Research Article 4351 Shared molecular mechanisms regulate multiple : canonical Wnt signals and components modulate -catenin isoform-1 and additional p120 subfamily members

Ji Yeon Hong1,2, Jae-il Park3, Kyucheol Cho1,2, Dongmin Gu1,2, Hong Ji1, Steven E. Artandi3 and Pierre D. McCrea1,2,* 1Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA 2Program in and Development, University of Texas Graduate School of Biomedical Science–Houston, TX 77030, USA 3Department of Medicine, Division of Hematology, Stanford University, School of Medicine, Stanford, CA 94305, USA *Author for correspondence ([email protected])

Accepted 6 September 2010 Journal of Cell Science 123, 4351-4365 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jcs.067199

Summary Wnt signaling pathways have fundamental roles in animal development and tumor progression. Here, employing Xenopus embryos and mammalian cell lines, we report that the degradation machinery of the canonical Wnt pathway modulates p120-catenin stability through mechanisms shared with those regulating -catenin. For example, in common with -catenin, exogenous expression of destruction complex components, such as GSK3 and axin, promotes degradation of p120-catenin. Again in parallel with -catenin, reduction of canonical Wnt signals upon depletion of LRP5 and LRP6 results in p120-catenin degradation. At the primary sequence level, we resolved conserved GSK3 phosphorylation sites in the amino-terminal region of p120-catenin present exclusively in isoform-1. Point-mutagenesis of these residues inhibited the association of destruction complex components, such as those involved in ubiquitylation, resulting in stabilization of p120-catenin. Functionally, in line with predictions, p120 stabilization increased its signaling activity in the context of the p120–Kaiso pathway. Importantly, we found that two additional p120-catenin family members, ARVCF-catenin and -catenin, associate with axin and are degraded in its presence. Thus, as supported using gain- and loss-of-function approaches in embryo and cell line systems, canonical Wnt signals appear poised to have an impact upon a breadth of catenin biology in vertebrate development and, possibly, human cancers.

Journal of Cell Science Key words: p120, catenin, , Wnt3a, LRP

Introduction (adenomatous polyposis coli) (Amit et al., 2002; Behrens et al., Wnt signaling pathways engage in multiple aspects of development 1998; Hedgepeth et al., 1999; Ikeda et al., 1998; Lee et al., 2003). and tumor progression (Grigoryan et al., 2008; Moon et al., 2004; In the absence of Wnt ligand(s), -catenin is phosphorylated by Nusse, 2005; Reya and Clevers, 2005). In what is defined as CK1 and GSK3 in association with a larger scaffolding complex canonical Wnt signaling, -catenin (also known as catenin beta-1) including axin and APC. Phosphorylated -catenin is then is generally considered the key element transmitting Wnt signals recognized and ubiquitylated by -TrCP (-transducin repeat- into the nucleus (McCrea et al., 1993; Moon et al., 2004; Sokol, containing protein), a substrate-recognition E3 subunit of ubiquitin 1999). However, recent vertebrate work from our group and others ligase, resulting in complex degradation through the ubiquitin- has indicated that a structurally related protein, p120-catenin (also mediated proteasome pathway (Aberle et al., 1997; Liu et al., known as catenin delta-1), also responds to Wnt signals, and 1999; Winston et al., 1999). By contrast, when extracellular Wnt unexpectedly, that a number of established -catenin targets ligands bind to the transmembrane and context-dependent receptor– are co-regulated by p120-catenin in conjunction with the co-receptor proteins, Fz (Frizzled) and LRP (low-density lipoprotein transcriptional repressor Kaiso (Park et al., 2005; Spring et al., receptor-related protein), intracellular Dsh (Dishevelled) becomes 2005). Despite these findings, with some recent debate centered on activated by unknown mechanisms (Zeng et al., 2008). Among kaiso (Iioka et al., 2009; Ruzov et al., 2009a; Ruzov et al., 2009b), other possibilities, activated Dsh together with LRP is thought to an important unanswered question concerns the nature of the sequester axin to the inner plasma membrane and inhibit the ability precise mechanism by which p120-catenin is regulated in response of GSK3 to phosphorylate -catenin directly, thereby promoting to the presence or absence of Wnt signals, what similarities such -catenin stabilization (Bilic et al., 2007; Cselenyi et al., 2008; mechanisms might share with those known to regulate -catenin Schweizer and Varmus, 2003; Tamai et al., 2004; Wehrli et al., and whether additional p120-subfamily members might also be 2000; Zeng et al., 2008; Zeng et al., 2005). This pool of -catenin subject to Wnt regulation. responds to additional signals before accessing the nucleoplasmic The metabolic stability of -catenin is regulated by destruction space, where -catenin relieves repression otherwise conferred by complex components including GSK3 (glycogen synthase kinase- the HMG (high-mobility group) proteins LEF (leukocyte enhancing 3), CK1 (casein kinase I isoform alpha), axin and APC factor) or TCF (T-cell factor) (Esufali and Bapat, 2004; Gottardi 4352 Journal of Cell Science 123 (24)

and Gumbiner, 2004; Mosimann et al., 2009; Wu et al., 2008). (encoding cyclin D1) and MMP7 (encoding matrilysin). At such Genes activated by the -catenin–LEF (TCF) complex (canonical- promoters, p120 recognizes and associates with kaiso, a Wnt target genes) are numerous, with well-known examples transcriptional repressor of the BTB/POZ zinc-finger family that including the gene encoding the homeobox protein siamois recognizes consensus sequences (KCSs) in DNA (Park et al., 2005; (Xenopus), as well as MYC and CCND1 (encoding cyclin-D1) Spring et al., 2005). Once formed, the p120–Kaiso complex is (Brannon et al., 1997; He et al., 1998; Nusse, 1999; Tetsu and thought to dissociate from the gene promoter. Kaiso-directed gene McCormick, 1999). In addition to the extensive involvement of the repression is thus relieved, and increased transcriptional activity Wnt pathway in embryogenesis and adult-tissue homeostasis, ensues. In addition to Kaiso–KCS interactions with DNA, for pathological pathway activation is linked to multiple human which conflicting reports have arisen in certain gene contexts diseases, such as those characterized by bone abnormalities and (Iioka et al., 2009; Ruzov et al., 2009a; Ruzov et al., 2009b), Kaiso cancer (Chen and Alman, 2009; Oving and Clevers, 2002). further recognizes methyl-CpG islands present in gene control Members of the p120-catenin subfamily include p120-catenin, regions that are associated with repressive states (Daniel, 2007; ARVCF-catenin, -catenin and p0071 (Hatzfeld, 2005; McCrea Daniel et al., 2002; Prokhortchouk et al., 2001; Yoon et al., 2003). and Park, 2007). p120 subfamily members bear limited structural However, at these sites in particular, no indications have yet arisen resemblance to the -catenin subfamily members -catenin and that p120-catenin acts to relieve Kaiso-mediated repression, and (-catenin). The most obvious similarity is that each further uncertainty concerns the relationship between the roles of contains a central Arm (Armadillo) domain consisting of either Kaiso at CpG-island versus sequence-specific DNA-binding sites. nine (p120 subfamily members) (Choi and Weis, 2005) or 12 (- Our most recent work has focused upon upstream elements of catenin and plakoglobin) Arm repeats. Through such Arm domains, the p120–Kaiso pathway. A central question that we address in this members of each catenin subfamily were first found to bind to the report is the specific means by which p120-catenin signaling cytoplasmic tails of cadherin cell–cell adhesion proteins. However, activity is controlled and the extent to which the mechanisms while p120 subfamily members competitively associate with involved are shared with -catenin. Using both vertebrate embryo cytoplasmic cadherin membrane-proximal regions, where they and cell line systems, we find that regulatory overlaps do indeed contribute to cadherin stabilization (Reynolds and Carnahan, 2004; exist, as examined in-depth for p120-catenin, that are further likely Xiao et al., 2007), -catenin or plakoglobin bind more distally and to extend to additional p120 subfamily members, such as ARVCF- confer other attributes to the complex, such as indirect cytoskeletal catenin and -catenin. Thus, we propose that Wnt pathway signals association (Abe and Takeichi, 2008; Drees et al., 2005; Zhurinsky impinge through shared mechanisms on the stability of distinct et al., 2000). In addition to binding and modulating and p120 subfamily members, producing a networked catenin response engaging in nuclear activities, p120 subfamily members have now relevant to development and, potentially, disease. been well recognized to modulate small GTPases such as RhoA and Rac (Anastasiadis, 2007; Casagolda et al., 2010; Elia et al., Results 2006; Fang et al., 2004; McCrea and Park, 2007; Perez-Moreno et GSK3 modulates p120-catenin stability al., 2008; Wildenberg et al., 2006; Wolf et al., 2006). p120 Based upon our prior work that suggested similarities in p120- subfamily members can further be distinguished from -catenin or catenin and -catenin regulation (Park et al., 2006), we directly

Journal of Cell Science plakoglobin in that each transcript bears multiple potential examined the involvement of established components of the translational start sites and arises from differential splicing events. destruction machinery for -catenin in p120 regulation. Studies These characteristics have added layers of regulatory complexity were initially performed to identify the impact of expressing to studies of p120 subfamily proteins (Mo and Reynolds, 1996; exogenous GSK3 in early Xenopus embryos. As our prior Yanagisawa et al., 2008). findings showed that inhibiting GSK3 through LiCl increased Aside from the central Arm domains, which themselves share endogenous p120-catenin levels, it was expected that ectopic only modest homology, catenin amino- or carboxy-terminal regions expression of GSK3 should decrease p120-catenin levels (Park bear yet less resemblance across distinct members, even within a et al., 2006). Indeed, native GSK3 and CK1 reproducibly subfamily. These end domains engage in inter-protein associations, reduced the level of epitope-tagged Xenopus p120 isoform-1 while in an intra-molecular manner, might modulate Arm-domain (longest isoform; translation beginning at the most upstream ATG interactions (Castano et al., 2002; Choi et al., 2006; Gottardi and site of p120), whereas CK1 or a kinase-dead form of GSK3 Gumbiner, 2004; Mo et al., 2009; Solanas et al., 2004). In the (KD) (negative-control), did not have statistically significant context of the canonical Wnt pathway, the amino-terminal domain effects (Fig. 1A, and data not shown). Unexpectedly, lower as of -catenin is well known to harbor conserved GSK3 and CK1 opposed to intermediate or sometimes even high GSK3 sites that, when phosphorylated, allow for -TrCP recognition expression (5 pg versus 10 pg, or in some cases 100 pg mRNA) (Aberle et al., 1997; Liu et al., 1999; Winston et al., 1999). Thus, produced modestly greater reductions in ectopic or endogenous this domain participates in determining the extent and activity of p120-catenin isoform-1 levels (Fig. 1A and supplementary the cytoplasmic:nuclear signaling pool of -catenin. Additionally, material Fig. S1A,B), as discussed later in relation to other the -catenin subfamily member plakoglobin contains this findings (Fig. 5 and Discussion). Exogenous epitope-tagged p120- destruction box, and its protein stability is likewise modulated by catenin was used in these initial studies given that a large canonical Wnt signaling (Karnovsky and Klymkowsky, 1995; proportion of endogenous p120 is bound to cadherin, which, as Kodama et al., 1999). established for -catenin, would be expected to exhibit less Previous work arising from our and independent groups has sensitivity to Wnt-pathway regulation. We next conducted in outlined a new role for p120-catenin in nuclear signaling. One vitro kinase assays with p120, CK1 and GSK3. As occurs for - context examined has been the response of p120 to canonical Wnt catenin (Liu et al., 1999), phosphorylation upon p120 was faint signals, where, together with -catenin, p120 modulates expression but reproducible, suggesting that it is a direct GSK3 and/or of select Wnt gene targets such as Siamois (Xenopus), CCND1 CK1 target (supplementary material Fig. S2 and data not shown). Shared mechanisms regulate multiple 4353 Journal of Cell Science

Fig. 1. GSK3 modulates the levels of p120-catenin protein. (A)Myc–p120-catenin mRNA (0.5 ng) was microinjected into each blastomere of two-cell-stage embryos with the indicated levels (in vitro transcribed mRNA) of GSK3 kinase, GSK3 kinase-dead mutant (KD), CK1 or CK1. Embryos were harvested at stages 10–11 for immunoblotting (IB) with antibody against Myc, with serving as an internal loading control. The right panel quantitates Myc–p120 protein levels normalized to actin, using data from four independent experiments. Shown are means + s.e.m. (B)Failure of gastrulation (blastopore closure) following expression of exogenous p120-catenin (0.5 ng mRNA injection), versus rescue upon coexpression with GSK3 mRNA (5 pg) or more-partial rescue using coexpressed axin (5 pg). Shown are means + s.e.m. (C)HeLa cells were incubated in media containing LiCl (25M, 4 hours), and cytoplasmic versus nuclear fractionation was determined (Pierce). Endogenous p120-catenin was detected using a monoclonal antibody directed against p120 (pp120). (D)Either Venus–sh- random or Venus–sh-GSK3 and Myc–xp120-catenin were co-transfected into HeLa cells, as indicated. Forty eight hours after transfection, cells were assayed for Myc–p120 using immunofluorescence or immunoblotting (left versus right panels). For immunofluorescence, cells were fixed with 4% PFA and probed for Myc– p120 followed by Texas-red-conjugated anti-mouse visualization. Arrowheads indicate p120-catenin. For immunoblotting, served as an internal loading control. (E)HA–p120-catenin was expressed in 293T cells. Following a 1 hour incubation with 1.48ϫ106Bq of 35S-Met and -Cys, cells were treated at the times indicated with 0.5% NP-40 lysis buffer. HA–p120 was immunoprecipitated using antibodies against HA and resolved by SDS-PAGE followed by autoradiography (band densities quantitated using ImageJ). These data were collected from two independent experiments. Shown are means ± s.e.m.

We next tested for a functional relationship between p120 and a carefully titrated dose of GSK3 was co-injected with p120, we GSK3 using a phenotypic assay in vivo. p120-catenin was observed significant rescue of the gastrulation phenotypes (Fig. overexpressed, and, as anticipated, gastrulation failures arose in a 1B; compare the first two bars). Kinase-dead GSK3 did not significant fraction of embryos (Fang et al., 2004). However, when produce such rescue effects (GSK3 KD, negative control), whereas 4354 Journal of Cell Science 123 (24)

a titrated dose of axin displayed modest rescuing activity. Consistent prolonged the half-life of endogenous p120-catenin (Fig. 1E). with their respective rescuing effects, GSK3 or axin acted to Taken together, these data (Fig. 1) suggest that p120 is subject to reduce exogenous p120 levels when they were coexpressed with modulation by GSK3. p120 in vivo (Fig. 7A; compare lanes 1–3). To complement over-expression assays, we next employed loss- p120-catenin is regulated by the ubiquitin-proteasome of-function tests. As morpholinos to deplete GSK3 have not been pathway characterized for work in Xenopus, we instead employed a proven Given that -catenin is phosphorylated by CK1 and GSK3 and short-hairpin shRNA to block GSK3 function in HeLa cells (Kim degraded through the proteasomal pathway, we tested whether et al., 2006). As predicted, p120-catenin protein levels were elevated p120 is similarly regulated. To assist in following the signaling upon the depletion of endogenous GSK3, as assessed using pool of p120, we employed MDA-MB-231 and MDA-MB-435 immunofluorescence analysis as well as immunoblotting (Fig. 1D). cells, which are largely E-cadherin deficient, in addition to 293T Noteworthy in our immunofluorescent images is the increased cells, which express E-cadherin. MDA-MB-231, MDA-MB-435 presence of p120 in both the cytoplasmic and nuclear and 293T cells were incubated with the proteasome inhibitor compartments, relative to negative-control shRNA-transfected cells. MG132, and endogenous p120 was detected by immunoblotting. To better resolve the signaling pool of p120-catenin, we employed Consistent with the pulse–chase data from MG132-treated HeLa cell fractionation of the cytoplasmic and nuclear populations. In cells (Fig. 1E), p120-catenin was stabilized by MG132 in a dose- common with -catenin, cytoplasmic p120-catenin was stabilized dependent manner in all cell lines tested (Fig. 2A). To detect when cells were incubated in the presence of the GSK3 inhibitor endogenous p120-catenin, we employed two previously LiCl, whereas a lesser effect was seen for p120 in the nuclear characterized monoclonal antibodies against p120, 6H11 and pp120 fraction (Fig. 1C). Additionally, the sensitivity of p120 to GSK3 (Ireton et al., 2002). 6H11 recognizes an amino-terminal epitope, was supported in pulse-chase data, where LiCl or MG132 treatment selectively resolving the isoform generated from the most-upstream

Fig. 2. Proteasome-mediated degradation appears to modulate the levels of p120-catenin. (A)MDA-231, MDA-435 and 293T cells were treated with varying doses of the proteasome Journal of Cell Science inhibitor MG132 (0, 2, 5, 10, 25M). After 6 hours, cells were harvested for immunoblotting (IB) with an antibody against p120 (6H11 and pp120). (B)Myc–p120-catenin was co-transfected into HeLa cells with HA–ubiquitin. After 24 hours, cells were treated with LiCl (25M) for 4 hours to block GSK3 function. Cells were harvested and HA–ubiquitylated proteins immunoprecipitated, followed by Myc–p120 immunoblotting. Endogenous -catenin was used as a positive ubiquitylation control. (C)MDA-435 and 293T cells were treated with varying doses of the CK1 inhibitor D4476 (0, 2, 5, 10, 25M). p120-catenin was visualized using an antibody directed against this protein (pp120), and endogenous -catenin served as an endogenous positive control. (D)Single cells of two-cell embryos were injected with Myc–p120-catenin and Myc–-catenin mRNA (0.1 ng mRNA each) and treated for 24 hours with varying concentrations of the CK1 inhibitor D4476 (10, 50, 200M) in the presence of Fugene6. An antibody against Myc was used to detect both p120-catenin and -catenin. (E)HeLa cells were grown on coverslips, transiently transfected with Myc–kaiso and treated with MG132 (10M) and LiCl (50M). Cells were fixed with 4% PFA for 10 minutes, blocked with 5% goat serum in PBS and immunostained with antibody against Myc. Shared mechanisms regulate multiple catenins 4355

translational start site, known as isoform-1. pp120 resolves Additionally, we immunoprecipitated the E3 ubiquitin ligase - additional isoforms, as it is directed against a carboxyl-terminal TrCP, resolving its association with p120 relative to negative epitope present across most p120 translation products. In the cell controls (Fig. 3D). To test endogenous p120 binding to GSK3, lines used here, pp120 detects two products thought to be isoform- we immunoprecipitated endogenous GSK3 and resolved the p120– 1 and -3, based upon their relative SDS-PAGE migration (Mariner GSK3 complex in 293T and MDA-435 cells (Fig. 3E). Thus, in et al., 2004; Paredes et al., 2007) and reactivity to 6H11 (isoform- an in vivo context, p120-catenin directly or indirectly interacts 1) versus pp120 (isoform-1 and -3, etc.). Inhibition of the with some of the key proteins known to regulate -catenin stability. proteasome pathway through dose-dependent titration of a chemical inhibitor (MG132) raised p120 isoform-1 levels in all cell lines The amino-terminal domain of p120 associates with GSK tested, with an observable yet lesser impact upon isoform-3, as and CK1 was evident in MDA-435 cells (Fig. 2A). This response will be The amino-terminal domain of -catenin provides the platform for addressed later in more depth (Fig. 7). Likewise, as CK1 is a its successive association with CK1 and GSK3 and harbors the priming kinase for GSK3 in the destruction process for-catenin, serine and threonine sites that become phosphorylated (Liu et al., we assayed the effect of incubating Xenopus embryos in the 1999; Orford et al., 1997). As a first step in assessing p120-catenin presence of the CK1 inhibitor D4476. Consistently, D4476 exposure structure–function relationships, we searched for potential GSK3 increased exogenous p120 levels, as was expected and observed phosphorylation sites. Three evolutionally conserved GSK3 for -catenin (positive control) (Fig. 2D). In keeping with our phosphorylation regions were resolved, with one being amino- amphibian embryo and exogenous expression results, endogenous terminal and two residing in the domain. p120 isoform-1A in mammalian cells was stabilized when MDA- Interestingly, the amino-terminal region of p120 contained a 3 15 435 or 293T cells were treated with D4476 (Fig. 2C). Again DSX3SX2SX3S motif comparable to the GSK3-sensitive site analogous to -catenin (Aberle et al., 1997), p120 ubiquitylation present in -catenin (Fig. 5A) (Winston et al., 1999). To map the was clearly evident upon its coexpression with HA–ubiquitin in interaction of p120 with GSK3 and CK1, we generated a series HeLa cells (Fig. 2B). Likewise, as predicted if dependent upon of p120 deletion constructs (a–f) (Fig. 4A) and performed co- GSK3, such p120 ubiquitylation dropped when cells were immunoprecipitations from Xenopus embryo extracts. Constructs incubated in the presence of LiCl (Fig. 2B). As expression of containing the amino-terminal region of p120 associated with exogenous p120 recruits the transcription repressor Kaiso from the GSK3 (fl, b, c and e), whereas p120 constructs lacking this region nucleus to the cytoplasm (Kelly et al., 2004; Spring et al., 2005), (a, d and f) did not (Fig. 4B). We then examined ubiquitylation of we next asked whether endogenous p120 stabilization correlates the p120 deletion mutants following expression in HeLa cells. with a similar outcome. As expected, in the coordinate presence of Correspondingly, only those lacking the amino-terminal region of MG132 and LiCl, Kaiso relocalization (Fig. 2E) was coincident p120 were negative for ubiquitylation (Fig. 4C). Based on such with known effects upon p120 stabilization (Fig. 1C and Fig. 2A), findings, we next tested whether the CK1 priming kinase of - although it is important to note that these inhibitors produce many catenin (primes for GSK3) interacts with the analogous region of effects aside from p120 stabilization. LiCl or MG132 alone p120-catenin. Employing a similar co-immunoprecipitation strategy, produced a reproducible but considerably weaker Kaiso we observed that CK1 associates with the amino-terminal domain

Journal of Cell Science relocalization, consistent with their lesser protection of p120 relative of p120 (c), but not with a construct (d) lacking this region (Fig. to the combined treatment (data not shown). Together, data from 4D). Thus, in keeping with our earlier findings, these results Figs 1 and 2 suggest that endogenous p120-catenin is subject to suggest that p120 and -catenin engage in shared protein some of the same regulatory processes established for -catenin in interactions reflecting their similar biochemical and likely the context of canonical Wnt signaling. functional responses to Wnt signals.

Association of destruction complex components with p120 point mutant differs in destruction-complex p120-catenin responsiveness Several in vivo and in vitro reports show that -catenin associates To test whether the potential GSK3 or CK1 sites identified in with axin, -TrCP, GSK3 and CK1 (Ikeda et al., 1998; Liu et the amino-domain of p120 are in fact relevant to its sensitivity to al., 1999; Liu et al., 2002). Based upon the implied functional destruction complex components including GSK3, CK1 and - relationships we observed for p120-catenin (Figs 1 and 2), we TrCP, we generated a compound point mutant (p1204SA: SerrAla: tested whether p120 might likewise engage in such associations. Ser6, Ser8, Ser11, Ser15) (Fig. 5A). Full-length p120-catenin (fl: As a considerable fraction of endogenous p120 is complexed with isoform-1) versus p1204SA mutant were expressed in Xenopus cadherins as opposed to being within an accessible signaling pool, embryos, and their protein stability examined in response to co-immunoprecipitations were performed from Xenopus embryo coexpressed GSK3. Whereas p120-fl levels were consistently extracts using ectopically expressed proteins. We first tested for reduced upon GSK3 coexpression (Fig. 5B, compare lanes 1 and the association of p120 with GSK3, and, although weakly apparent 2; see also Fig. 1A and Fig. 7A), the levels of the mutant were, by in immunoblots, the p120–GSK3 complex was reproducibly contrast, not lowered (Fig. 5B, compare lanes 3 and 4). resolved relative to controls and GSK3 KD (Fig. 3A, see also Fig. In fact, surprisingly, the levels of p120 were reproducibly 4B). We next assessed axin, which interacts with multiple increased upon p1204SA coexpression with GSK3. Apparently, in components of the destruction complex for -catenin. In producing p1204SA, a direct or indirect protective effect of GSK3 precipitations conducted in either direction (reverse precipitation was unmasked. This possibility might also have been reflected in not shown), axin associated with p120 (Fig. 3B; Kaiso negative the unexpected lesser impact upon p120 levels and degradation of control). p120 was also found in complex with CK1, the priming intermediate (10 pg or in some cases higher) GSK3 levels relative kinase that acts upon -catenin immediately before GSK3 (Fig. to lower (5 pg) GSK3 levels (Fig. 1A and supplementary material 3C; kaiso and C-cadherin serving as negative controls). Fig. S1A,B). We hypothesize that, at intermediate or higher GSK3 4356 Journal of Cell Science 123 (24)

Fig. 3. p120-catenin associates with components of the destruction complex. (A)HA–p120-catenin (1 ng) was co-injected with either Myc–GSK3 or Myc–GSK3 KD (0.5 ng) into both blastomeres of two- cell embryos. Myc–GSK3 immunoprecipitates (IP) were immunoblotted (IB) with antibody against HA to detect p120. (B)HA–axin (0.5 ng) was microinjected with Myc–p120-catenin (0.5 ng) or Myc–Kaiso (0.5 ng) into both blastomeres of two-cell embryos, subsequently harvested at gastrulation. Anti- HA antibody (axin) immunoprecipitates were blotted with antibody against Myc to detect p120-catenin versus Kaiso (negative control). WCL, whole-cell lysate. (C)HA– CK1 was injected into both blastomeres of two-cell embryos along with Myc–p120, Myc–C-cadherin or Myc–Kaiso, and embryos harvested at early–mid gastrulation (stage 10–11). This was followed by anti- HA immunoprecipitation (CK1) and then anti-Myc (p120, or negative controls C- cadherin or kaiso) or anti-HA immunoblotting (CK1). (D)The association of Myc–-TrCP (0.5 ng) and HA–p120-catenin (1 ng) was resolved using methods analogous to those used in C. (E)293T and MDA-435 cells were grown in 10 cm dishes, and lysates immunoprecipitated for endogenous GSK3 (BD Transduction). The association of endogenous p120-catenin (6H11) with

Journal of Cell Science GSK3 was resolved by immunoblotting (- catenin positive control).

levels, wild-type p120 might become susceptible to direct or putative amino-terminal CK1–GSK3 phosphorylation site of indirect GSK3 effects that are protective in nature p120 (Fig. 5A). Indeed, using a co-immunoprecipitation and (phosphorylation or other events), whereas lower GSK3 activity immunoblotting approach, we consistently resolved the interaction largely acts at the negative-regulatory sites that we resolved. Indeed, of p120 with -TrCP, whereas our phosphorylation mutant p1204SA distinct from a further predicted conserved site (Ser199) just exhibited considerably reduced -TrCP co-association (Fig. 5C, upstream of the Arm domain of p120, other potential but more compare lane 2 with lanes 4 and 6). These observations are distal GSK sites have been reported in p120 (Xia et al., 2003). consistent with the known biology of -catenin, wherein Evidence supporting the modification by GSK3 of such additional phosphorylation is required for -TrCP recruitment (Liu et al., predicted sites was not obvious, however, in our in vitro kinase 1999). Given this outcome, we suspected that p1204SA would prove assays (supplementary material Fig. S2, and data not shown). less susceptible to ubiquitylation relative to wild type and tested Alternative possibilities include GSK3 phosphorylation of LRP, this possibility in HeLa cells through coexpression with HA– leading to inhibition of the destruction complex (either axin or ubiquitin. Indeed, while p120 displayed a strong ubiquitylation GSK3) and -catenin stabilization (Cselenyi et al., 2008; Tamai signal, poly-ubiquitylation upon p1204SA was greatly reduced. et al., 2004; Zeng et al., 2008; Zeng et al., 2005). Whatever the Interestingly, mono- or di-ubiquitylated forms showed lesser underlying mechanistic explanation, mutation of the conserved differences when comparing p120 versus p1204SA (Fig. 5D). four amino-terminal serine residues of p120 (p1204SA: SerrAla: Consistent with reduced ubiquitylation, pulse–chase data indicated Ser6, Ser8, Ser11, Ser15) protects p120 from GSK3-mediated that p1204SA has an extended half-life relative to native p120 degradation, consistent with their relevance in determining p120 (approximately 3 hours versus 1.5 hours) (Fig. 5E). At the functional levels and activities. level, p120-catenin has a number of in vivo activities, including In addition to candidate kinase sites, study of the primary the capacity to relieve Kaiso-mediated repression of target genes sequence of p120 pointed to a conserved potential -TrCP containing sequence-specific binding sites (KCS). Thus, we next recognition motif, DSEXXS (Orford et al., 1997; Winston et al., examined whether the transcriptional activity of p1204SA is higher 1999). In common with -catenin, this motif resides adjacent to the than that of wild-type p120, using Wnt-11 as a direct endogenous Shared mechanisms regulate multiple catenins 4357

gene readout (Kim et al., 2004). Employing semi-quantitative RT- bind membrane-distally) and their regulation of small GTPases PCR (Fig. 5F), and real-time PCR (Fig. 5G), p1204SA promoted (-catenin and plakoglobin apparently lack this functionality). Wnt-11 expression (relief of kaiso-mediated repression) to a greater Given our p120-catenin findings, we wished to know whether extent than wild-type p120-catenin, even though p1204SA was other p120 subfamily members might interact and respond to reproducibly expressed at lower levels (Fig. 5B, compare lanes 1 components of the destruction complex. Probing Xenopus embryo and 3). Furthermore, the p1204SA mutant proved more effective in extracts, we first tested whether one of the main constituents of promoting the expression of xSiamois (supplementary material the destruction complex, axin, had the capacity to diminish Fig. S3), another direct p120–Kaiso (as well as -catenin–TCF– ARVCF and -catenin levels. Serving as positive controls, - LEF) gene target (Park et al., 2005). These data indicate that catenin and p120 levels were reduced in the presence of axin stabilized p120 (p1204SA) exhibits more potent gene-regulatory (Fig. 6A and Fig. 1B). Likewise, ARVCF and -catenin were effects than wild-type p120-catenin, analogous to the -catenin significantly decreased upon axin coexpression, whereas negative context (Lyons et al., 2004; Yost et al., 1996). controls (xKazrin and EWS) showed no response to axin (Fig. 6A). We further asked whether ARVCF-catenin and -catenin Shared mechanisms modulate the metabolic stability of associate with axin, as we demonstrated above for p120, and as multiple p120-catenin subfamily members known to occur for -catenin. Indeed, in common with p120- p120-catenin is the prototypical member of the p120-catenin catenin and -catenin (positive controls), both ARVCF and - subfamily that comprises p120-catenin itself, ARVCF-catenin, - catenin associated with axin, relative to negative controls such as catenin and p0071-catenin (McCrea and Park, 2007). These p120- xDyrk and xKaiso (Fig. 6B and 6C). Although full-length - catenin family members have certain shared features, such as a catenin exhibited less association with axin than did -catenin, central Armadillo domain (nine repeats as opposed to the 12 in shorter forms of -catenin, which likely arose from endogenous -catenin or plakoglobin), their association with cadherin proteolytic processing or incomplete translation, showed strong membrane-proximal regions (-catenin and plakoglobin instead association with axin (Fig. 6C). Journal of Cell Science

Fig. 4. Mapping of the association of p120 with GSK3, CK1 and ubiquitin. (A)Myc-tagged p120- catenin deletion constructs (a–f). The table summarizes the relative p120-construct:GSK3 association, as shown in B. (B)HA–GSK3 (0.5 ng mRNA) was coexpressed with varying p120–catenin deletion constructs (a–f) (0.5 ng), followed by HA–GSK3 immunoprecipitation and Myc-construct immunoblotting (IB). (C)Either Myc– p120-catenin (fl) or Myc–DN-p120-catenin (construct d) was co-transfected with HA-tagged ubiquitin into HeLa cells. Following HA–ubiquitin immunoprecipitation, co- associated (versus not associated) Myc–p120 constructs were visualized by immunoblotting. (D)Myc–p120 constructs (fl, c and d) (0.5 ng mRNA) were co-injected with HA–CK1 (0.5 ng) in both blastomeres of two-cell embryos. Gastrula embryo extracts were immunoprecipitated for HA–CK1 and blotted for co- associated (versus not associated) constructs of Myc– p120. Right panel, lysate indicates Myc–p120 construct expression. 4358 Journal of Cell Science 123 (24)

Fig. 5. Phosphorylation-dependent p120-catenin ubiquitylation and proteasomal degradation. (A)Cross-species sequence alignment of p120- catenin regions harboring conserved predicted GSK3 phosphorylation and ubiquitylation sites. Highlights shown in each region include conserved serine residues, and a DSEXXS motif for -TrCP recognition. (B)HA–p120 (0.3 ng mRNA), versus the HA–p1204SA point mutant (4SrA, see Fig. 4A, 0.3 ng), was co-injected with HA–GSK3 (0.1 ng) into both cells of two-cell embryos. Gastrula embryo lysates (stages 11–12) were immunoblotted (IB) for the HA–p120 constructs, revealing a differing response to GSK3. (C)HA–p120 versus HA–p1204SA (0.5 ng) were co-injected with Myc– -TrCP (0.5 ng) into one blastomere of two-cell embryos. Gastrula embryo lysates were immunoprecipitated for Myc–-TrCP and assayed for co-associated HA–p120 or HA–p1204SA. (D)Myc–p120 or Myc–p1204SA was co-transfected with HA–ubiquitin into HeLa cells. HA–ubiquitin was immunoprecipitated and then immunoblotting used to detect ubiquitylated Myc–p120 versus Myc–p1204SA. (E)The half-lives of HA–p120 versus HA–p1204SA were monitored by pulse– chase analysis. Following a 1 hour pulse–chase with [35S] Met and Cys, HeLa cells transfected with HA–p120 and HA–p1204SA were harvested at the times indicated, and anti-HA immunoprecipitates were resolved by SDS-PAGE and visualized by autoradiography (quantitation of band intensities employed Image J and was normalized to the zero time-point). These data are representative of two independent experiments. (F)Myc–GSK3 (5 pg mRNA) or Myc–kaiso (0.25 ng) was microinjected with HA–p120 versus HA–p1204SA (0.25 ng) into one blastomere of two- cell embryos. Total mRNA injection loads were Journal of Cell Science equalized using -galactosidase mRNA. Gastrula embryo cDNA (stages 10–12) was subject to RT- PCR to assay endogenous xWnt-11 transcript levels. Band intensities are indicated relative to the -gal control (set at 1), following normalization to the internal loading control (encoding histone H4). (G)Myc–kaiso or -gal (negative control) was injected alone (0.25 ng), or Myc–kaiso was co- injected with HA–p120 (WT) versus HA–p1204SA (0.25 ng), into one blastomere of two-cell embryos. Gastrula embryo cDNA was assayed by real-time RT-PCR for xWnt-11 transcript levels. ODC, ornithine decarboxylase.

Our previous work showed that p120-catenin binds and is impact upon the p120-catenin than the -catenin trajectory of the stabilized by the intracellular protein Frodo, allowing the p120- Wnt pathway. These data leave open the possible existence of catenin–kaiso pathway to modulate certain Wnt target genes (Park additional unknown modulators of p120 subfamily members in et al., 2006). We thus tested whether depletion of Frodo might also response to Wnt signals. have an impact upon -catenin but found that, although p120 levels became lowered as expected, -catenin levels were Canonical Wnt signals modulate p120-catenin isoform-1 maintained (Fig. 6D). This was consistent with our subsequent levels finding that Frodo does not appear to bind to -catenin, in contrast Generally, destruction of -catenin is blocked upon activation of to its known interaction with p120 (Fig. 6E). In summary, our the canonical Wnt pathway. In addition to more recently reported results indicate that components of the destruction complex known mechanisms (Wu et al., 2008), canonical Wnt signals activate to act upon -catenin are further involved in regulating the levels intracellular Dishevelled and LRP5 or LRP6, which inhibit the of p120 subfamily members and that Frodo has a more obvious destruction complex by membrane recruitment of the core Shared mechanisms regulate multiple catenins 4359

component axin, followed by LRP phosphorylation and further p120 levels through inhibition of the destruction complex. We co- axin recruitment (Cselenyi et al., 2008; Zeng et al., 2008). injected p120-catenin together with GSK3 in the presence versus Membrane recruitment of axin, together with associated GSK3, absence of the Wnt8 ligand, which exhibits canonical and, in some permits enhancement of the cytoplasmic signaling pool of -catenin contexts, non-canonical activity, and also assessed the impact of by a mechanism that remains somewhat unclear, facilitating its coexpressing Frodo with GSK3. Although the role of Frodo in nuclear entry and activation of Wnt target genes such as xSiamois, Wnt signaling remains unclear, it associates with Dsh (Gloy et al., MYC and CCND1. Extending this mechanism conceptually to 2002), and we reported earlier its positive modulation of p120- p120-catenin, we wished to confirm that Wnt signals increase catenin levels (Park et al., 2006). As expected, Wnt8 as well as Journal of Cell Science

Fig. 6. Axin promotes the degradation of members of the p120-catenin subfamily. (A)The indicated Myc-tagged p120 subfamily members (1 ng mRNA each) were co-injected with Myc–axin (0.1 ng) into both blastomeres of two-cell embryos, and gastrula embryo lysates were Myc-immunoblotted (IB). HA–kazrin and HA–EWS (1 ng each) were co-injected with Myc–axin (0.5 ng) as negative controls for the effects of axin, whereas actin and GAPDH served as internal loading controls. (B)The indicated HA-tagged p120 subfamily members (1 ng each) were co-injected with Myc–axin (1 ng) into both blastomeres of two-cell embryos. Gastrula (stage 11–12) embryo lysates were immunoprecipitated for Myc–axin, followed by immunoblotting for HA-tagged p120 subfamily catenins. The bottom panel confirms axin immunoprecipitations. HA–kaiso and HA–Dyrk served as negative controls. (C)HA–axin was co-injected with either Myc-tagged -catenin or -catenin into both blastomeres of two-cell embryos. HA–axin immunoprecipitates were immunoblotted with antibody against Myc to detect -catenin or - catenin. (D)Either HA–p120-catenin (0.25 ng) or Myc–-catenin mRNA (0.25 ng) was co-injected with Frodo morpholino [10 ng versus standard morpholino (STD-MO) or negative control], into both blastomeres of two-cell embryos. Embryos were harvested at stage 11–12 for immunoblotting with antibody against Myc (-catenin) or HA (p120). Actin served as a loading or negative control. (E)Either Myc–-catenin (0.5 ng) or Myc–p120-catenin (0.5 ng) was co-injected with HA–Frodo (0.5 ng) into both blastomere of embryos at the two-cell stage. Embryos were harvested at early-mid gastrula stages (10–11) and lysates immunoprecipitated for HA–Frodo followed by immunoblotting. WCL, whole-cell lysate. 4360 Journal of Cell Science 123 (24)

Frodo reproducibly protected p120-catenin from the negative effects catenin. Wnt8, as anticipated, reduced GSK3-facilitated of GSK3 (Fig. 7A, compare lanes 5 and 7 with lane 2). destruction of p120 (Fig. 7B). Wnt8 is generally thought to activate Furthermore, consistent with earlier results showing the partial the canonical Wnt/-catenin pathway, whereas Wnt11 and Wnt5a capacity of axin to rescue developmental phenotypes arising from are portrayed as non-canonical in most contexts. However, Wnt11 overexpression of p120 (Fig. 1B), we found that axin promoted and Wnt5a have also been shown to activate the canonical pathway p120-catenin degradation (Fig. 7A, compare lane 3 with 1). The in axis development of Xenopus embryos (Kofron et al., 2007; Tao ability of Wnt8 to counter axin-mediated destruction of p120 was et al., 2005). Indeed, in our hands, even endogenous -catenin shown to be dose dependent (Fig. 7C). Frodo, by contrast, protected experienced a modest protective benefit when Wnt11 or Wnt5a p120 from the effects of axin (Fig. 7A, compare lane 8 with 3), as was coexpressed with GSK3 (Fig. 7B). As anticipated, relative to Frodo also protected p120 from the negative impact of coexpressed Wnt8 and Wnt11, the non-canonical Wnt5a had a lesser capacity GSK3 (Fig. 7A, compare lane 5 with 2). At the low levels of to protect HA–p120 from the effect of GSK3 (Fig. 7B). GSK3 and axin that are expressed exogenously, effects upon Owing to the complexity of reported Wnt ligand and Frizzled endogenous -catenin were not observed (whereas an effect of receptor effects in Xenopus embryos during axis specification higher GSK3 expression upon endogenous -catenin is shown in (among others), we employed a knockdown approach in Fig. 7B). conjunction with a cell line system to investigate the response of To evaluate the protective effects of differing Wnt ligands, we p120-catenin to other Wnt signaling components such as axin and expressed Wnt8, Wnt 11 or Wnt5a together with GSK3 and p120- LRP5 and LRP6. MDA-231, HeLa and 293T cells were used to

Fig. 7. Upstream Wnt pathway components modulate the levels of p120-catenin. (A)Co-injection of Frodo or Wnt8 counter the effectiveness of Myc–GSK3 (5 pg) or Myc–axin (0.1 ng) to reduce HA–p120 (0.5 ng) levels. Embryos expressing up to two exogenous constructs (as noted) in addition to HA–p120 were harvested at gastrulation, and the corresponding lysates immunoblotted (IB) with antibody against HA to detect p120. Actin was used as an internal loading control. Total mRNA injection loads were equalized using -galactosidase. (B)Exogenous Wnts (0.5 ng of Wnt8, Wnt11 or Wnt5a mRNA) were co- injected with both HA–GSK3 (0.1 ng) and HA–p120 (0.25 ng) into both blastomere of two-cell embryos. At stage 12, Wnt protection (versus no protection) from GSK3 impact upon HA–p120 was assessed by immunoblotting. Actin served as an internal loading control. (C)An increasing dose of Wnt8 mRNA was co- Journal of Cell Science injected with both HA–p120 (0.5 ng) and Myc–axin (0.5 ng) into two blastomeres of two-cell embryos that were later collected at gastrulation (stage 11, with 20 embryos per condition). The protein levels of p120-catenin were assessed by immunoblotting. (D)MDA-231, HeLa and 293T cells were transfected with siRNAs directed against axin-1 and axin-2 (50 pmol), as indicated, for 48 hours. Endogenous p120-catenin levels were monitored through anti-p120 immunoblotting (6H11). Each experiment was repeated at least three times. nc, negative control. (E)HeLa cells were transiently transfected with siRNA for LRP5 or LRP6 (50 pmol), along with either Myc-tagged LRP5 or LRP6, and effects assessed by means of Myc- immunoblotting. (F)MDA-435 cells were seeded in six- well plates, followed by transfection with LRP5 and LRP6 siRNAs. The effect of LRP5 and LRP6 depletion on p120- catenin was monitored using distinct antibodies directed against p120 (pp120 or 6H11). The asterisk (*) indicates a nonspecific band serving as an additional negative control. (G)The stability of p120-catenin in HeLa cells was resolved as described for F. (H)Myc–p120 full-length (isoform-1) or DN-p120 were transiently transfected into 293T cells for 24 hours, followed by MG132 or D4476 treatment (6 hours) at the doses indicated. The levels of p120 were monitored through Myc-immunoblotting. (I)293T cells were transiently transfected with mouse p120 isoform-1A or isoform-3A. p120-catenin isoforms were monitored in the presence of BIO or D4476 (6 hours), employing an antibody against Myc. Shared mechanisms regulate multiple catenins 4361

test the effect of knocking-down axin-1 and axin-2 using proven p120 isoform-1 protein levels are subject to regulation by siRNAs (Pan et al., 2008). Complementing our overexpression the destruction complex experiments conducted in Xenopus embryos, depletion of axin-1 Our recent work has indicated that the levels of p120 protein and axin-2 stabilized endogenous p120-catenin (Fig. 7D). We respond to canonical Wnt signals through Frodo, resulting in the further examined the effect of knocking down LRP5 and LRP6. modulation of certain Wnt/-catenin target genes harboring both Being positive modulators of canonical Wnt signaling, we would Kaiso- and TCF-binding sites (Park et al., 2006; Park et al., 2005). expect knockdown of LRP5 and LRP6 to produce an impact We reasoned that additional pathway components might act upon opposite to that of depletion of axin. Indeed, using a previously p120-catenin, prompting us to address potential mechanisms. In characterized siRNA directed against LRP5 and LRP6 (Pan et al., this report, we indicate that protein components involved with the 2008), p120 levels were reduced in MDA435 and HeLa cells (Fig. physiologic destruction of -catenin also promote p120-catenin 7F,G). Interestingly, the pp120 antibody, which detects most p120- degradation – with the prime examples being axin and GSK3. In catenin isoforms, revealed that the longer isoform of p120-catenin, mapping studies, the amino-terminal region of p120-catenin was presumably p120-catenin isoform-1, was more responsive to found to be required for conferring sensitivity to, and for interaction depletion of LRP5 and LRRP6. As isoform-2 and other higher with, destruction complex proteins (CK1 and GSK3). isoforms initiate translation at a primary sequence position beyond Importantly, we observed that the degradation mechanism applies the destruction motif of p120, these endogenous data appear to principally to p120 isoform-1, which harbors amino-terminal GSK3 support the view that the most amino-terminal region of p120, and CK1 sites that are not present in isoforms arising from present in isoform-1, favors its regulation by conical Wnt signals. translational initiation events at later primary-sequence positions. This view was additionally supported through use of proteasome We mutated four potential phospho-residues predicted to comprise and CK1 (D4476) inhibitors, which resulted in increased levels of a CK1 and/or GSK3 consensus region. In contrast to the wild p120 isoform-1 (Fig. 2). Consistently, lesser effects on isoform-3 type, the levels of these constructs when coexpressed with GSK3 were seen following proteasome (MG132) or CK1 inhibition (Fig. were no longer reduced. Consistent with the existence of an 1C, Fig. 2A,C). adjoining putative ubiquitylation site dependent upon prior CK1 To further assay possible differences between the p120 isoforms, or GSK3 phosphorylation, the p120 point-mutant lost most of its we expressed full-length xp120 (similar to human isoform-1) or an capacity to bind to -TrCP and was largely free of ubiquitylation. N-terminal deletion mutant (DNp120; similar to human isoform-4) Furthermore, we observed that components of the canonical Wnt in 293T cells. While a prior report pointed to the occurrence of two pathway such as certain Wnt ligands and LRP5 and LRP6, in Xenopus p120 isoforms (iso1 similar to hp120 isoform-1; and iso2 addition to the little-understood component Frodo, confer protective similar to hp120 isoform-3), the Xenopus iso2 (similar to hp120 effects upon p120, whereas a core component of the destruction isoform-3) is not yet functionally characterized, whereas Xenopus complex, axin, negatively modulates p120 in both Xenopus and isoforms similar to hp120 isoform-2 or isoform-4 have not yet cell line systems. Our results point to the view that the levels of been reported. As noted earlier, DNp120 lacks the potential GSK p120 protein are regulated through mechanisms analogous to those phosphorylation site S199, and it does not associate with either operating on -catenin in vivo. Intriguingly, other members of the GSK or CK1 (Fig. 4B,D), and nor is there evidence for p120-catenin subfamily, namely -catenin and ARVCF-catenin,

Journal of Cell Science ubiquitylation (Fig. 4C). Thus, while human isoform-3 and -4 are were also responsive (apparent substrates) of the destruction functionally distinct (Roczniak-Ferguson and Reynolds, 2003; complex. While we did not examine these latter catenins in depth, Yanagisawa et al., 2008), we largely used DNp120 (Xenopus we note that, in common with -catenin and p120, each contains surrogate isoform-4) in comparing Wnt pathway responses relative a number of conserved potential GSK3 sites. Indeed, independent to those of isoform-1. When comparing Xenopus isoform-1 (full- support for the responsiveness of -catenin to GSK3 and length) and DNp120, only isoform-1 responded to CK1 or ubiquitylation has arisen recently from the laboratory of K. Kim proteasome inhibition (Fig. 7H). Finally, we evaluated mouse (Oh et al., 2007). In that work, -catenin responded positively to isoform-1 versus isoform-3 upon GSK3 inhibition using another GSK knockdown. Likewise, in keeping with our findings centered agent, BIO, in 293T cells. Isoform-1 became modestly but upon p120, -catenin became ubiquitylated in the presence of reproducibly stabilized, whereas isoform-3 appeared unresponsive MG132, with ubiquitylation being reduced upon GSK inhibition. under the same conditions (Fig. 7I). Inhibition of CK1similarly Mutation of a potential GSK3 phosphorylation site (Thr1078 produced a reproducible, if modest, response in isoform-1 (Fig. residue) likewise resulted in less ubiquitylation, although 7I). Collectively, our data indicate that p120 and -catenin, and interestingly, the site was mapped to the carboxyl-, as opposed to probably additional p120 subfamily members, share regulatory the amino-terminal, region of -catenin. Together with our data mechanisms responsive to Wnt pathway activity. Furthermore, presented here and that published previously (Oh et al., 2009), we with respect to p120, it is isoform-1 that is most clearly modulated, envisage that the degradation machinery of the canonical Wnt in keeping with its capacity to associate with components of the pathway modulates multiple members of the catenin family, with destruction complex and to be phosphorylated by CK1 or GSK3. multiple catenins forming a Wnt-responsive network extending considerably beyond -catenin alone. Discussion Interestingly, an independent group recently reported the The canonical Wnt pathway has been viewed as having -catenin involvement of p120-catenin in canonical Wnt signaling (Casagolda as the primary signal transduction mediator in response to Wnt et al., 2010). In response to Wnt-ligand, p120 was found to promote pathway activation. While plakoglobin (-catenin), a member of Dishevelled phosphorylation through the association of p120 with the -catenin subfamily, has been implicated in context-dependent CK1 and E-cadherin (cadherin-1). This was shown to result in Wnt gene regulation (Karnovsky and Klymkowsky, 1995; Sadot et increased stability of -catenin. This study complements our own al., 2000), little emphasis has been directed toward the possible as cadherin-dissociated p120 (perhaps more than one isoform) roles of p120-catenin subfamily members. represents a distinct signaling-pool origin versus that which we 4362 Journal of Cell Science 123 (24)

propose arises from Wnt-pathway inhibition of the destruction generation of distinct polypeptides, as does the presence and use complex, which exhibits selectivity toward isoform-1. In all cases, of four alternative translation start sites in humans (van Hengel and our collective work supports the concept that p120-catenin van Roy, 2007). Recent evidence indicates that differing isoforms participates in canonical Wnt signaling. confer distinct, or even opposing, functions with respect to cell Although our results demonstrate responsiveness of p120 (and adhesion and invasion (Liu et al., 2009; Miao et al., 2009; probably also ARVCF- and -catenin) to canonical Wnt pathway Yanagisawa et al., 2008). The p120 isoform-1, initiated from the components and signals, central questions remain to be addressed. first translational start site, encompasses the destruction motif First is the issue of why, in addition to -catenin, p120 and other subject to Wnt-pathway regulation and, correspondingly, is more subfamily members would be modulated coordinately. In the case responsive to depletion of LRP5 and LRP6, or the inhibition of of p120-catenin, we among other groups have gathered evidence proteasomes, GSK3 or CK1. In the context of ectopically expressed that p120 acts in combination with -catenin at shared and p120 isoforms, isoform-1 was considerably more sensitive than developmentally significant gene promoters, such as Xenopus p120 isoform-3 or an N-terminal deletion mutant DNp120 (the Siamois and to a lesser extent Wnt11. In the case of Siamois, for latter similar to human isoform-4). Endogenous Xenopus isoforms example, gene activation occurs to an additively larger extent when equivalent to human isoform-2 and -4 have not yet been reported de-repression by -catenin of TCF or LEF occurs in combination or characterized. However, in certain cell lines, we observed an with p120-mediated de-repression of the Kaiso transcriptional additional response of isoform-3, which might conceivably arise repressor (Kim et al., 2004; Park et al., 2005). Thus, the Wnt/p120- from its association with isoform-1, indirect effects upon the catenin and Wnt/-catenin pathways could be required for cadherin–catenin complex or the existence of additional destruction coordinate regulation of Wnt signaling in context dependent motifs within p120. Although exogenous isoform-3 and DNp120 manners. Requiring further study to resolve, independent work (similar to human isoform-4) displayed similarly unapparent or questions whether Siamois and Wnt11 are gene targets of Kaiso mild responses to inhibition of GSK, CK1 or the proteasome, these (Ruzov et al., 2009a; Ruzov et al., 2009b), whereas another isoforms have distinctive features, including their relative independent study has supported the responsiveness of Siamois to distribution in differing intracellular localizations (Roczniak- kaiso depletion in the presence of weak Wnt pathway stimulation Ferguson and Reynolds, 2003). Relative to other p120 isoforms, (Iioka et al., 2009). Whatever the outcome of this discussion, our expression of isoform-1 is associated with cells having results here, while directed toward upstream p120 regulation, mesenchymal characteristics and also associated with epithelial continue to be consistent with Siamois and Wnt11 gene cells that have undergone progression toward invasive and responsiveness to p120 and Kaiso (Fig. 5F and 5G, and transformed cell states (Yanagisawa et al., 2008). We imagine that supplementary material Fig. S3). We hypothesize that the regulatory events determining the choice of translational initiation Wnt/p120-catenin pathway, and probably the Wnt/ARVCF- and sites would provide another layer of control for the responsiveness Wnt/-catenin pathways that require further elucidation, act in of p120 to canonical Wnt signals. At present, little is known parallel with the Wnt–-catenin signaling trajectory to modulate concerning how the particular translational initiation sites in the certain canonical Wnt gene targets (or cytoplasmic downstream p120 transcript are selected or, similarly, what governs the effectors) in a context-dependent manner. Interestingly, -catenin differential splicing decisions of the p120 transcript, which together

Journal of Cell Science has also been reported to modulate Kaiso function at gene promoters produce multiple p120 isoforms. (Bienz, 1998; Rodova et al., 2004), and ARVCF appears present in Apart from nuclear functions, p120 subfamily members play some cell and tissue nuclei (Mariner et al., 2000). While there is additional key roles at cell–cell junctions and other locations, no known action of ARVCF in the nuclear compartment, we have regulating cadherin stability and trafficking (Davis et al., 2003; recently resolved its association with Kazrin, a structurally novel Ireton et al., 2002; Xiao et al., 2003), as well as modulating small and incompletely characterized cortical or junctional protein, that GTPases (e.g. Rac1 and RhoA; see Introduction). Thus, in we and others find additionally shuttles into the nucleus (Cho et considering the potential consequences of Wnt signaling for al., 2010) (Borrmann et al., 2006; Groot et al., 2004; Sevilla et al., members of the p120 subfamily, we must include possible effects 2008). Together, a coordinate response of catenins to the destruction that are not transcriptional in nature but, instead, represent more machinery of the canonical Wnt pathway could be imagined to immediate effects upon cell adhesion, motility or cytoskeletal result in a networked gene-regulatory outcome downstream of Wnt activity. signals. With respect to potential upstream modulators of catenins, Frodo and the closely related Dapper functionally and physically Materials and Methods interact with Dishevelled, and each appears to modulate Wnt cDNA constructs signals positively or negatively in context-dependent manners Standard recombinant DNA techniques were used to construct pCS2-based plasmids harboring Xenopus laevis: Myc–p120-catenin, HA–p120-catenin (Fang et al., 2004), (Cheyette et al., 2002; Gloy et al., 2002; Hikasa and Sokol, 2004; Myc–-catenin, HA–-catenin, Myc–ARVCF, HA–ARVCF (Fang et al., 2004), Teran et al., 2009). Furthermore, Frodo acts in downstream Wnt- Myc–-catenin and HA–-catenin (Gu et al., 2009). Myc–GSK3-pXT7 and signaling capacities (Hikasa and Sokol, 2004). In this study, we did catalytically inactive mutant (K85R) (S. Y. Sokol, Mount Sinai School of Medicine) (Dominguez et al., 1995), HA–CK1-pCS2 (Xi He, Harvard Medical School) (Liu not resolve a -catenin–Frodo complex, although we confirmed et al., 2002), Myc–axin-pCS2 (P. S. Klein, University of Pennsylvania) (Hedgepeth the expected interaction of p120-catenin with Frodo (Park et al., et al., 1999), xWnt8 (S. Y. Sokol, Mount Sinai Scholl of Medicine) (Sokol et al., 2006). As Frodo binds to Dishevelled, we conjecture that Frodo 1991), xWnt11 (R. Keller, University of Virginia) (Du et al., 1995; Ku and Melton, (and Dapper) exhibits some selectivity for the p120 signaling 1993) and xWnt5a (R. Harland, University of California, Berkley) (Wallingford et al., 2001) were kindly provided as indicated. GSK3 was subcloned into the pCS2- trajectory in upstream Wnt contexts and that analogous, but HA vector for coimmunoprecipitation. Generated previously were pCS2 plasmids presently undefined, proteins might work together with Dishevelled harboring Myc–p120 deletion constructs, Myc–Frodo and HA–Frodo (Park et al., to modulate the trajectory of other catenins. 2006). The quadruple p120-catenin point mutant (S6, S8, S11 and S15r4SA) was generated by PCR amplification of HA–p120-catenin (in vector pCS2) as a template As noted earlier, the initial p120-catenin transcript (pre-RNA) is with 5Ј mutated primer as follows: p1204SA-F, 5Ј-GGAATTC ATGGATGAG - subject to inter- and intra-exonic splicing events that allow for the CCAGAGGCTGAAGCTCCGGCCGCTATATTGGCCGCAGTGAGAGCT-3Ј; and Shared mechanisms regulate multiple catenins 4363

p1204SA-R, 5Ј-AAGGCCTGACAC GCTGAT CT TCA GCA TCACCAAG ATTCA - Research Project Grant (003657-0008-2006). DNA sequencing and GTGATCCTCCAGCACTTACGGA-3Ј. other core facilities were supported by a University of Texas M.D. Anderson Cancer Center National Cancer Institute Core Grant (CA- Embryo culture, microinjections and in vitro transcription Fertilization, embryo culture and microinjections were performed in accordance 16672). J.Y.H. was supported from a William Randolf Hearst with standard methods (Fang et al., 2004). Embryos were microinjected with capped Foundation Student Research Award and a Developmental Award from mRNA synthesized in vitro (mMessage mMachine, Ambion). All pCS2-based the UT SPORE in Lung Cancer (P50 CA070907). Deposited in PMC constructs were linearized by using NotI before in vitro transcription. Gastrulation for release after 12 months. phenotypes, principally failure of blastopore closure, were visually scored at embryonic stages 11–12. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/123/24/4351/DC1 Immunoprecipitation and immunoblot analysis Immunoprecipitation and immunoblotting employed monoclonal antibodies directed References against Myc (9E10), HA (12CA5), p120-catenin (mouse or human pp120, BD Abe, K. and Takeichi, M. (2008). EPLIN mediates linkage of the cadherin catenin Transduction; mouse or human 6H11, Santa Cruz) and GSK3 (mouse, BD complex to F-actin and stabilizes the circumferential actin belt. Proc. Natl. Acad. Sci. Transduction). Polyclonal antibodies included -catenin and p120-catenin raised in USA 105, 13-19. our laboratory (Park et al., 2006) and Frodo provided by the laboratory of S. Y. Aberle, H., Bauer, A., Stappert, J., Kispert, A. and Kemler, R. (1997). beta-catenin is Sokol. For immunoprecipitations, procedures were largely performed as described a target for the ubiquitin-proteasome pathway. EMBO J. 16, 3797-3804. previously (Fang et al., 2004). Whole-embryo lysates were prepared using 0.5% Amit, S., Hatzubai, A., Birman, Y., Andersen, J. S., Ben-Shushan, E., Mann, M., Ben- Triton X-100 buffer [0.5% Triton X-100, 10 mM HEPES (pH 7.4), 150 mM NaCl, Neriah, Y. and Alkalay, I. (2002). Axin-mediated CKI phosphorylation of beta-catenin 2 mM EDTA, 2 mM EGTA], inclusive of a proteinase inhibitor cocktail (Sigma). at Ser 45, a molecular switch for the Wnt pathway. Genes Dev. 16, 1066-1076. Interference from the IgG heavy chain in immunoblot analyses was reduced by Anastasiadis, P. Z. (2007). p120-ctn: a nexus for contextual signaling via Rho GTPases. employing TrueBlotTM anti-mouse IgG IP Beads and Mouse TrueBlot Biochim. Biophys. Acta 1773, 34-46. ULTRA:horseradish peroxidase (HRP) anti-mouse IgG (eBioscience). Embryos were Behrens, J., Jerchow, B. A., Wurtele, M., Grimm, J., Asbrand, C., Wirtz, R., Kuhl, lysed in 0.5% Triton X-100 buffer (20 l per embryo), in the presence of a proteinase M., Wedlich, D. and Birchmeier, W. (1998). Functional interaction of an axin homolog, inhibitor cocktail (Sigma). After centrifugation (5488 g, 20 minutes), the supernatant conductin, with beta-catenin, APC, and GSK3beta. Science 280, 596-599. fraction was denatured in 5ϫ SDS sample buffer (200 mM Tris-Cl [pH 6.8], 40% Bienz, M. (1998). TCF: transcriptional activator or repressor? Curr. Opin. Cell Biol. 10, glycerol, 8% SDS, 0.08% Bromophenol Blue), followed by incubation at 95°C for 366-372. 5 min. Half-embryo equivalents were resolved by SDS-PAGE and transferred onto Bilic, J., Huang, Y. L., Davidson, G., Zimmermann, T., Cruciat, C. M., Bienz, M. and nitrocellulose membranes. Immunoblotting and antibody incubations took place in Niehrs, C. (2007). Wnt induces LRP6 signalosomes and promotes dishevelled-dependent 2% bovine serum albumin–TBST [25 mM Tris-HCl (pH 7.8), 125 mM NaCl, 0.5% LRP6 phosphorylation. Science 316, 1619-1622. Tween20]. SuperSignal WestPico (Pierce Biotechnology) reagents were utilized to Borrmann, C. 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D., Valls, G., Lugilde, E., Vinyles, M., Casado-Vela, J., along with Venus–sh-random or Venus–sh-GSK3 (Kim et al., 2006). Twenty four Solanas, G., Batle, E., Reynolds, A. B., Casal, J. I. et al. (2010). A p120-catenin/CK1e complex regulates Wnt signaling. J. Cell Sci. 123, 2621-2631. hours after transfection, cells were fixed with 4% PFA and immunostained with Castano, J., Raurell, I., Piedra, J. A., Miravet, S., Dunach, M. and Garcia de Herreros, antibody against Myc (9E10). Fluorescence images (Nikon ECLIPSE E800 A. (2002). Beta-catenin N- and C-terminal tails modulate the coordinated binding of microscope) were recorded using SPOT advanced software. For immunofluorescence proteins to beta-catenin. J. Biol. Chem. 277, 31541-31550. staining of kaiso (Fig. 2E), HeLa and MDA-231 cells were grown on glass coverslips. Chen, Y. and Alman, B. A. (2009). Wnt pathway, an essential role in bone regeneration. 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