Emerging Roles for P120-Catenin in Cell Adhesion and Cancer

Emerging roles for p120-catenin in cell adhesion and cancer

Albert B Reynolds*,1 and Agnes Roczniak-Ferguson1

1Department of Cancer Biology, Vanderbilt University, 771PRB, 2220 Pierce Ave, Nashville, TN 37232-6840, USA

Although originally identified as a Src substrate, p120- to be ‘cytoskeletal’ proteins, but cDNA sequencing catenin (p120) is now known to regulate cell–cell adhesion revealed domains closely associated with protein tyr- through its interaction with the cytoplasmic tail of osine kinase (PTK) signaling (e.g., tyrosine kinase, SH2, classical and type II cadherins. New evidence indicates and SH3 domains), providing the first clue that these that p120 regulates cadherin turnover at the cell surface, major Src targets were not chosen randomly. The thereby controlling the amount of cadherin available for identified genes/proteins (Tensin, Focal Adhesion Ki- cell–cell adhesion. This function is necessary but not nase/FAK, p130CAS, p120ctn, p110AFAP, and cortac- sufficient to promote strong adhesion, which is further tin) are now well established as bone fide Src effectors controlled by signals acting on the amino-terminal p120 (Figure 1). Importantly, they have in common the regulatory domain. p120 also modulates the activities of ability to regulate the actin cytoskeleton, suggesting a RhoA, Rac, and Cdc42, suggesting that along with other collective role modulating events such as cell motility Src substrates, p120 regulates actin dynamics. Thus, p120 and adhesion. Thus, although the exact function of c-Src is a master regulator of cadherin abundance and activity, is to this day not entirely clear, the lesson from the and likely participates in regulating the balance between substrates is that Src-kinases directly or indirectly adhesive and motile cellular phenotypes. This review regulate the actin cytoskeleton. summarizes recent progress in understanding mechanisms Although not recognized at the time, these observa- of p120 action, and discusses new implications with tions were, in fact, the first clue that p120 itself would respect to roles for p120 in disease and cancer. somehow be involved in regulation of the actin Oncogene (2004) 23, 7947–7956. doi:10.1038/sj.onc.1208161 cytoskeleton. In this context, however, p120 was initially puzzling because sequence analysis of the p120 cDNA Keywords: cell adhesion; p120 catenin; cadherin; Rho; revealed an ARM repeat domain (Reynolds et al., 1992) Rac; tumor suppressor rather than the obvious PTK signaling motifs associated with the other substrates. However, the recently identified functions of p120 in cadherin-mediated adhesion (Ireton et al., 2002; Davis et al., 2003; Xiao et al., 2003a) and RhoGTPase regulation (Anastasiadis et al., Introduction 2000; Noren et al., 2000; Anastasiadis and Reynolds, 2001; Grosheva et al., 2001) are indeed consistent with In the late 1980s, attempts to understand the mechanism the collective role of Src substrates. Although p120 is of v-Src transformation were clouded by the prevailing not widely considered a regulator of actin dynamics, it view that the robust activation of v-Src kinase activity has become increasingly instructive to think of p120 in was likely to lead to promiscuous phosphorylation of this overall context. This review summarizes recent many cellular proteins. Indeed, viewed in that light, the progress in understanding mechanisms of p120 action, substrates revealed by antiphosphotyrosine antibodies and discusses implications of these data with respect to might turn out to be irrelevant cytoskeletal proteins, roles for p120 in disease and cancer. targeted more for their abundance than any Src-relevant function. Nonetheless, strategies to affinity purify A Src substrate turned catenin phosphorylated substrates using phosphotyrosine anti- As alluded to above, p120ctn was originally singled out bodies, and to identify them en masse by generating of a lineup of several prominently phosphorylated Src monoclonal antibodies led remarkably to a relatively substrates from v-Src-transformed chicken embryo comprehensive panel of antibodies specific for tyrosine fibroblasts (Reynolds et al., 1989). Detection of p120 phosphorylated proteins (Kanner et al., 1990). Subse- was initially the result of a technical breakthrough, quent cDNA cloning studies identified the genes namely the development of antiphosphotyrosine anti- encoding these proteins (Wu et al., 1991; Reynolds bodies, which made possible for the first time, the simple et al., 1992; Schaller et al., 1992; Flynn et al., 1993; Sakai and direct visualization of tyrosine phosphorylated et al., 1994). In retrospect, fears of promiscuity were proteins by Western blotting. p120 was unique because largely unfounded. The substrates did, in fact, turn out unlike the other substrates, it was prominently phosphorylated by v-Src, but not by a transformation *Correspondence: AB Reynolds; E-mail: [email protected] defective v-Src variant (v-Src/G2A) rendered cytoplasmic p120-catenin, cell adhesion and motility AB Reynolds and A Roczniak-Ferguson 7948

α-catenin TJ actin β-catenin RhoA Adherens Junction AFAP Rac1

p120 C E-cadherin Src kinases Cdc42

D F CAS A Regulation K Src kinases Focal of transcription Adhesion T C β α

T Tensin (p210) TJ: tight junction CAS Crk associated substrate (CAS p130) D: desmosome

FAK Focal adhesion kinase (FAK p125)

p120-catenin (p120ctn)

AFAP Actin filament associated protein (AFAP p110)

C Cortactin (p85)

Src (and Src-like) kinases: Fyn, Yes, Fer

Figure 1 Src substrates regulate the actin cytoskeleton. Shown are some of the major Src substrates originally identiﬁed using antibodies to phosphotyrosine. Major Src substrates are found in adherens junctions (p120, b-catenin), focal adhesions (FAK, CAS, Tensin), or bound to components of the actin cytoskeleton (AFAP, cortactin). They form the regulatory backbone of an actin-based system controlled by Src family proteins, Rho GTPases, and a complicated network of scaffolding GEF/GAPs that bind and regulate Rho GTPases (not shown). The black double arrowheads depict the concept that together, these proteins regulate the intercellular crosstalk necessary to coordinate cell motility and adhesion. Rho GTPases are recruited from the cytoplasm to sites that control these events through actin rearrangements

by mutation of its amino-terminal glycine residue. Thus, suggesting that p120 kinases are membrane associated. it was postulated that p120 phosphorylation might play Likewise, membrane-uncoupled Src mutants appear to a key role in the transformation process (Reynolds et al., have little access to cadherin-associated p120 (Reynolds 1989). et al., 1989). Thus, phosphorylation of p120 by Src Gene cloning revealed that p120 lacked domains requires that both proteins localize to the cell mem- obviously linked to PTK signaling (Reynolds et al., brane, the former via cadherin binding, the latter by 1992). Instead, it contained a central region with linked myristoylation of it N-terminal glycine residue. Despite Armadillo repeats showing approximately 22% homol- these provocative observations, p120 tyrosine phosphor- ogy to b-catenin and its cousin plakoglobin, known ylation does not appear to be essential for Src-induced cadherin-binding partners (Reynolds et al., 1992; Peifer transformation, as p120 siRNA knockdown cell lines et al., 1994). This observation led to the key finding that can be transformed efficiently by Src (A Reynolds, p120 interacts directly with E-cadherin, consistent with unpublished). Ironically, though much has been learned its localization to cell–cell junctions (Reynolds et al., about p120, the role of p120 tyrosine phosphorylation 1994; Shibamoto et al., 1995; Staddon et al., 1995). In remains unknown. retrospect, the discovery was at least partly fortuitous: among the multitude of ARM domain proteins identified in recent years, only b-catenin, plakoglobin, and A core function for p120: regulation of cadherin turnover p120 family members interact with cadherins. It is now clear that cadherins are both necessary and p120 is the prototypic member of a p120 subfamily of sufficient for localization of p120 to the cell membrane ARM domain proteins that include ARVCF, delta- (Thoreson et al., 2000). In cadherin-deficient cells, p120 catenin, p0071, and the more distantly related plako- is stranded in the cytoplasm and unphosphorylated, phillins (reviewed in Anastasiadis and Reynolds, 2000).

Oncogene p120-catenin, cell adhesion and motility AB Reynolds and A Roczniak-Ferguson 7949 p120 associates with most (if not all) classical (type I) 1998; Wheelock and Johnson, 2003; Nelson and Nusse, and nonclassical (type II) cadherins (Reynolds et al., 2004). 1996) via p120 Arm repeats 1–7 (Ireton et al., 2002) and Although p120 probably participates in these actions, the juxtamembrane domain (JMD) of the cadherin recent evidence suggests that its core function in the cytoplasmic tail (Figure 2) (Yap et al., 1998; Thoreson complex is to regulate cadherin turnover (Ireton et al., et al., 2000). The importance of the interaction is 2002; Davis et al., 2003). Thus, p120 directly inﬂuences underscored by the fact that the JMD is the most highly adhesive strength by controlling the amount of cadherin conserved cadherin domain. b-Catenin or plakoglobin available at the cell surface for adhesion. Under normal interact tightly in a mutually exclusive fashion with the circumstances, cadherin turnover occurs constitutively so-called catenin binding domain (CBD) of cadherins, as part of an ongoing endocytosis mechanism that and mediate the physical connection of the cadherin participates in the control of cadherin-mediated adhe- complex with the actin cytoskeleton through a-catenin. sion (Le et al., 1999; Le et al., 2002; Xiao et al., 2003b) Together, these ‘catenins’ regulate dynamic cell–cell (reviewed in Bryant and Stow, 2004). It now appears adhesion by modulating events such as cadherin that p120 is a determining factor in regulating this clustering and the strength of the cadherin connection dynamic process (Ireton et al., 2002; Davis et al., 2003; with the actin cytoskeleton (reviewed in Yap, Xiao et al., 2003a). An important clue comes from

METR PTPµ Cadherin EGFR LAR RPTPs RPTKs VEGFR VEPTP FGFR Fe r D E S P HP 1 Phosphorylation? -1 PTP1B p120

β-catenin

α-catenin

Degradation

Recycling Trafficking RhoA Stress Fibers Focal Contacts Rac1 ER and Golgi Lamelipodia Increased Cdc42 motility and p120 catenin Filipodia invasion JNK, p38α, p38γ β-catenin ? ? Kaiso α-catenin Cell cycle, SRF AP-1 proteases, etc. actin

Figure 2 Roles for p120 in adhesion and motility. The catenins (a-, b- and p120) directly coprecipitate with cadherins and constitute core components of the cadherin complex. Specific receptor tyrosine kinases (RPTKs) and receptor tyrosine phosphatases (RPTPs) are functionally (and probably physically) coupled to cadherin complexes and participate in dynamic regulation of adhesion. The p120 amino-terminus physically associates with the Fer tyrosine kinase (Fer), as well as Src kinases Fyn and Yes (not shown). The cytoplasmic PTP’s DEP-1 and SHP-1 also bind the p120 amino terminus and regulate the tyrosine phosphorylation status of cadherin complex components. A core function for p120 is to promote adhesion through binding and stabilizing cadherin at the cell surface. It is postulated that regulatory signals control p120 affinity for cadherin, thereby controlling internalization and/or recycling of cadherin complexes (depicted by miniaturized versions of cadherin complex). Regulatory interactions in the p120 amino terminus further modulate p120 activity. p120 can also modulate the activities of Rho GTPases in the cytoplasm, where it is thought to promote cell motility. In E-cadherin-deficient cells, all cellular p120 is stranded in the cytoplasm, and may promote cell motility and invasiveness via several Rho-directed actions as shown. p120 is found at low levels in the nucleus of normal cells, and at increased levels in cadherin- deficient cells. Its role in the nucleus is unknown, but it binds to the transcription factor Kaiso, and in theory at least, could also promote invasive behavior in cadherin-deficient cells

Oncogene p120-catenin, cell adhesion and motility AB Reynolds and A Roczniak-Ferguson 7950 observation of p120-deficient SW48 carcinoma cells, Of course, other models are possible. For example, it which are poorly organized and loosely associated due has yet to be formally demonstrated that the internaliza- to poor cell–cell adhesion. Restoring p120 expression tion mechanism for unbound cadherins in p120 siRNA- efficiently rescues proper epithelial morphology by treated cells is the same as that used for endocytosis of stabilizing E-cadherin and increasing its abundance cadherin when p120 is present. Thus, it is possible that approximately 10-fold (Ireton et al., 2002). Interestingly, p120 remains bound during the process and plays a the mechanism is post-translational: it requires direct direct role in modulating the internalization and/or p120 interaction with E-cadherin, which in turn sorting endosomal machinery. Although the simplest increases E-cadherin half-life substantially with little or interpretation is that p120-binding/dissociation controls no effect on E-cadherin mRNA levels. These data show the initial internalization event, it is also possible that that p120 is essential for cadherin stability. p120 reassociation after cadherin internalization causes Follow-up studies with p120-specific siRNA show the cadherin to recycle back to the cell surface, rather that this phenomenon is relevant in multiple cell lines than exiting the system through destruction pathways. and applies to many if not all p120-associated cadherins Regardless of the exact triggering event, p120 is an (Davis et al., 2003; Xiao et al., 2003a). Importantly, essential cadherin surface retention signal, and partici- titration of p120 levels by controlling p120 siRNA levels pates directly or indirectly in trafficking decisions at the reveal that p120 acts as a rheostat or set point cell surface that control cadherin turnover. mechanism for determining the overall cellular levels of E-cadherin. Surprisingly, p120 is not required for trafficking of nascent E-cadherin to the cell surface p120 regulatory domain: critical and complex roles in (Davis et al., 2003), and in MDCK cells at least, does adhesion not appear to associate with E-cadherin prior to its arrival (Miranda et al., 2003). At the surface, however, p120 is expressed as multiple isoforms that arise by nascent E-cadherin is immediately turned over if p120 is alternative splicing and usually coexist in characteristic absent (Davis et al., 2003). Cadherins are also stabilized ratios that differ from one cell type to another (Figure 3). by close p120 family members ARVCF, d-catenin, and p120 structure and isoform nomenclature has been p0071 (which also bind classical cadherins), but not the reviewed previously (Anastasiadis and Reynolds, 2000). more distantly related plakophillins, which bind desmo- Briefly, p120 consists of a central Armadillo Repeat somal cadherins (Davis et al., 2003; Xiao et al., 2003a). Domain (Arm domain), flanked by amino-terminal However, p120 is ubiquitously expressed at high levels, sequences that perform regulatory functions, and a whereas expression of the immediate p120 family carboxy-terminal tail whose role is unknown (Figure 3). members is mostly cell type specific, and relatively low Isoforms 1–4 arise from alternative splicing events that in most p120-expressing cells. These observations result in differential usage of four distinct ATG start indicate that p120 is an essential modulator of codons. Exons A, B, and C are internal alternatively cadherin-mediated adhesion, and acts in part by directly spliced sequences. The role of exon A is unknown, but determining the amount of cadherin available at the cell exon B contains a functional nuclear export signal surface to participate in adhesion. (NES) (van Hengel et al., 1999). Exon C is rarely Extrapolating the siRNA data to normal cells where present. p120ctn1 (isoform 1) features a 100 amino-acid p120 is present at relatively high levels suggests models N-terminal coiled-coil domain that is conserved in all whereby cadherin endocytosis is controlled by p120, p120 family members, and is strongly preferred in highly which in turn is modulated by signaling to the p120 motile cells such as fibroblasts and macrophages regulatory domain (e.g., phosphorylation). One possi- (Reynolds et al., 1994). p120ctn3 (isoform 3) lacks the bility is that regulatory events modulate p120–cadherin coiled-coil domain, and is preferentially expressed in affinity (Figure 2). The p120 on/off rate could be more sessile cell types such as epithelial cells. p120ctn4 dynamically modulated such that p120 binding (isoform 4) can be detected at the mRNA level by RT– retains cadherin at the cell surface, and p120-dissocia- PCR but is rarely, if ever, observed at the protein level tion promotes cadherin internalization. Based on and may not be physiologically relevant in cells. It lacks coimmunoprecipitation data, the affinity of the p120– both the coiled-coil and regulatory domains, and when cadherin interaction appears to be relatively low expressed in cells, strongly supports cadherin stability (Reynolds et al., 1994), which may in fact reflect the and adhesion but cannot be regulated (Aono et al., 1999; dynamic nature of this interaction. The mechanism for Ireton et al., 2002). Thus, p120 isoform 4 is used targeting unbound cadherin for internalization may frequently as an experimental tool to evaluate the involve competition of other proteins with p120 for consequences of losing the regulatory N-terminal binding to the cadherin JMD. For example, a Src- sequences. Depending on ones perspective, isoform 4 is associated pathway promotes binding of the ubiquitin either dominant active for cell–cell adhesion or domi- ligase Hakai to a tyrosine phosphorylated motif in the nant negative for cell motility. JMD, leading to E-cadherin ubiquitination and inter- Interestingly, in MDCK cells (and probably most nalization (Fujita et al., 2002). Presenilin-1 also com- other epithelial cells), Snail-induced epithelial to me- petes with p120 for binding to the JMD, and may senchymal transition (EMT) causes a striking transition initiate cadherin turnover through a proteolytic mechan- from ‘epithelial’ to ‘mesenchymal’ p120 isoform patterns ism (Baki et al., 2001). (Ohkubo and Ozawa, 2004), consistent with a motility/

Oncogene p120-catenin, cell adhesion and motility AB Reynolds and A Roczniak-Ferguson 7951 Coiled Regulatory Coil domain Armadillo repeat domain Tail

p120ctn 1ABC 1 2 3 4 5 6 C 7 8 9 10 A B

Alternative ** ATG start sites 123 4 tyrosine phosphorylation sites * NLS Y96, Y112, Y228, Y257, Y280, Y291, Y296, Y302 NES

Ser/Thr phosphorylation sites S122, S252, S268, S288, T310, S312, S873, T910 Figure 3 p120 structure and function. p120 contains a central Armadillo repeat domain consisting of 10 tandemly linked imperfect 42 amino-acid repeats (orange). Repeats 1–5 and 7 are essential for cadherin binding. Cadherin binding via p120 Arm repeats positively regulates adhesion by stabilizing cadherin at the cell surface. The role of the carboxy-terminal tail is unknown, but it contains at least two serine/threonine phosphorylation sites (open lollipops), and alternatively spliced exons A and B (red). The role of exon A is unknown, but exon B contains a functioning nuclear export signal (NES). Locations of nuclear localization signal (NLS, *) and nuclear export sequences (NES, diamonds) are shown. Exon C is rarely present and its function is unknown. The amino-terminal end contains two distinct regions, the coiled-coil domain (blue), and the regulatory domain (green). Alternative splicing in this region gives rise to isoforms 1, 2, 3, and 4, each initiating at distinct ATG start codons (arrows). The most common isoforms are 1 and 3. Isoform 4 is rarely, if ever, observed. Isoform 1 is found preferentially in motile cells, suggesting a role for the coiled-coil domain in cell motility. Isoform 3 is preferred in more sessile cells (e.g., epithelia). The regulatory domain contains the vast majority of tyrosine (red lollipops) and serine/threonine (open lollipops). Exact amino-acid locations of the known sites are listed. Their individual roles are unknown, but collectively, they participate in the dynamic regulation of p120 adhesive function invasion-related role for isoform 1. The isoform switch growth factors such as EGF and HGF (Cozzolino is not reversed by forced re-expression of E-cadherin, et al., 2003), as these activities are inhibited in a indicating that the effect is Snail-induced and not dominant-negative fashion by p120 isoform 4. The Fer directly caused by E-cadherin downregulation. Thus, tyrosine kinase binds constitutively to the regulatory developmental programs that coordinate epithelial to domain (Kim and Wong, 1995; Piedra et al., 2003; Xu mesenchymal transition can control p120 isoform- et al., 2004), and can thus interact with cadherin switching. During tumor progression, it is likely that complexes through binding p120. Fer appears to p120 isoform switching is a common occurrence when inactivate a cadherin-bound phosphatase PTP1b, which cells undergo transition to metastasis. in turn leads to increased tyrosine phosphorylation and Phosphorylation undoubtedly regulates p120 func- dissociation of b-catenin from the cadherin complex) tion, probably serving to link various signaling path- (Xu et al., 2004). Ironically, given p120’s past, Src family ways to the modulation of cadherin-based adhesion, but members Fyn and Yes, but not Src, interact directly separating out the roles of p120 phosphorylation has with p120 (Piedra et al., 2003), and appear to promote been challenging because p120 is phosphorylated at cell–cell adhesion in some systems (Calautti et al., 1998, numerous sites by various Src- and receptor-tyrosine 2002), but inhibit cell–cell adhesion in others, in part via kinases (Reynolds et al., 1989, 1992, 1994; Downing and tyrosine phosphorylation of b-catenin (Piedra et al., Reynolds, 1991; Kanner et al., 1991), and by serine/ 2003). The tyrosine phosphatases PTPm (Zondag et al., threonine kinases as well (Mariner et al., 2001; Xia et al., 2000), DEP1 (Holsinger et al., 2002), and SHP-1 2003). Although roles for individual phosphorylation (Keilhack et al., 2000; Mariner et al., 2001), also sites have not been established, almost all of the tyrosine associate with p120, in some cases via SH2 interactions and serine/threonine phosphorylation sites map to the with phosphorylated tyrosines embedded in the p120 regulatory domain (Mariner et al., 2001; Xia et al., 2003) regulatory domain. The binding of several such proteins (Figure 3), which in turn modulates several p120 to p120 suggests an important role for p120 as a scaffold activities. For example, overexpression in ﬁbroblasts of for PTPs. A number of cytokine and lipid signaling p120 isoforms 1 and 3 but not 4, induce a striking pathways modulate p120 phosphorylation on serine/ dendritic or branching phenotype (Reynolds et al., 1996) threonine residues, and PKC-dependant pathways have linked to regulation of RhoGTPases (reviewed in been implicated (Ratcliffe et al., 1997, 1999; Wong et al., Anastasiadis and Reynolds, 2001). Thus, the regulatory 2000; Konstantoulaki et al., 2003). These data implicate domain is probably required for the physical or p120 phosphorylation in roles such as the regulation of functional coupling of p120 to Rho GTPase activity. vascular permeability. Finally, two labs have described a p120 isoform 1 promotes and is apparently essential for novel interaction between the p120 regulatory domain motility and scattering activities induced by RTK and kinesin, a microtubule-associated motor protein

Oncogene p120-catenin, cell adhesion and motility AB Reynolds and A Roczniak-Ferguson 7952 (Chen et al., 2003; Yanagisawa et al., 2004). Kinesin been directly proven. By overexpression of mutated DN- may affect both the targeting and function of p120 at cadherin constructs lacking either the p120 or b-catenin several cellular locations. binding sites, Xiao and colleagues show that down- Stabilizing cadherin is not by itself sufficient to induce regulation of endogenous VE-cadherin is caused by strong adhesion. In the p120-deficient cell line SW48, sequestration of p120, but not b-catenin (Xiao et al., reconstitution of cell–cell adhesion with p120 isoform 4 2003a). Thus, it is likely to be instructive to re-examine causes significantly better rescue of epithelial morphol- the extensive literature based on blocking cadherin ogy than p120 isoforms 1 and 3, indicating that the action by DN-cadherin expression with the view that amino-terminal sequences confer a partial negative these data most likely reflect loss of p120 function. effect on adhesion (Ireton et al., 2002). However, Interestingly, in vivo experiments have been carried because the principle adhesion defect in these cells out previously in mice using DN-cadherin transgenes derives from p120 deficiency, even isoforms 1 and 3 (Hermiston and Gordon, 1995; Perl et al., 1998). confer reasonable rescue of adhesion (Ireton et al., Transgenic expression of DN-cadherins in the mouse 2002). Interestingly, Colo205 cells are almost completely small intestine induces a Crohn’s-like inflammatory nonadhesive despite normal levels of the known bowel disease (IBD) which eventually progresses to cadherin complex components. In contrast to SW48, adenomas (Hermiston and Gordon, 1995). Moreover, a epithelial morphology in colo205 is efficiently rescued by pathology study of p120 expression in IBD revealed loss isoform four, but not at all by isoforms 1 or 3 (Aono of p120 in 100% of active ulcerative colitis, and 75% of et al., 1999), even though all three isoforms effectively active Crohn’s (Karayiannakis et al., 1998). The mouse stabilize E-cadherin (unpublished data, A Reynolds). study implies a role for p120 downregulation in IBD and Therefore, cadherin stabilization by itself does not cancer, and is consistent with the human condition, translate automatically to stable cell–cell adhesion. which is known to predispose to cancer. Together with The defect in colo205 adhesion is not caused by the the role of p120 in regulating cadherin stability, these presence or absence of p120 per se, but is instead the observations raise the important possibility of a broader result of an as-yet unidentified and probably constitutive role for p120 as a target molecule in inflammatory signaling pathway that requires or acts through the p120 processes and disease. amino-terminal regulatory domain to effectively block The effects of a DN-cadherin transgene have also adhesion. Together, these data indicate that p120 is been described in the context of the polyoma large T- essential for cadherin stabilization, but that adhesive antigen model for tumor progression in the pancreas strength is then further modulated by signals acting on (i.e., Rip1Tag2) (Perl et al., 1998). Pancreas-directed the amino-terminal regulatory domain. Apparently, expression of large T-antigen induces adenomas that both p120 levels and tightly controlled signaling are occasionally become invasive. Invasiveness, though essential, as defects in either can by themselves derail relatively infrequent, is associated with E-cadherin loss adhesion in ways that appear to promote cancer. in 100% of cases, and double transgenic expression of E- Clearly, the regulatory domain of p120 is crucial and cadherin efficiently blocks the invasive phenotype. will take some time to understand in detail. New Interestingly, double transgenic expression of a DN- monoclonal phosphospecific antibodies have been gen- cadherin causes 25% of the tumors to metastasize to the erated to several of the major p120 phosphorylation lymph nodes, an effect never seen in the absence of DN- sites (Mariner et al., 2004) (X Xia, unpublished), and cadherin. Unfortunately, these experiments do not rule should remove one of the key obstacles complicating out potential secondary effects of DN-cadherin expres- research efforts. These tools, coupled with p120 siRNA sion (e.g., sequestration of b-catenin), but they none- knockdown and addback systems (Davis et al., 2003) theless strongly suggest that p120 itself functions as a solve important technical problems and will accelerate tumor and/or metastasis suppressor in vivo. Experiments progress in understanding this complex domain. are underway using conditional p120 knockout mice to directly determine whether the DN-cadherin-induced metastatic phenotype is in fact due to p120 inactivation Evidence for p120 roles in tumor suppression and disease (Reynolds lab). The suggestion that p120 might act as a tumor and/or The siRNA data described above offer a plausible metastasis suppressor is strongly bolstered by observa- explanation for the elusive mechanism of action asso- tions of frequent regional loss of p120 in a variety of ciated with expression of extracellular domain deleted human tumors (reviewed in Thoreson and Reynolds, dominant-negative (DN) cadherins. Such constructs 2002). It has been known for some time that E-cadherin typically downregulate endogenous cadherins (Kintner, itself is frequently downregulated in tumors, and that E- 1992; Zhu and Watt, 1996; Nieman et al., 1999; Troxell cadherin-deficiency plays a causative role in the transi- et al., 1999), an observation that is not explained by tion to metastasis (Frixen et al., 1991; Vleminckx et al., regulation of cadherin transcription. The siRNA data 1991; Birchmeier and Behrens, 1994; Perl et al., 1998). show clearly that cadherins are degraded in the absence Importantly, there are also now at least 18 reports in the of p120 by a post-translational mechanism. Thus, DN- pathology literature describing regional downregulation cadherins appear to work by sequestering p120, making or loss of p120 in almost all of the major tumor types it unavailable for binding and stabilization of resident (e.g., prostate, lung, colon, breast, bladder, and others) endogenous cadherins. Indeed, this concept has now (reviewed in Thoreson and Reynolds, 2002). Since E-

Oncogene p120-catenin, cell adhesion and motility AB Reynolds and A Roczniak-Ferguson 7953 cadherin is degraded in the absence of p120, the data nonetheless prominent morphologic changes character- raise the question as to whether and how often E- ized by abnormal extension of lamellipodia. In vitro cadherin-deficiency might be caused by prior loss of experiments with purified proteins indicate that p120 p120 rather than various other events known to cause lacks GEF or GAP activity, but inhibits RhoA GTPase p120-independent downregulation of E-cadherin. activity in a Guanine Nucleotide Dissociation Inhibitor The current literature does not discriminate between (GDI)-like manner (Anastasiadis et al., 2000). That is, these issues, in part because most studies do not attempt it appears to sequester Rho in its inactive GDP-bound to correlate p120 and E-cadherin loss. Studies in cell form. The implied physical interaction has now been lines show that E-cadherin loss by mechanisms inde- demonstrated directly in a drosophila cell system by pendent of p120 (e.g., promoter methylation, direct E- GST pulldown assays, and by direct coimmunoprecipi- cadherin gene mutation, downregulation by transcrip- tation from cell lysates (Magie et al., 2002). While the tion factors) do not significantly affect p120 levels. RhoA interaction is apparently direct, effects on Instead, p120 mislocalizes to the cytoplasm (Thoreson Rac appear to occur indirectly through Rac GEF’s such et al., 2000) where, as described later, it may directly as Vav-2 (Noren et al., 2000). Together, these promote cell motility and metastasis through interac- effects may account for the observation that p120 tions with RhoGTPases. Thus, p120 independent E- expression can promote cell motility in NIH3T3 cells cadherin downregulation should be reflected in the (Grosheva et al., 2001) and growth factor-dependant pathology literature by regional E-cadherin loss and motility and scattering in epithelial cells (Cozzolino cytoplasmic p120. Indeed, this condition has been et al., 2003). reported frequently in carcinomas (reviewed in Thor- In Drosophila Rho1 mutants, cadherin and catenin eson and Reynolds, 2002), and parallels the scenario in localization is disrupted (Magie et al., 2002), as has been cadherin-deficient carcinoma cell lines (Thoreson et al., reported in mammalian cell lines (Braga et al., 1997; 2000). Kuroda et al., 1997; Takaishi et al., 1997; Jou and In contrast, as discussed previously, siRNA-induced Nelson, 1998). Magie and colleagues report that both p120 downregulation in cultured cells directly causes p120 and a-catenin coimmunoprecipitate with Rho1. degradation of E-cadherin (and associated a-andb- Interestingly, p120 coprecipitates preferentially with catenins) and can cause near complete loss of cell–cell GDP-bound Rho (Magie et al., 2002), in strong support adhesion (Davis et al., 2003). This observation predicts of the notion that p120 acts very much like GDI, despite that p120 downregulation in tumors should cause a little or no similarity between the proteins (Anastasiadis corresponding loss of all members of the cadherin et al., 2000). In constrast, a-catenin binds equally well to complex. Indeed, a recent prostate cancer study finds both GDP- and GTP-bound Rho, and b-catenin fails to regional p120 downregulation in 70% of high-grade bind either one (Magie et al., 2002). Importantly, p120 tumors, and does in fact report a very high incidence of RNAi phenotypes in the drosophila embryos were codownregulation of all components of the cadherin consistent with inhibition of Rho. Based on evidence complex (Kallakury et al., 2001). Coupled with the now that p120 binding to Rho is mutually exclusive of E- extensive literature on p120 downregulation in tumors cadherin binding (Anastasiadis et al., 2000), it is possible of all types, these data suggest that E-cadherin-loss in at that p120 recruits inactive Rho from the cytoplasm, least a subset of cases is due to prior p120-down- and then passes it off to local RhoGEFs as it binds regulation, as apposed to other well-described mechan- to E-cadherin (Anastasiadis et al., 2000; Magie et al., isms associated with E-cadherin-deficiency. Nonetheless, 2002). Such a mechanism could localize Rho-GTPase direct evidence is lacking, and very little is known activity to the interface between cadherins and the about the timing, mechanism, or consequences of p120 actin cytoskeleton, where it is needed to promote downregulation during tumor progression. Clearly, junction assembly or disassembly. Precedent for such a given the master role of p120 in regulating E-cadherin mechanism comes from studies of GDI, where binding abundance and function, these are now paramount next of Rho-GDI complexes to the cytoplasmic domain generation issues for understanding the role of p120 in of various receptors (e.g., CD44) is coupled to cancer. release of Rho, which is subsequently activated by a local GEF (Sasaki and Takai, 1998; Olofsson, 1999). This mechanism ensures that the GTPase is activated Regulation of Rho-GTPases by p120: a role as metastasis at the right time and place to coordinate receptor promoter? clustering. Several lines of evidence also suggest a role for p120- Several lines of evidence strongly support a role for p120 Rho interactions in the cytoplasm. The best character- in regulating Rho GTPases. High-level p120 over- ized of these are the ability of overexpressed p120 to expression in fibroblasts induces a dramatic branching induce cellular branching (Reynolds et al., 1996; phenotype (Reynolds et al., 1996), that has recently been Anastasiadis et al., 2000), and/or increased cell motility attributed to potent inactivation of RhoA (Anastasiadis (Noren et al., 2000; Grosheva et al., 2001; Cozzolino et al., 2000; Noren et al., 2000), and/or activation of et al., 2003). The branching activity is clearly indepen- Rac1 (Noren et al., 2000; Grosheva et al., 2001) dent of cadherin function since it occurs in cells known (reviewed in Anastasiadis and Reynolds, 2001). p120 to lack cadherins. Moreover, coexpression of DN- overexpression in epithelial cells induces less striking but cadherin constructs block p120-induced branching by

Oncogene p120-catenin, cell adhesion and motility AB Reynolds and A Roczniak-Ferguson 7954 sequestering p120, implying that the binding of p120 to ities (unpublished data, J Daniel, P McCrea). These E-cadherin blocks its interaction with Rho. Although observations are intriguing, but the function of p120 in the branching phenotype reflects nonphysiologic levels the nucleus remains the most mysterious of p120’s many of p120 expression, it nonetheless implies that cytoplas- roles. mic p120 interacts with and modulates Rho activity (reviewed in Anastasiadis and Reynolds, 2001). Lower levels of p120 overexpression cause increased cell Mammals and frogs need it, flies and worms don’t motility in fibroblasts, an activity associated with Rac activation (Grosheva et al., 2001; Noren et al., 2001), Surprisingly, p120 is not essential in Caenorhabditis possibly via the Rac-GEF Vav-2 (Noren et al., 2001). elegans or Drosophila, even though the genomes of these Moreover, p120-Rho GTPase interactions may be organisms lack potentially redundant p120 family important for growth factor dependant cell motility members. Although p120-deficiency exacerbates the and scattering in epithelial cells (Cozzolino et al., effects of mutations in the other catenins and cadherins, 2003). both worms (Pettitt et al., 2003) and flies (Myster et al., These observations have led ourselves and others to 2003) are viable when p120 is removed, and p120- postulate a role for cytoplasmic p120 in promoting uncoupled E-cadherin can substitute effectively for wild- metastasis in E-cadherin-deficient cells. Indeed, loss of type E-cadherin in flies (Pacquelet et al., 2003). In adhesion by itself does not necessarily translate into contrast, knockout of p120 in mice causes early increased motility and invasiveness. On the other hand, embryonic lethality (W Birchmeier, unpublished), de- the abnormally high cytoplasmic levels of p120 asso- spite the presence in the genome of several potentially ciated with E-cadherin-loss could directly dysregulate redundant p120 family members. In mice, even condi- multiple events mediated by Rho GTPases (Figure 2). In tional p120 knockout that is specifically restricted to the addition to the well-characterized activities of Rho GI-tract is lethal within a few weeks after birth (A GTPases in cytoskeletal regulation, these proteins can Reynolds, unpublished). Both p120 and ARVCF are also signal to the nucleus via pathways linked to JNK, essential genes in Xenopus, and can cross rescue one p38a, and p38g, thereby directly influencing transcrip- another in gene depletion experiments. Interestingly, tional events relevant to malignancy. depletion of either gene can also be rescued by carefully It is worth noting that the transgenic DN-cadherin titrated DN-Rho or DA-Rac (Fang et al., 2004). experiments described previously appear to contradict Clearly, higher and lower organisms differ with regard this model. DN-cadherin expression appears to seques- to their reliance on p120, suggesting that p120 has ter and inactivate p120, and yet metastasis is strongly evolved both additional family members and increased promoted in the T-antigen/DN-cadherin double trans- complexity to accommodate the developmental and genic animals. Clearly, more work is needed to clarify organizational demands of higher organisms. aspects of these models that are not yet fully under- stood. Conclusions

A role for p120 in the nucleus? Ironically, though much is now known about p120, the role of p120 tyrosine phosphorylation remains myster- Nuclear localization of p120 is increased in E-cadherin- ious. p120 does not appear to be central to transforma- deficient cells (van Hengel et al., 1999; Roczniak- tion by Src, and yet several lines of evidence suggest that Ferguson and Reynolds, 2003), but whether this p120 may very well turn out to be both a tumor contributes to the metastatic phenotype is unclear. The suppressor and metastasis promoter. Strong evidence structural regions of p120 that influence nuclear indicates that a core function of p120 is to regulate localization have been described (van Hengel et al., cadherin turnover at the cell surface. p120 levels act as 1999; Roczniak-Ferguson and Reynolds, 2003), and both sensor and determinate of cadherin levels, provid- there is evidence that interactions between p120 and ing a crucial rheostat or set point mechanism that kinesin can strongly affect p120 nuclear trafficking controls cadherin availability. Stabilizing cadherin, (Yanagisawa et al., 2004). p120 interacts with Kaiso however, is by itself insufficient for strong adhesion, as (Daniel and Reynolds, 1999), a member of the BTB/ aberrant signaling through the p120 amino-terminal POZ family of transcription factors, but the functional regulatory domain can still disrupt adhesion. Src consequences are unknown. In addition to a CAST- substrates in general appear to coordinate crosstalk derived DNA-binding sequence (Daniel et al., 2002), between integrin and cadherin systems via regulation of Kaiso binds preferentially to CpG-methylated DNA the actin cytoskeleton. The ability of p120 to regulate (Prokhortchouk et al., 2001; Daniel et al., 2002) and Rho GTPases fits with this overall scheme and may be mediates DNA methylation-dependent repression important for coordinating the inter-related activities of through interaction with the N-CoR complex these proteins in cellular adhesion and motility. These (Yoon et al., 2003). BTB-POZ proteins typically have apparent roles of p120 in controlling adhesion may important roles in development and cancer, and ultimately be important in a variety of inflammatory though inconclusive, evidence is building that the diseases and cancer, providing several interesting p120–Kaiso interaction mediates cancer-relevant activ- challenges for future work.

Oncogene p120-catenin, cell adhesion and motility AB Reynolds and A Roczniak-Ferguson 7955 References

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