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Biochimica et Biophysica Acta 1863 (2016) 102–114

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Biochimica et Biophysica Acta

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Review Phosphorylation and isoform use in p120-catenin during development and tumorigenesis

Ji Yeon Hong a,⁎,Il-HoanOhb, Pierre D. McCrea c,⁎

a Division of Cardiology, Department of Medicine, Severance Biomedical Science Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea b The Catholic University of Korea, Catholic High Performance Cell Therapy Center, 505 Banpo-dong, Seocho-Ku, Seoul 137-701, Republic of Korea c Department of Genetics, University of Texas MD Anderson Cancer Center, University of Texas Graduate School of Biomedical Science, Houston, TX 77030,USA

article info abstract

Article history: P120-catenin is essential to vertebrate development, modulating cadherin and small-GTPase functions, and Received 21 February 2015 growing evidence points also to roles in the nucleus. A complexity in addressing p120-catenin's functions is its Received in revised form 12 October 2015 many isoforms, including optional splicing events, alternative points of translational initiation, and secondary Accepted 13 October 2015 modifications. In this review, we focus upon how choices in the initiation of protein translation, or the earlier Available online 23 October 2015 splicing of the RNA transcript, relates to primary sequences that harbor established or putative regulatory phosphorylation sites. While certain p120 phosphorylation events arise via known kinases/phosphatases and Keywords: fi Structure-function have de ned outcomes, in most cases the functional consequences are still to be established. Kinases In this review, we provide examples of p120-isoforms as they relate to phosphorylation events, and thereby to Phosphatases isoform dependent protein–protein associations and downstream functions. We also provide a view of upstream Cadherins pathways that determine p120's phosphorylation state, and that have an impact upon development and disease. Small-GTPases (RhoA Because other members of the p120 subfamily undergo similar processing and phosphorylation, as well as related Rac1) catenins of the plakophilin subfamily, what is learned regarding p120 will by extension have wide relevance in Wnt signaling vertebrates. Nuclear signaling © 2015 Elsevier B.V. All rights reserved. Development Cancer

1. Introduction more membrane-distal site on the tail, indirectly linking the cadherin complex to the contractile cortical-actin cytoskeleton, an interaction Together with cell interactions taking place with the extracellular that contributes to cell adhesion and motility [91,135]. matrix (e.g. integrin-mediated), varied cell–cell adhesion complexes As noted, p120-catenin was originally described as a Src kinase contribute to development and tissue homeostasis. In vertebrate substrate, and then as a component of the cadherin–catenin complex. epithelial cells, three major types of cell–cell contacts tend to be P120-catenin promotes cadherin stability, lowering the complex's sus- discussed, including adherens, tight and desmosomal junctions. Given ceptibility to endocytosis, ubiquitination, and proteosomal destruction our focus upon p120-catenin, we note that it was initially characterized [25,33,56,92,149,150]. With the exception of αE-, αN- and αT-catenin, as a substrate of the Src kinase [113], and shortly thereafter was found which are structurally related to vinculin, all catenins including p120 in association with one of the principal transmembrane components contain a central Armadillo-repeat domain that facilitates protein– of epithelial adherens junctions, E-cadherin [1,100,111]. Adherens protein interactions. The Armadillo domain is only modestly homologous junctions are complex both with respect to the number of components between p120 subfamily catenins (45–55% primary sequence identity), involved, and the kinds of outside-in and inside-out (etc.) signals trans- and the amino- and carboxyl-terminal regions are yet more divergent. duced or facilitated. Two of an assortment of proteins able to bind to the In addition to binding and modulating E- or other classic-cadherins, cytoplasmic carboxyl-tails of classic cadherins include p120-catenin and p120-catenin regulates small-GTPases (e.g. Rac1 and RhoA). This can β-catenin. Within the p120 subfamily of catenins are ARVCF-, δ-and occur while p120 is associated with or dissociated from the larger p0071-catenin; each competes with the other as well as with p120- cadherin–catenin complex [6,69–71,144]. P120 can bind small-GTPases catenin itself to bind a membrane-proximal site on the cadherin tail as a guanine-nucleotide dissociation inhibitor/GDI (e.g. for RhoA), or [17,54,86]. β-catenin (or the related γ-catenin/plakoglobin) binds to a associate indirectly via GEFs, GAPs or other small-GTPase effector pro- teins. P120-catenin further associates with microtubules and motor proteins [23,43,155].Afinal intriguing property of p120-catenin relates ⁎ Corresponding authors. E-mail addresses: [email protected] (J.Y. Hong), [email protected] to its nuclear entry and association with transcriptional regulators such (P.D. McCrea). as Kaiso [30,31,108],Glis2[51] and REST/CoREST [77]. In particular,

http://dx.doi.org/10.1016/j.bbamcr.2015.10.008 0167-4889/© 2015 Elsevier B.V. All rights reserved. J.Y. Hong et al. / Biochimica et Biophysica Acta 1863 (2016) 102–114 103 p120-isoform 1 appears to participate in vertebrate canonical Wnt sig- of p120's isoform-specificity, its phosphorylation and protein–protein naling [49]. In summary, p120 binds and regulates the cadherin–catenin interactions help to determine p120's intracellular localizations and complex (cell–cell junctions), small-GTPases and cytoskeletal factors functions. (cell–cell junctions and elsewhere), and transcription factors (nucleus). Thus, we will address in this review how p120's phosphorylation and 2. Isoforms of p120-catenin isoform status is relevant to an array of cellular processes. P120-catenin's phosphorylation, isoform status and intracellular lo- Considering combinatorial possibilities that result from four distinct calization vary with the cell type and tissue (reviewed in [70,107,114]. translation-initiation sites and four alternatively spliced exons, human With respect to nuclear localization, p120 was initially suggested to p120-catenin possess in theory 64 primary-sequence isoforms (Fig. 1) contain two nuclear localization signals (NLS) [2,7], and three potential [62]. P120-isoform 1 is the product of the most upstream translational nuclear-export (NES) regions (Fig. 1) [137]. The NLS sequence between initiation. It contains an amino-terminal coiled-coil domain, absent Armadillo repeats 5 and 6 modulates p120's (isoform 1) nuclear locali- from isoforms 2 to 4. Isoform 4 is missing almost the entire amino- zation [63], although other findings have instead pointed to p120's terminus of p120-catenin, thus lacking the coiled-coil as well as a regu- (isoform 3) Armadillo region in nuclear entry (repeats 3 and 5) or latory region harboring many potential or confirmed phosphorylation export (repeat 8) [114]. For example, the Armadillo domain of p120 sites [147]. P120-isoform 2 and -isoform 3 lack smaller regions, perhaps appears needed for it to shuttle into the nucleus in response to Rac1 most noteworthy being the amino-terminal coiled-coil region that [76]. Also favoring p120 nuclear entry is the transcription factor Glis2, enables some of p120's interactions (Fig. 1). The differential functions especially in combination with Src phosphorylation of p120 [51]. of p120's isoforms are just beginning to be revealed, such as in the regu- At cell–cell contacts such as adherens junctions, p120 interacts with lation of RhoA activity [154]. As noted, varying p120-isoforms are E- and other cadherins, with either's phosphorylation affecting their expressed in different ratios among varying cell types or contexts [94]. association [36,38,44,104,138]. Thus, as we will discuss in the context With the exception of macrophages [156], non-adherent (e.g. B- and T-)

Fig. 1. P120-catenin structural features. Structural features of human p120-catenin in relation to its direct interaction partners, alternative translational start sites (yellow boxes 1–4), alternatively spliced regions (tan boxes A-D), and subcellular localization signals (NLS black boxes; NES red boxes). An alternatively spliced region D is rarely excluded. The Armadillo do- main is depicted in green; and in the case of p120-catenin and related sub-family members, it has nine repeat units each of about forty-two amino acids. Indicated are a number of putative nuclear localization sequences (NLS) [2,63,114], and nuclear export sequences (NES) [137]. An amino-terminal coiled-coil region is shown in light blue and exists solely in p120-isoform 1. Additional information on the functional differences between p120-catenin isoforms is included in the review text, while representative references relating to the indicated protein inter- actions of p120-catenin are included within the figure itself. 104 J.Y. Hong et al. / Biochimica et Biophysica Acta 1863 (2016) 102–114 cells express little p120-catenin. Interestingly, during epithelial-to- 3.1. Phosphorylation specific to p120-isoform 1 mesenchymal transition/EMT, (e.g. promoted via the expression of Snail, c-Fos, SIP1/ZEB2, Slug, Twist or Zeppo1), switches have been ob- Recent work has identified four conserved serine phosphorylation served from p120-isoform 3 to isoform 1 [20,99,118,125]. Mechanisms sites at the very amino-terminus of p120-isoform 1 (S4, 8, 11, 15) governing the ratio of p120's primary-sequence isoforms have been (Fig. 1). The closely-spaced serines comprise a “destruction box” similar partially clarified through work upon RNA splicing factors such as to that in β-catenin [49] (reviewed in [129]). These sites become ESRP1 and ESRP2 [141] (Fig. 3B). Studies also point to p120-isoform 1 phosphorylated by CK1α and GSK3β when canonical Wnt pathway more so than isoform 3 in cancer cell invasion and metastasis [89,125, activity is lacking, resulting in reduced catenin stability and thus 127,131,153,154,158,161]. reduced signal transduction to the nucleus (Fig. 3A). In the case of In addition to four distinct translation initiation sites, p120 contains p120, nuclear partners include the transcriptional regulators Kaiso [30, four alternative spliced exons, denoted A, B, C and D (Fig. 1). Exons A 31,108],Glis2[51], and REST/CoREST [77]; these associated factors are and C are respectively 21 and 6 amino acids long and not well character- each distinct from the transcriptional regulators binding β-catenin ized, while exon B's 29 amino acids appear to encode a nuclear export (e.g. TCF/LEF). Of p120's partners, only Kaiso has been shown thus far sequence (NES) [2]. Exon D (24 amino acids) is almost always present to respond (via p120) to canonical Wnt signals [49,101,102,121].As [107], with for example, early reports including its inclusion in fetal would be expected, isoform 1 is the most sensitive to the activity of and adult brain tissues [3]. No potential phosphorylation sites exist canonical Wnt pathway components such as LRP5/6. An additional within these splice insert regions, although potential sites are nearby. phosphorylation site in isoform 1 was resolved immediately after the For example, S879 and S873 adjoin exon A, and S916 resides between coiled-coil domain (S47), phosphorylated by the kinase Dyrk1A. exons A and B (Fig. 1). Conceivably, the presence versus absence of Interestingly, in opposition to CK1α and GSK3β, the phosphorylation one of these exons could modulate phosphorylation-dependent interac- of p120-isoform 1 by Dyrk1A enlarges the “signaling pool” of p120 to pro- tions nearby. mote its activity in the nucleus, as well as conceivably, p120's modulation P120-catenin's function is also modulated through subcellular local- of small-GTPases and the cytoskeleton. In Xenopus and human cell lines, ization, likely to be influenced by primary-sequence elements present Dyrk1A's phosphorylation of p120-isoform 1 enhances canonical Wnt (or absent) in distinct p120-isoforms. An example was provided above signaling [50]. For example, in a classic assay of Wnt activity in vivo, with p120 nuclear export (cytoplasmic localization) being enhanced ventral co-expression of a phospho-mimic of p120-isoform 1 (T47D) by the NES in isoform B (Fig. 1). Residues that instead reside within enabled an otherwise sub-phenotypic dose of exogenous β-catenin to p120's amino-terminus may also have a role in determining p120 local- produce ectopic dorsal axes in Xenopus embryos. Recent work has fur- ization. For example, a larger proportion of isoform 3 than 1 (Fig. 1) thermore identified DIPA as a binding partner specific for p120-isoform enters the nucleus in cells in contact with matrix and grown in 3-D 1 [69,84,85], and an amino-terminal region preceding p120's coiled-coil [125]. In turn, isoform 1 may be directed to cell–cell contacts and domain is an important binding site for kinesin (Fig. 1). Given that other elsewhere via interactions at its very amino-terminus [85]. As discussed proteins associate with p120's amino-terminal region, such as NLBP, later, distinguishing phosphorylation sites reside in amino-terminal PLEKHA7 and RPTPμ, future tests must assess the isoform specificity of (translational) isoforms of p120, as well as potentially, sequences these interactions [23,64,71,72,85,87,109]. flanking p120's optional splice inserts. E-cadherin binds p120's central Armadillo domain at adherens 3.2. Phosphorylation specific to p120-isoforms 1 and 2 junctions in normal epithelial cells, where p120 promotes cadherin clustering and adhesive function [157]. This occurs in part via p120's en- There is only one potential phospho-tyrosine (Y96) between the hancement of cadherin stability at cell–cell contacts [25,33,56,57,92, second and third translational initiation sites of p120 (Figs. 1 & 2) [4], 149,150]. In turn, multiple p120-isoforms as well as other Armadillo as well as three serines (S81, S92 and S97). These sites, however, are catenins would be predicted to be modulated via sequestration only weakly scored when using algorithms to predict phosphorylation by cadherins. It is interesting that in the case of the structurally related (e.g. NetPhosph 2.0). Currently, no evidence suggests Y96 affects p120 β-catenin, reports have suggested to varying extents that its amino- function, but Y96 is phosphorylated by an unknown kinase in Src or carboxyl-terminal tails fold in cis to affect associations of the transformed cells [82]. Fer, a member of the Src tyrosine-kinase family, Armadillo-domain in trans [93,26]. If this likewise applies to p120, preferentially associates with the two longer isoforms of p120 [115]. then one can imagine that p120-isoforms 1 to 4, with distinctions in While only one tyrosine residue selectively resides in isoforms 1 and 2 length and phosphorylation potential, may vary in their capacity to (Y96), it could conceivably provide an opportunity for tyrosine kinases modulate p120's Armadillo-domain interactions. such as Fer to regulate p120-isoforms 1 and 2 in the context of cadherins, small-GTPases or other protein complexes. Further study is warranted of 3. Phosphorylation of P120-catenin the kinase(s) and phosphatase(s) involved at these tyrosine and serine/ threonine sites. Phosphorylation is one of many modifications impinging upon protein interactions and subcellular localization. Known as well as 3.3. Phosphorylation specific to p120-isoforms 1, 2 and 3 putative phosphorylation sites exist in the amino-terminal region of p120-catenin [4]. To our knowledge, no other secondary modifications Many known or potential phosphorylation sites reside in the region with the exception of ubiquitination [49] have been evaluated. As between the respective initiations of isoforms 3 and 4 (“regulatory addressed below with respect to canonical Wnt signaling, key residues domain” mentioned in previous reviews [4]) [107,121,148] (Fig. 2). are phosphorylated in the amino-terminus of p120-isoform 1 (Fig. 1) Therefore, some of these sites are anticipated to provide shared effects [49]. With regards to other parts of p120, such as the Armadillo or upon isoforms 1–3. P120 associates with kinases such as Fer, Fyn and carboxyl-tail domains, other kinases or phosphatases are capable of Yes [65,105,151], as well as interacts with a number of phosphatases making modifications in less isoform-specific manners. such as DEP1 [48], PTP-PEST [38],RPTPμ [162],RPTPrho[13],andSHP-1 Since the varied isoforms of p120 modulate cadherins, small-GTPases [61], which are implicated in either modulating cadherin dependent cell and gene targets, they are relevant to development, homeostasis and adhesion or motility, small-GTPases, or nuclear gene programs. disease progression. Such isoforms localize differentially and according As mentioned previously, the larger sequence region upstream of to context, with phosphorylation altering p120's protein interactions, p120's Armadillo domain includes binding sites for partners such as cytoplasmic-nuclear shuttling and nuclear activity. It is thus relevant to NLBP [64],PLEKHA7[72,87,109],RPTPμ [162],DIPA[69,84,85],andGα12 probe the isoform-specific roles of p120 as a function of phosphorylation. [8]. While validation remains needed in most cases, the phosphorylation J.Y. Hong et al. / Biochimica et Biophysica Acta 1863 (2016) 102–114 105

Fig. 2. Phosphorylation of p120-catenin. Outlined in the text are proposed kinases, phosphatases and signaling mechanisms modulating the level of p120-catenin phosphorylation. As reflected in this figure, multiple residues are modified in response to serine, threonine (Red) or tyrosine (Blue ovals) kinases. Black circles indicate potential phosphorylation sites that are not yet identified as phosphorylated, and that are not yet known to have functional effects. All four human translational start site isoforms are depicted (p120-isforms 1–4), so that one can easily compare which phospho-residues in p120 pertain to only one versus multiple isoforms. Given the number of phosphorylation sites involved, relevant references are included in the review text, as opposed to this legend.

state of the amino-terminal regulatory region is expected to modulate of receptor tyrosine kinases/RTKs such as EGFR, VEGFR and PDGFR some of these interactions or be a consequence of them, and while a num- [37,39,55,83].P120Y228islikelytobephosphorylatedbyEGFRsignal- ber of phospho-sites currently appear to have lesser functional ing, but this modification occurs without obvious effects upon cell–cell implications [83], this could change upon applying other assays. Here, junctions when evaluated using epithelial cell cultures [83],and we will first discuss tyrosine and serine/threonine phosphorylation appears not to be mediated via Src kinase. Thus, it is not currently events common to isoforms 1–3. known if a particular p120-isoform(s) is preferentially regulated by an RTK. With regard to non-receptor tyrosine kinases, as noted earlier, 3.3.1. Tyrosine phosphorylation Fer-kinase associates to an amino-terminal region of p120 (amino P120 was first identified 20 years ago in a screen for novel substrates acids 131–156), between p120's 3rd and 4th translational start sites of the Src kinase [113]. Depending on the context, heightened tyrosine (Fig. 1) [65], with Src and Fyn sites mapped relatively nearby to Y112, phosphorylation of cadherins and catenins has correlated with either Y217 and Y228. Recently, the non-receptor tyrosine kinase PTK6 diminished [19] (e.g. via Fer kinase) [52,68,74,115,116],enhanced[16, (Protein receptor tyrosine kinase 6) was found in association with 19,28,97,130], or little effect upon cell–cell junctions. Similar to tyrosine p120-catenin, although its phosphorylation of p120-catenin has yet to phosphorylation, serine/threonine modification of p120 likewise modu- be characterized [45]. lates junction functions to context-dependent extents [12,98]. In addition to the indirect activities of tyrosine kinases, there is also the question of direct RTK phosphorylation of p120, and if particular 3.3.1.1. Src-family kinases. P120 is enriched in potential Src-family kinase phospho-tyrosine residues display isoform-specificity. Unfortunately, sites within its amino-terminal region (e.g. Y112, Y217, Y228) (Fig. 1). specific residues directly phosphorylated by EGFR, PDGFR or VEGFR This is one of two areas where RhoA binds [19,154]. Fyn's phosphoryla- have yet to be resolved. Recent work using anti-VEGF antibodies points tion of p120 at Y112 diminishes p120's association with RhoA such that to Y228 as being associated with glioblastoma progression [55,138]. RhoA is no longer inhibited (its activity rises), whereas Src phosphory- This residue is phosphorylated by Src [82], often a downstream RTK lation of p120-catenin on Y217 and Y228 has the opposite effect, since effector. Seven additional Src phosphorylation sites were identified, Src enhances p120's interaction to further inhibit RhoA. Recently, with mutation of all eight sites (Y96, Y112, Y228, Y257, Y280, Y291, the p120 dephospho- as opposed to phospho-form of Y112 was found Y296, Y302) eliminating p120's interaction with the phosphatase to associate with Rac and Vav2 to promote Rac activity and thereby SHP-1 [82]. Since p120-isoform 4 bound more poorly to SHP-1 than β-catenin nuclear localization, while Y217 dephosphorylation had did isoforms 1–3, it suggests that the greater number of Src-targeted similar effects with regards to Rac [136] (Table 1). In common with tyrosine residues within the amino-terminus of p120 may enable a Src kinase, Fer also enhances p120:RhoA association, and both kinases stronger SHP-1 association [61]. As phosphatases modulate many favor p120's association with E-cadherin [19] (Table 1). In another re- biological outcomes including those at cell–cell contacts, differential re- port, p120's Y217 was shown to function in v-Src mediated reduction cruitment of SHP-1 (etc.) as determined by the p120-isoform present, of cell–cell adhesion in L-cells [100]. Collectively, these findings indicate may be a means to modulate those outcomes. that functionally important phospho-residues reside with the amino- In embryonal carcinoma cells, nerve growth factor or TrkA-receptor terminal region of p120-catenin, lacking in isoform 4 (Fig. 1). stimulation leads to p120's (as well as β-catenin's) tyrosine phosphory- lation, with these effects inhibited by the Src inhibitor PP2 [28].The 3.3.1.2. Receptor tyrosine kinases. The phosphorylation of adherens residues involved are not known, a reflection of the work still needed junction components has also been studied in relation to the activities to identify and characterize p120's phospho-tyrosines. 106 J.Y. Hong et al. / Biochimica et Biophysica Acta 1863 (2016) 102–114

3.3.2. Protein tyrosine phosphatases of p120's entire upstream region, as occurs naturally in generating iso- Given their regulation of p120's phosphorylation state, phospha- form 4, eliminates multiple phosphorylation sites and associations in tases are as central to our discussion as kinases. Phosphatases such as this region, but not that with cadherins which instead involves p120's SHP-1, SHP-2, DEP1 and RPTPμ act upon p120-catenin. For example, neighboring Armadillo domain [57,98]. For example, PDGFR activation the tyrosine phosphatase SHP-1 binds p120 following EGF-stimulated leads to p120's phosphorylation at S879 via a PKCα-dependent path- p120-phosphorylation as mentioned [61], and SHP-1 effectively de- way [15]. Upon thrombin stimulation and thereby PKCα activation, phosphorylates Y296 and presumably additional residues following phosphorylation at S879 promotes p120's dissociation from VE- the activity of Src [42] (Table 1). The tyrosine phosphatase DEP1 also as- cadherin. As might be predicted, S879 point-mutagenesis enhances sociates with p120, although the phosphorylation sites it acts upon are p120:VE-cadherin association and junction function, with a reduction not known [48]. The RPTPμ tyrosine phosphatase binds p120 in a man- in cadherin endocytosis. Unlike PKCα's negative impact upon the asso- ner independent of p120's central Armadillo domain (Fig. 1) [162].In ciation of p120 with VE-cadherin, and thus upon cell–cell interactions most cases, the p120 tyrosine residues targeted by a particular phospha- [138], atypical-PKC promotes adhesion. This occurs through atypical- tase has yet to be determined. Better knowledge of both phosphatase PKC phosphorylation of Numb, and Numb's dissociation from the and kinase activity directed towards p120 should be useful in under- carboxyl-tail of p120. Because Numb otherwise recruits α-adaptin, standing p120's functions in junctional, cytoplasmic and nuclear con- Numb's loss reduces the extent of cadherin internalization [119].Itap- texts. More complete or refined assays may also be required to assess pears, therefore, that different PKC family members modify carboxyl- functional effects, since in many cases, negative experimental results tail residues within p120, exhibiting distinct effects upon p120 and may not accurately reflect the in vivo significance of the phospho- cadherin function. residues examined, or that of the kinases or phosphatases acting upon Depletion of the phosphatase PTP-PEST results in enhanced p120 p120. phosphorylation (Y335). This leads to reduced p120 association with E-cadherin, with correspondingly greater p120 modulation of small- 3.3.3. Serine/threonine phosphorylation GTPases and the cytoskeleton (via Vav2 & cortactin), resulting in height- P120 is phosphorylated upon numerous serine/threonine residues, ened cell motility [38]. Since Y335 is present in all p120-isoforms with for example, effects reported for the PKC isoforms PKCα,PKCδ (Fig. 2), the downstream functional consequences of this relationship [60] and PKCε [32]. PKC promotes phosphorylation of S879, and the de- with PTP-PEST might at first seem to be shared. However, as noted ear- phosphorylation of S268, S288, T310 and T916 [15,138,147,148,160]. lier, p120-isoform 1 is the most effective in inhibiting RhoA, such that S268 is selectively present in p120-isoforms 1–3 and its phosphoryla- differences in isoform expression levels is likely to produce functional tion is dependent upon one PKC isoform, PKCε. Phosphorylation of distinctions. S288 in response to PKC promotes the binding of p120-isoform 3 to the transcription repressor Kaiso, its movement from the nucleus to cy- 4. P120-catenin isoforms and phosphorylation in development toplasm and presumably gene target modulation [160] (Fig. 3C). Nearby to S288, residues S268 and S269 are likewise phosphorylated. Here, P120-catenin has essential pleiotropic roles in vertebrate develop- CK1ε responds to canonical Wnt pathway activation, with p120 phos- ment and homeostasis, while interestingly, it plays modulatory but phorylation via CK1α somehow promoting p120's release from non-essential roles in invertebrates raised under laboratory conditions cadherin [18] (Table 1). This increases p120's signaling pool, with (Drosophila & C. elegans) (reviewed in [86]). For example, in mice, the S268 and S269 phosphorylation promoting p120's association with whole animal knock-out of p120 is early embryonic lethal [34]. Addi- the Kaiso transcriptional regulator to activate (relieve repression of) tionally, several tissue specific knock-out mice have been generated, gene targets [36], as well as activating Rac. The author's model proposes such as in salivary gland, skin, teeth, dorsal forebrain, intestine, mam- interaction of the cadherin-complex with the Wnt-pathway receptor mary gland, eye, pancreas and endothelial cells [9,34,47,75,103,126]. complex, integrating their signaling to the nucleus. As the serine resi- Defects include those appearing at adherence junctions and in small- dues discussed (S268, S269 and S288) are present only in isoforms 1– GTPase functions (and likely gene-regulatory responses), with gross ef- 3, they alone would be expected to participate in such a pathway. fects being manifest in aberrant tissue morphogenesis. P120 ablation in While EGFR, VEGFR or PDGFR activation enhances p120's tyrosine different organ system reveals a variety of effects. In tissues such as skin, phosphorylation [37,39], VEGFR has also been noted to promote the de- salivary gland, mammary gland, dental enamel, kidney and vasculature, phosphorylation of p120 upon serine/threonine [145]. This effect is p120 removal results in reduced E-cadherin levels, sometimes in con- thought to occur through downstream effects upon PKC and ultimately junction with inflammation [9,34,73,81,126].Duringembryoniceyede- an unidentified phosphatase. Dephosphorylation of p120 also occurs in velopment, p120 interacts functionally with Shroom3 in apical cells exposed to potentiators of PKC (e.g. phorbol esters), with effects constriction, with p120 removal disrupting lens pit invagination [75]. observed upon S268, T310 and T916 in addition to other residues In addition, p120 in the context of neural crest cells contributes to [139,147,148]. P120 is alternatively phosphorylated upon serine/threo- forming the eye and craniofacial skeleton [27,134,152]. In certain con- nine residues following activation of RTKs (e.g. PDGFR), with the effects texts other effects are observed. For example, p120 loss in the skin, in- again being in part due to the downstream engagement of PKCα [15, testine and pancreas promotes immune cell infiltration and 138]. For example, S879 is directly or indirectly phosphorylated by proinflammatory cytokine release [47,103,126]. Reports also suggest PKCα. S879 is just upstream of the A splice-isoform insert in p120- p120 is tied to inflammation via effects upon small-GTPases, NF-κBac- catenin (Figs. 1 & 2), so their juxtaposition may be relevant to either tivity and Kaiso [21,53,110]. Recently, p120 ablation in mouse pancreas isoforms's functional activities in vivo. progenitor cells produced embryonic defects in tubulogenesis, branching morphogenesis and acinal cell differentiation, with concur- 3.4. Phosphorylated residues shared across p120-isoforms rent inflammation [47]. Aberrant tubule formation was also reported upon p120 loss during glomerulogenesis [81].InXenopus,p120 Less is known concerning phosphorylation events in the Armadillo- knock-down embryos exhibit gastrulation defects [41],withdisrup- domain or carboxyl-tail of p120. The Armadillo or carboxyl-tail region of tions arising due to defects in cadherin, small-GTPase and nuclear (e.g. p120 interacts with multiple proteins such as MUC1, REST/CoREST, Kaiso) functions [49,66,102]. More information on p120's functions Numb and p190RhoGAP, with these associations presumably being during embryonic development is provided in prior reviews [86,106, shared among isoforms 1–4 [77–79,119,159]. 107]. In both p120's amino- and Armadillo-regions, phospho-serine, Although p120-catenin is widely expressed, specific isoforms are -threonine and -tyrosine sites are present. Interestingly, the removal more apparent in certain tissue contexts. While not exclusive, the J.Y. Hong et al. / Biochimica et Biophysica Acta 1863 (2016) 102–114 107 shorter p120-isoforms 3 or 4 are abundant in epidermis, palate and 5. P120-catenin isoforms and phosphorylation in cancer tongue epithelia, and in the ducts of excretory glands, while longer isoforms are favored at vascular-endothelial cell–cell junctions (blood In multiple cancers such as adenocarcinoma, breast cancer, bladder vessels), junctions of serosal epithelium lining internal organs, brain cancer and squamous cell carcinoma, reduced or relocalized p120 choroid plexus, pigment epithelium of retina, and intercalated discs presence correlates with reduced E-cadherin presence, altered small- of cardiomyocytes [2,90,95]. The isoform specific roles of p120 in GTPase activity and poor prognosis [58,112,124,131,143]. Recently, development are largely unknown. As discussed in this review, partial p120-catenin cytoplasmic localization was correlated with poor differences in function presumably derive from isoform-specific distinc- prognosis in human colon carcinoma and esophageal squamous cell tions in the presence versus absence of regulatory phospho-residues. carcinoma [11,22]. Through p120's function in stabilizing cadherins, Given that p120's phosphorylation-status is likely to have roles in consideration has been given to p120's activities as a tumor suppressor cadherin mediated cell–cell adhesion, cytoskeletal functions via the [34,73,80,96,120,121,128,133]. For example, conditional deletion of modulation of small-GTPases, as well as signaling processes mediated p120 in the mouse oral cavity, esophagus and forestomach results in by canonical Wnt components or other pathways, it is appropriate to invasive squamous cell cancer in addition to desmoplasia and inflam- postulate that defined phosphorylation or dephosphorylation events mation [128]. P120's relationship to E-cadherin or other partners upon p120 are required in development. is made more interesting by its isoforms having differing affects upon As mentioned earlier, a number of reports indicate p120's involve- tumor progression. Given its longer N-terminus, p120-isoform 1 ment in canonical Wnt signaling. Canonical Wnt signaling, which is (relative to isoform 3) exerts greater inhibition of RhoA. This can modulated by β-catenin, is central in development as well as tumori- enhance cell migration and invasion [123,127], with for example, p120- genesis. In recent reports, the kinases GSK3β,CK1α and Dyrk1A isoform 1 expression being predictive of renal tumor micrometastasis phosphorylate p120-isoform 1 at defined residues to regulate p120's [154]. Since some phosphorylation sites are specific to p120-isoform 1, stability, which determines the size of its signaling pool [49,50].The they may in turn be relevant to normal or tumor cell migration. Other same upstream canonical Wnt ligands and destruction-components work likewise points to distinctions in p120-isoform expression and that act upon p120-isoform 1 also regulate β-catenin. An enlarged localization in pathology, as in the case of metastatic lung cancer [40, signaling-pool of p120 promotes its binding to the transcription factor 123,161], breast invasive lobular carcinoma [122,123], and colon [11], Kaiso, and perhaps other transcriptional regulators. Although divergent ovarian [24] and pancreatic cancer [132]. Some reports indicate that views have arisen [14,117], the association of p120 with Kaiso associa- cytosolic p120-catenin localization correlates with hyperplasia and the tion has been tied to the de-repression (activation) of p120/Kaiso progression of E-cadherin deficient tumors (or E-cadherin mutant sequence-specific gene targets (KBS: TCCTGCNA) [66,102]. Further, tumors), and is predictive of poor prognosis [127] [11,22]. while a surprise at first, the promoters of certain genes harbor binding Given that differing p120-isoforms exhibit distinct localizations sites for both Kaiso and TCF/LEF, apparently to permit the coordinate according to context [2,114], choices involving isoform expression or regulation of such genes by p120-catenin and β-catenin in response the presence of E- versus other cadherins is expected to play a role in to upstream Wnt-signals, potentially during embryogenesis [102] determining the roles of p120 in cancer. For example, in contrast to (Fig. 3A). Given that GSK3β and Dyrk1A phosphorylation sites were p120's association with E-cadherin, that with P- or N-cadherin in identified towards the amino-terminus of p120-isoform 1 (Table 1), some instances promotes motility more so than adhesion [111,132]. p120-mediated Wnt-responsiveness would be expected in those tis- Therefore, p120's contributions to pathology are likely to be determined sues expressing isoform 1. Apart from this relationship, p120 partici- in part by what cadherins are expressed in the tumor [121]. pates in canonical Wnt signaling in other ways. For example, upon Multiple phosphorylation sites reside in p120's amino- and carboxy- upstream Wnt-pathway stimulation, p120 is phosphorylated on S268 terminal regions and may have a role in tumorigenesis. Utilizing and S269 by CK1α, while being dephosphorylated on tyrosine Y112 phospho-specific antibodies, cancer cell lines have been evaluated to and Y217. The two serine phosphorylation events release p120 from assess p120's phosphorylation state [83,147] (Table 1). E-cadherin E-cadherin [18]. Upstream Wnt-activity further leads to phosphory- contributes to determining p120's phosphorylation in certain cancer lation upon E-cadherin and its release from the signalosome complex, fa- cell types, presumably by recruiting p120 into complexes or regions voring GSK3β internalization (removal) and the activation of β-catenin rich in kinase activity [71,114,147]. target genes [35,36,140]. Since S268, S269, S288, Y112, Y217 and Y228 Recently, upon Ras-PKCε activation in breast cancer cells, p120's exist in p120-isoforms 1–3, (but not in isoform 4), these effects may be phosphorylation at S268 was found to contribute to cellular foci forma- more general. The PKCα kinase instead phosphorylates p120 at S879, tion (Table 1). Another report found that p120-isoform 3 undergoes promoting the dissociation of p120 from VE-cadherin in endothelial S288 phosphorylation and greater Kaiso binding, correlating with cells, and the disassembly (enhanced permeability) of junctions [138]. enhanced lung-cancer cell invasion in vitro [160,161]. While the work As noted, PKC activity has also been observed to lower p120 phosphory- above suggested that p120-isoform 3 was the prime modulator of lation (S268), presumably indirectly and occurring via an unknown phos- Kaiso function, findings from our group instead point to p120-isoform phatase [148]. Since S879 and T916 exist in all isoforms of p120, their 1 being most responsive to canonical Wnt signals. Upon pathway examination might prove informative when considering isoform-wide activation and in a manner analogous to β-catenin, p120-isoform 1 is effects. stabilized at the protein level. The model is that isoform 1 then enters In another context, the dephosphorylation of p120 takes place via the nucleus to relieve (activate) Kaiso repressed gene targets [49,50]. RPTPμ, which acts upon tyrosines and is needed for proper p120 lo- Intriguingly, recent work on the whole genome level has indicated calization, as linked to adipocyte differentiation [67].Thus,inre- that Kaiso is largely associated with transcriptional activation as sponse to largely uncharacterized upstream signals, kinases and opposed to repression [14]; if this proves the case, it will be important phosphatases produce effects upon p120 that relate to its cellular lo- to establish if p120 additionally has a role in this context. As part of calization and protein associations, and thereby to larger pathway earlier mentioned work, p120 phosphorylation at Y228 is predictive of functions. While most of the current work has taken place in cell glioblastoma multiforme (GBM) invasiveness [55], and is also elevated lines, it points to experiments that must now be undertaken in de- in invasive renal and breast tumors [71].Further,T916phosphorylation velopmental models. With respect to the focus of this review, is high in these tumors; and while the functional role of phospho-T916 which examines phospho-residues that have isoform-selective ef- was not resolved, it was found to obstruct the epitope of the widely fects, the developmental impact of a kinase or phosphatase will par- employed pp120 monoclonal antibody. Thus, many past studies are likely tially depend on the particular p120-isoforms being expressed at to have underestimated p120 levels in tumors. Further, tyrosine phos- that time in a tissue. phatases such as PTP-PEST promote Y335's dephosphorylation and the 108 ..Hn ta./Bohmc tBohsc ca16 21)102 (2016) 1863 Acta Biophysica et Biochimica / al. et Hong J.Y.

Table 1 Phenotypes related to p120-catenin phosphorylation. Summary of observed phenotypes that relate to changes at a particular residue (column 5); or changes to the kinase or phosphatase acting upon p120-catenin in the context of that residue(s) (column 3). Indicated are the residue or residues involved (column 1), the kinase or phosphatase involved (column 2), the signaling pathway implicated or proposed (column 4), and the p120-isoforms that are potentially affected. References are included (column 7; not comprehensive), with additional references present within the review text.

Serine/Threonine Serine/Threonine If undertaken, phenotype related to the kinase or Signaling pathway(s) Phenotype related to the phosphorylation site(s) P120 References residue kinase phosphatase, such as overexpression or depletion isoform(s) containing phos. Site

S6, S8, S11, S15 CK1α & GKS3β Gastrulation defects resulting from p120 Wnt 4S- N 4A p120 point mutant is stabilized, w/ 1 Hong...McCrea, J Cell Sci, 2010 overexpression are partially rescued via GSK3β greater signaling capacity. overexpression. T47 Dyrk1A Dyrk1A overexpression produces gastrulation defects Wnt P120 phospho-mimic point mutant is more 1 Hong...McCrea, J Cell Sci, 2012 in Xenopus embryos, as well as facilitates β-catenin stable, w/ enhanced signaling capacity. induced duplicate axis formation. S252, T310 GSK3β N/A N/A N/A 1, 2, 3 Xia...Reynolds, Biochemistry, – 2003 114 S268, S288, T310, T916 PKC activation via PMA N/A Serum starvation, LPA, Cadherin-p120 association was not altered by 1, 2, 3 Xia...Reynolds, Biochemistry, results in the dephos. thrombin, PMA, VEGF mutation of these phsopho-sites. 2003 Xia...Reynolds, Exp Cell of these four residues produces p120 dephos. via Phosphorylation of p120 required its membrane Res, 2006 Wong...Staddon, effects upon PKC. assn. Biochem J, 2000 S268 PKCε PKCε overexpression leads to S268 phos., overcomes N/A PKCε phenotype correlated with S268 1, 2, 3 Dann...Klippel, Oncogene, contact inhibition and results in cellular foci phosphorylation, but mutant not tested. 2014 formation. PKCε depletion reverts Ras-induced mesenchymal morphology. S268, S269 CK1α N/A Release of p120 from cadherin Phospho-deficient point mutant S268A, S269A 1, 2, 3 Del Valle-Perez...Dunach, Mol and LRP5/6, Wnt pathway. failed to rescue gastrulation defect induced via Cell Biol, 2011 Vinyoles, Mol p120 depletion. Cell, 2014 Valls...Dunach, J Cell Sci, 2012 S288 PKC S288 phosphorylation is high In lung cancer cell lines, P120-isoform3 recruits Kaiso Cell invasion is regulated by S288 in lung cancer 3 Zhang...Wang, Int J Oncol, 2011 primary cells and tissues. to the cytoplasm. cell lines (S288E mutant was utilized). S252, T310 GSK3β (in vitro) N/A N/A N/A 1, 2, 3 Xia...Reynolds, Biochemistry, 2003 S879 PKCα PKCα induced p120 dissociation from VE-cadherin Thrombin/Par-1 and P120 point mutants (S879A or S879D) indicate 1, 2, 3, 4 Vandenbroucke St. and AJ disassembly. LPS-induced lung vascular direct effects (2012), Amant...Malik, Cir Res, 2012 PKCα depletion reduces thrombin-induced decreases permeability (2012) Correlated to the PDGF signaling (2009). Brown...Reynolds, Exp Cell Res, in transendothelial electric resistance. PDGF (2009) 2009 T916 N/A N/A N/A T916 phosphorylation is high in renal cell 1, 2, 3, 4 Kourtidis...Anastasiadis, PLoS carcinoma, but T916 is not required in One, 2015 transforming ability Tyrosine residue Tyrosine kinase Y112 Fyn Overexpression in NIH3T3 cells reduces stress fiber Phosphorylation lowers p120 In contrast to wild-type p120, the Y112E mutant 1, 2, 3 Castano...Dunach, Mol Cell formation. assn. w/ RhoA, Rac and Vav2, has little effect upon stress fibers. Biol, 2007 in preference to E-cadherin binding. Y112, Y217 Src/Fyn N/A Wnt pathway, p120 release P120 Y112E and Y217E phosphomimic mutants 1, 2, 3 Valls...Dunach, J Cell Sci, 2012 from cadherin and LRP5/6 to have reduced assn. w/ Rac1 and Vav2. Mutant enable assn. with Vav2 and failed to rescue gastrulation delay upon p120 Rac1. depletion (Y112E). Y217 Src v-Src overexpression reduces E-cadherin mediated v-Src mediated signaling P120 Y217F mutant increases cell aggregation. 1,2,3 Ozawa...Ohkubo, J Cell Sci, cell aggregation. 2001 Y217, Y228 Src N/A Src and cadherin-related Src phos. of p120 Y217 and Y228 promotes p120 1, 2, 3 Castano...Dunach, Mol Cell pathways assn. w/ and inhib. RhoA. Biol, 2007 Y228 N/A Certain carcinoma cell lines (e.g. HCT116, HT29 and EGFR activation induces p120 Mutation of Y228 and 7 other tyrosine residues 1, 2, 3 Mariner...Reynolds,J Cell Sci, A431) show high levels of Y228 phosphorylation. Y228 phosphorylation. in p120 did not prevent junction formation or 2004 compaction of cultured cells. ..Hn ta./Bohmc tBohsc ca16 21)102 (2016) 1863 Acta Biophysica et Biochimica / al. et Hong J.Y. Y228 Src Activation of Src family kinase is common in VEGF/Src family kinases. Y228 phosphorylation is high in highly or 1, 2, 3 Huveldt...Anastasiadis, PLoS Glioblastoma multiforme (GBM). moderately invasive GBM. One, 2013 Y228 N/A P120 Y228 phosphorylation is high in renal cell Src family kinases. Mutation of p120 simultaneously at 8 tyrosine 1, 2, 3 Kourtidis...Anastasiadis, PLoS carcinoma and breast cancer cells. sites (including Y228) reduces tumorigenic One, 2015 potential. Tyrosine residue Phosphatase Y296 SHP-1 N/A EGF/Src N/A 1, 2, 3 Frank...Bohmer, J Biol Chem, 2004 Y335 PTP-PEST Silencing PTP-PEST reduces p120-E-cadherin PTP-PEST is essential for P120 point mutant Y335F fails to associate with 1, 2, 3, 4 Espejo...Sastry, J Cell Sci, 2014 association and increases p120's cytoplasmic pool. junction assembly via effects Vav2, activate Rac1 or induce epithelial Espejo...Sastry, Am J Physiol, Ectopic expression of PTP-PEST suppresses cell upon p120 and small-GTPases. migration (HCT116). 2010 migration in colon carcinoma cells. Stable knock-down PTP-PEST reduces junction integrity, enhances Rac1 and reduces RhoA activity, and generates a mesenchymal-like phenotype in colon carcinoma cells. – 114 109 110 J.Y. Hong et al. / Biochimica et Biophysica Acta 1863 (2016) 102–114 cytosolic localization of p120 in colon carcinoma cells [38].Recently,it Further work with cell lines as well as lung and breast tumor samples was determined via deep sequencing that the absence of p120's splice re- has indicated that E-cadherin is a key determinant of p120-catenin local- gion B correlates with some colorectal cancers [146]. Potential phospho- ization and activity [29,127,161]. In epithelial cells lacking E-cadherin, residues do not exist in splice region B, although sites present nearby p120 is often present in the cytoplasm where in common with p120 (S879, T916) may conceivably respond to the presence/absence of splice still at junctions [69,71,144], it interacts with small-GTPases modulating region B, and be relevant to tumorigenesis. Taken together, the phosphor- the cytoskeleton [5,11,103,107,122,125,161] (reviewed in [70,121]), or ylation status of p120-catenin is likely to prove relevant to cancer pro- reaches the nucleus to engage in gene control [30,31,49,59,77,114,140, gression, with a number of the involved residues being isoform specific. 160]. Acting through small-GTPases such as RhoA and Rac1 in EMT or P120 may participate in epithelial-to-mesenchymal transitions cancer, p120-isoform 1 might be predominant in promoting motility (EMTs) in tumor progression, with for example, a switch from short- and invasiveness [154]. Since certain phospho-residues exist only to-long p120-isoforms occurring in response to factors including in p120-isoform 1 (S6, S8, S11, S15, T47), and as noted modulate p120 Zeppo1 (ZNF703) [125]. In some tumor cells lacking E-cadherin, and stability in response to Wnt-signals or the kinase Dyrk1A, they may in cells over-expressing Src, p120-catenin facilitates cell invasion and prove worthy of further examination in the context of tumor formation anchorage-independent growth via effects upon Rho/ROCK signaling. or metastasis. Studies of breast, colon and lung cancer cells indicate that heightened levels of cytosolic p120 contribute to the invasiveness of tumors 6. Summary deficient for E-cadherin [11,22,123,161]. For example, cytosolic p120- catenin facilitates anchorage-independent tumor growth and metasta- In summary, the collective evidence is that isoform and phosphory- sis in invasive lobular carcinoma cells via affects upon RhoA [122]. lation use in p120-catenin is relevant to cell behaviors in development, Considering that p120's phosphorylation status is responsive to being homeostasis and pathology. Among others, upstream inputs include the localized at the plasma membrane via cadherin, and its release from presence versus absence of cadherins, growth factor signaling and cadherin is further responsive to phosphorylation, p120's cadherin- possibly canonical Wnt signals. Future study of p120 biology will need and phospho-modulated movements to the cytoplasm potentially to focus more on factors and mechanisms that determine isoform contribute to the EMT process. Thus, changes in p120's intracellular expression or retention, localization and activity, and the relevance of phosphorylation, localization and isoform use in EMT are proposed to phosphorylation as well as additional covalent modifications in modu- facilitate cancer progression (reviewed in [70,88,121]). lating p120's functional roles. Based upon a recent report showing

Fig. 3. Isoform-specific mechanisms of p120-catenin. Model of isoform-specific roles of p120-catenin. A. Canonical Wnt signals, initiated by Wnt-ligand binding to the LRP5/6 and Frizzled receptors, act to prevent destruction of p120-isoform 1, such that it is more stable and therefore active, as in the de-repression of the transcriptional repressor Kaiso depicted in (C) [49]. Canonical Wnt-signals have a lesser protective effects upon shorter p120-isoforms, since the shorter isoforms 2–4 lack the destruction box found only in isoform 1. That is, in the absence of canonical Wnt signals, (e.g. lack of Wnt-ligand binding to Frizzled/LRP5/6 co-receptors), p120-isoform 1 is acted upon by the destruction complex (containing components such as Axin, APC, GSK3beta and CK1alpha), and delivered to the proteasome for elimination (not shown). B. Switches in p120-catenin isoforms have been observed upon ectopic expression of Snail or Zeppo1, suggesting the potential role of p120-isoforms during epithelial-mesenchymal transition (Fig. 3B) [99,125]. In addition, epithelial cell type specific regulators ESRP1 and ESRP2 modulate p120's splicing events [142]. Since p120-isoform 1 more effectively inhibits RhoA than isoform 3, consequent effects arise that relate to cytoskeletal modulation, and thereby cell process-extension, motility or invasion. C. P120-catenin binds and displaces the Kaiso repressor from its consensus binding sites, leading to gene activation [49,50,160].Forsimplicity, beta-catenin and alpha-catenin (etc.) are not depicted, and N-cadherin is shown without associated catenins. J.Y. Hong et al. / Biochimica et Biophysica Acta 1863 (2016) 102–114 111 that the pp120 antibody, which is widely used in p120 biology, does not [12] C. Bertocchi, M. Vaman Rao, R. 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