Here, we provide evidence that the core function of p120 in cadherin complexes is to regulate cadherin turnover. Previously, we showed that the stabilizing effect of p120 on E-cadherin in a p120-deficient SW48 cell line involved a post-transcriptional mechanism and required direct p120–E-cadherin interaction (Ireton et al., 2002
). However, it was not clear whether this phenomenon was generally applicable beyond SW48 cells, nor could we determine the underlying mechanism. Here, using siRNA and/or p120 reconstitution, we show that E-cadherin levels depend absolutely on p120 expression. Importantly, this set point mechanism is common to other (probably all) p120-binding cadherins because p120 knockdown also induced significant down-regulation of P-, VE-, and N-cadherins. The timing and location of p120 action argue strongly that p120 regulates adhesion via controlling cadherin turnover at the cell surface. These observations have crucial implications for roles of p120 in cadherin function and cancer.
We believe that the only exception to the requirement for p120 occurs in cells that express p120 family members such as ARVCF or δ-catenin. This qualifier is based in part on cell lines such as HCT116 where the observed reduction in E-cadherin levels after p120 siRNA expression did not perfectly parallel the extent of p120 loss. Indeed, although ARVCF is typically difficult to detect in many epithelial cell lines, it is expressed at moderate levels in HCT116 cells (unpublished data). Our data show that ARVCF and δ-catenin efficiently compensate for p120 loss when ectopically expressed in A431 cell lines expressing p120 siRNA. Despite significant structural and sequence similarity, plakophilin-3 had no effect, presumably because it does not bind classical cadherins. These data strongly imply that surface cadherin stability is invariably dependent on the binding of either p120 or a closely related family member, and the presence of variable levels of p120 family members likely accounts for the discrepancy in cell lines where p120 knockdown does not cause a corresponding loss of resident classical cadherins.
The fact that p120 availability limits cadherin levels has several crucial implications. For example, overexpression of dominant-negative cadherins frequently down-regulates expression of endogenous cadherins (Kintner, 1992
; Fujimori and Takeichi, 1993
; Zhu and Watt, 1996
), but the mechanism is unknown. Our data strongly suggest that a key action of dominant-negative cadherins is the sequestering of endogenous p120, thereby driving the turnover and degradation of endogenous cadherins. In addition, cadherin levels in cells may ultimately be controlled by factors that regulate p120 levels, and competition for interaction with p120 is likely to be physiologically relevant in cells that express more than one cadherin.
In theory, the absence of cadherins in p120-deficient cells indicates either a failure to normally synthesize cadherins or an efficient means of eliminating them when p120 is not present. However, our pulse-chase data indicate that p120 is not required for normal synthesis or transit of cadherin to the cell surface. Instead, p120 absence dramatically accelerates cadherin degradation after its arrival at the surface, indicating a role in regulating cadherin turnover at the membrane (modeled in ). Our data do not precisely distinguish the point at which p120 acts to prevent degradation. The simplest explanation is that p120 limits degradation by regulating internalization. Only cadherin-bound p120 is phosphorylated (Thoreson et al., 2000
), and p120 phosphorylation is the most likely means of regulating p120–cadherin affinity and/or p120 activity in the complex. We cannot rule out the less likely possibility that once internalized, p120 might control the next step, which targets the endocytosed cadherin for either degradation or recycling back to the surface. Regardless, it is likely that the ultimate destruction of the cadherin in p120-deficient A431 cells resides mainly in the proteosome, and to some extent in the lysosome.
Figure 7. Model for p120 function in regulating cadherin turnover. The low affinity of p120 for cadherins, as judged by coimmunoprecipitation experiments, probably reflects the ability of p120 to rapidly alternate between cadherin-bound and -unbound states. (1) (more ...)
Under normal circumstances, cadherin turnover is constitutive and endocytosis is a crucial mechanism for down-regulating cadherin adhesiveness (Le et al., 1999
; Xiao et al., 2003
). Previously, we postulated that p120 acts as a switch, inducing the assembly or disassembly of cadherin complexes through transient signaling events (probably tyrosine and serine phosphorylation), which in turn might regulate cadherin clustering. Our new data strongly favor a mechanism whereby dynamic assembly and disassembly of cadherin complexes is driven primarily by regulation of cadherin turnover rather than physical clustering (). A plausible explanation is that the rate of cadherin turnover is dictated by events at the cell surface that transiently increase or decrease p120 affinity for cadherins. The off state favors internalization/degradation, whereas the on state favors retention/recycling. The low affinity of p120 for cadherins, as judged by coimmunoprecipitation experiments (Thoreson et al., 2000
), probably reflects the ability of p120 to rapidly alternate between cadherin-bound and -unbound states. It is worth noting that α- and β-catenins are largely passive players in this model. Because their stability is controlled by cadherin binding, their fate is ultimately tied to cadherin levels, which are clearly controlled by p120. Of course, turnover and clustering are not mutually exclusive mechanisms, but our current data suggest that turnover may take precedence.
Recent experiments in C. elegans
indicate that p120 is not essential in these organisms. Indeed, both worms (Pettitt et al., 2003
) and flies (Myster et al., 2003
) are viable when p120 is removed, and p120-uncoupled E-cadherin can substitute effectively for wild-type E-cadherin in flies (Pacquelet et al., 2003
). In contrast, the murine p120 knockout is embryonic lethal (unpublished data). Additionally, our current data show clearly that p120 is essential in mammalian cells. It is possible that mammalian p120 has evolved both additional family members and increased complexity to accommodate the developmental demands of higher organisms.
An unanswered question is the exact targeting mechanism for internalization and/or degradation of cadherins not associated with p120. Because direct binding of p120 to E-cadherin is required, it is possible that p120 binding blocks the interaction of an unknown binding partner (or event) that targets E-cadherin for degradation. Candidates include presenilin-1 (Baki et al., 2001
; Marambaud et al., 2002
) and Hakai (Fujita et al., 2002
), which are reported to compete with p120 for binding the cadherin juxtamembrane domain. Presenilin-1 binding promotes proteolytic degradation of E-cadherin (Baki et al., 2001
; Marambaud et al., 2002
), whereas Hakai is a ubiquitin ligase that binds tyrosine-phosphorylated E-cadherin, leading to its ubiquitination and destruction (Fujita et al., 2002
). Several tyrosine kinase receptors are turned over via a similar mechanism involving the oncogene and ubiquitin ligase Cbl, which binds tyrosine-phosphorylated residues via its classical SH2 domain (for review see Hicke, 1999
). However, we were unable to block E-cadherin destruction in the p120 siRNA cell lines with either presenilin or tyrosine kinase inhibitors (unpublished data). Moreover, the mechanism we describe is common to several cadherins, whereas the Hakai mechanism appears specific for E-cadherin. Nonetheless, our data favor a model where an E-cadherin–targeting event is triggered by the absence or transient off-loading of p120.
Finally, several lines of evidence suggest that this new role for p120 in regulating cadherin turnover may be important in cancer. In cell lines, E-cadherin loss leaves p120 stranded in the cytoplasm, but has little effect on p120 levels. It is well established that E-cadherin loss occurs frequently by mutation (Berx et al., 1998
) and by epigenetic mechanisms (Comijn et al., 2001
; Matsumura et al., 2001
) that probably do not involve p120. In contrast, p120 loss clearly represents a different scenario that directly induces loss of E-cadherin, and thus ultimately, the entire cadherin complex. It follows that p120 loss may precede cadherin loss in the reported subset of tumors that have been shown to lack both proteins (for review see Thoreson and Reynolds, 2002
). Accumulating evidence suggests that p120 down-regulation occurs frequently in colon, prostate, lung, bladder, breast, and several other malignancies (for review see Thoreson and Reynolds, 2002
). p120 is both mutated and underexpressed in the colon carcinoma cell line SW48, and indeed, E-cadherin is indeed strongly down-regulated in these cells, providing the first physiologically relevant example of this phenomena in a carcinoma cell line. However, no other p120-deficient cell lines have been described, and physical alterations in the p120 gene locus have not been associated with malignancy. Together, these observations suggest that p120 down-regulation in tumors occurs by an epigenetic mechanism that has yet to be identified, and raise the possibility that like E-cadherin, p120 acts as a tumor suppressor.
In conclusion, we show that p120 levels determine steady-state levels of functional cadherins by regulating cadherin turnover at the cell surface. This is likely the core function of p120 in the cadherin complex and suggests that cadherin adhesiveness is modulated, in part, by signaling events that dynamically influence p120–cadherin affinity. In addition, p120 is clearly at the top of the cadherin food chain in terms of who controls the overall fate of the complex. Together with reports of p120 down-regulation in a wide range of epithelial tumors, these data suggest a role for p120 as a tumor suppressor.