in immunoprecipitates of E-cadherin, but again only from lysates of cells in which both proteins had been coexpressed. Second, and importantly, BK2 did not recognize E-cadherin in a direct binding assay in vitro, using purified components under nondenaturing conditions. Third, BK2 did not immunoprecipitate E-cadherin from lysates of MCF10A cells, which do not express PTPμ to a level that can be detected by antibody BK2 but express substantial levels of E-cadherin. The authors present data from a similar experiment in COS cells, which they state lacks endogenous PTPμ. They report that cadherin is detected in BK2 immunoprecipitates whether or not PTPμ was expressed ectopically. However, using a number of antibodies to PTPμ, including BK2, we detected expression of this protein in COS cells (data not shown), and thus coprecipitation of cadherin would not be unexpected. The reason for this discrepancy is unclear, although the authors did not test for the presence of PTPμ in COS cell lysates by blotting with BK2. Fourth, in WC5 cells transformed by temperature-sensitive v-Src and expressing E-cadherin ectopically, immunoprecipitates of PTPμ from lysates of cells cultured at the nonpermissive temperature contained coprecipitating cadherin, whereas at the permissive temperature the levels of associated cadherin were reduced substantially (Fig. ). It is unlikely that BK2 would display cross-reactivity only in lysates from one temperature condition. Finally, we have demonstrated interaction between PTPμ and various members of the cadherin family using different antibodies that recognize at least three distinct epitopes in the phosphatase.
Zondag et al. (1996)
presented several additional arguments to question the validity of the association we observed between PTPμ and cadherin. For example, they cited their failure to detect the phosphatase in anticadherin immunoprecipitates as further evidence that PTPμ and cadherin do not interact. However, it is important to note that in these experiments the authors used a pan-cadherin antibody that is directed against the COOH-terminal sequence that contains the segment of E-cadherin that is required for interaction with PTPμ. Therefore, the absence of PTPμ from these immunoprecipitates could easily be explained by steric hindrance introduced by antibody binding to cadherin. In addition, the authors dismiss our demonstration of direct interaction in blot-overlay binding studies as the result of production of the cadherin and PTPμ fusion proteins in bacteria, which, they suggest, is likely to yield misfolded or denatured protein and result in a high risk of nonspecific protein–protein interactions. However, the PTPμ used as probe was produced in insect Sf9 cells, not bacteria, and was catalytically functional and therefore was not denatured or misfolded. Furthermore, we included controls in the experiment to show that, as would be expected, under identical conditions E-cadherin bound β-catenin (specifically the NH2
-terminal segment and not the COOH-terminal segment of β-catenin) but not α-catenin. Therefore, it is unlikely that our results can be explained by nonspecific association or improper protein folding.
The observation that PTPμ could interact with several cadherins prompted us to investigate the binding site for PTPμ on the cadherins. For these studies, we used a series of WC5 rat astrocyte-like cell lines, which express PTPμ endogenously and express ectopically mutant forms of E-cadherin that lacked various portions of the cytoplasmic segment. The results indicated that the COOH-terminal 38 residues, which overlap with the catenin-binding domain, were required for the interaction with PTPμ. A number of factors suggest that this is likely to be a direct binding site: (a
) We have demonstrated previously that the intracellular segment of PTPμ interacts directly with the intracellular segment of E-cadherin in vitro; (b
) we have shown here (Fig. ) that PTPμ and E-cadherin interact after coexpression in Sf9 cells and, considering the extent of overexpression achieved in this system, it is unlikely that the interaction is mediated by an endogenous Sf9 cell protein; and (c
) deletions of other portions of the E-cadherin cytoplasmic segment had little effect on the association with PTPμ in WC5 cells. Although we did not detect a direct interaction between PTPμ and β-catenin in vitro or in Sf9 cells, we have detected both cadherin and β-catenin in immunoprecipitates of PTPμ (Brady-Kalnay et al., 1995
). In light of data suggesting that E-cadherin functions as a dimer (Brieher et al., 1996
; Nagar et al., 1996
), it is possible that one E-cadherin molecule of the dimer may bind PTPμ while the other interacts with β-catenin. The observation that the COOH-terminal 38 residues of E-cadherin are required for interaction with PTPμ raises the possibility that the association may be regulated by β-catenin in vivo.
In summary, we believe that our data have established convincingly the existence of a complex between PTPμ and various members of the family of cadherins in a number of different cell systems. In addition, we believe that we have presented data to refute convincingly the assertions of Zondag and colleagues (1996) that the association we observe between PTPμ and cadherins is artifactual. Our observations highlight further the potential importance of reversible tyrosine phosphorylation in regulating the adhesive properties of the cadherin family of cell adhesion molecules.