Table of contents

Protein interactions of Armadillo homologs: Ubiquitin mediated degradation of ß-catenin

ß-catenin is a central component of the cadherin cell adhesion complex and plays an essential role in the Wingless/Wnt signaling pathway. In the current model of this pathway, the amount of ß-catenin (or its invertebrate homolog Armadillo) is tightly regulated and its steady-state level outside the cadherin-catenin complex is low in the absence of Wingless/Wnt signal. The ubiquitin-dependent proteolysis system is involved in the regulation of ß-catenin turnover. ß-catenin, but not E-cadherin, p120(cas) or alpha-catenin, becomes stabilized when proteasome-mediated proteolysis is inhibited and this leads to the accumulation of multi-ubiquitinated forms of ß-catenin. Ubiquitination is inhibited and the protein stabilized with the substitution of the serine residues in the glycogen synthase kinase 3ß (GSK3ß - Drosophila homolog: Shaggy ) phosphorylation consensus motif of ß-catenin. This motif in ß-catenin resembles a motif in IkappaB (inhibitor of NFkappaB) that is required for the phosphorylation-dependent degradation of IkappaB via the ubiquitin-proteasome pathway. Ubiquitination of ß-catenin is greatly reduced in Wnt-expressing cells, providing the first evidence that the ubiquitin-proteasome degradation pathway may act downstream of GSK3ß in the regulation of ß-catenin (Aberle, 1997).

Ubiquitin-mediated proteolysis has a central role in controlling the intracellular levels of several important regulatory molecules, such as cyclins, CKIs, p53, and IkappaBalpha. Many diverse proinflammatory signals lead to the specific phosphorylation and subsequent ubiquitin-mediated destruction of the NF-kappaB inhibitor protein IkappaBalpha. Substrate specificity in ubiquitination reactions is, in large part, mediated by the specific association of the E3-ubiquitin ligases with their substrates. One class of E3 ligases is defined by the recently described SCF complexes, the archetype of which was first described in budding yeast and contains Skp1, Cdc53, and the F-box protein Cdc4. These complexes recognize their substrates through modular F-box proteins in a phosphorylation-dependent manner. A biochemical dissection is described of a novel mammalian SCF complex, SCFbeta-TRCP, that specifically recognizes a 19-amino-acid destruction motif in IkappaBalpha (residues 21-41) in a phosphorylation-dependent manner. This SCF complex also recognizes a conserved destruction motif in beta-catenin, a protein with levels also regulated by phosphorylation-dependent ubiquitination. Endogenous IkappaBalpha-ubiquitin ligase activity cofractionates with SCFbeta-TRCP. Furthermore, recombinant SCFbeta-TRCP assembled in mammalian cells contains phospho-IkappaBalpha-specific ubiquitin ligase activity. These results suggest that an SCFbeta-TRCP complex functions in multiple transcriptional programs by activating the NF-kappaB pathway and inhibiting the beta-catenin pathway (Winston, 1999).

beta-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of beta-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. beta-catenin levels are regulated by the ubiquitin-dependent proteolysis system and beta-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3beta (GSK-3beta)/Axin kinase complex. FWD1 (the mouse homologue of Slimb/betaTrCP: see Drosophila supernumerary limbs), an F-box/WD40-repeat protein, specifically forms a multi-molecular complex with beta-catenin, Axin, GSK-3beta and APC. Mutations at the signal-induced phosphorylation site of beta-catenin inhibit beta-catenin association with FWD1. FWD1 facilitates ubiquitination and promotes degradation of beta-catenin, resulting in reduced cytoplasmic beta-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibits the ubiquitination process and stabilizes beta-catenin. These results suggest that the Skp1/Cullin/F-box protein FWD1 (SCFFWD1)-ubiquitin ligase complex is involved in beta-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated beta-catenin. FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of beta-catenin in response to external signals. SCFFWD1 may be critical for tumor development and suppression through regulation of beta-catenin protein stability (Kitagawa, 1999).

Regulation of beta-catenin stability is essential for Wnt signal transduction during development and tumorigenesis. It is well known that serine-phosphorylation of beta-catenin by the Axin-glycogen synthase kinase (GSK)-3beta complex targets beta-catenin for ubiquitination-degradation, and mutations at critical phosphoserine residues stabilize beta-catenin and cause human cancers. How beta-catenin phosphorylation results in its degradation is undefined. Phosphorylated beta-catenin is shown to be specifically recognized by beta-Trcp, an F-box/WD40-repeat protein that also associates with Skp1, an essential component of the ubiquitination apparatus. beta-catenin harboring mutations at the critical phosphoserine residues escapes recognition by beta-Trcp, thus providing a molecular explanation for why these mutations cause beta-catenin accumulation that leads to cancer. Inhibition of endogenous beta-Trcp function by a dominant negative mutant stabilizes beta-catenin, activates Wnt/beta-catenin signaling, and induces axis formation in Xenopus embryos. Therefore, beta-Trcp plays a central role in recruiting phosphorylated beta-catenin for degradation and in dorsoventral patterning of the Xenopus embryo (Liu, 1999).

In the ubiquitin-proteasome pathway, the ubiquitinated substrates either undergo degradation by the proteasome or stabilization through the action of the deubiquitinating enzyme. The deubiquitinating enzyme Fam (Drosophila homolog: Fat facets) is colocalizes with AF-6, one of the effectors of the Ras small GTPase, at cell-cell contact sites in epithelial cells and interacts with AF-6 in vivo and in vitro. Fam has deubiquitinating activity in vitro and prevents the ubiquitination of AF-6 in intact cells. The degradation of beta-catenin, which accumulates at the cell-cell contact sites as a cadherin/catenin complex, is thought to be regulated by the ubiquitin-proteasome pathway. These observations prompted an examination of the possible Fam regulation of the stabilization of beta-catenin. It was found that Fam interacts with beta-catenin both in vivo and in vitro. The Fam-binding site of beta-catenin maps to the region close to the APC or Axin-binding site of beta-catenin. Over-expression of Fam in mouse L cells results in an elevation of beta-catenin levels and in an elongation of the half-life of beta-catenin. In these L cells, Fam is colocalized with beta-catenin at the dot-like structures in the cytoplasm. These results indicate that Fam interacts with and stabilizes beta-catenin in vivo, presumably through the deubiquitination of beta-catenin (Taya, 1999).

The signaling activity of beta-catenin is thought to be regulated by phosphorylation of a cluster of N-terminal serines, putative sites for GSK3beta. In the prevailing model in the literature, GSK3beta-dependent phosphorylation of these sites targets beta-catenin for ubiquitin-mediated degradation. Wnt signaling inhibits GSK3beta activity and this blocks degradation, allowing beta-catenin to accumulate and signal. beta-Catenin activity is shown not to be regulated solely by protein stability. Mutations in the putative GSK3beta phosphorylation sites of beta-catenin enhance its signaling activity, but this cannot be accounted for by accumulation of either total or cadherin-free protein. Instead, the mutant protein has a threefold higher specific activity than the wild type both in vivo and in an in vitro signaling assay. It is concluded that the N-terminal serines convey a layer of regulation upon beta-catenin signaling in addition to the effects these sites exert upon protein stability (Guger, 2000).

The evidence suggests that the N-terminus of beta-catenin can regulate the specific activity of the protein in addition to its stability. The molecular nature of this additional form of regulation remains unknown. It is speculated that the putative GSK3beta sites may directly control import and/or export of beta-catenin to/from the nucleus, or that they regulate beta-catenin's transcriptional activity once it is inside the nucleus. In many previous studies, nuclear localization of beta-catenin often correlates with an increase in the amount of cytosolic beta-catenin protein. Hence it maybe assumed that the import of beta-catenin into the nucleus is regulated by the amount of available (cadherin-free) beta-catenin. However, the import of beta-catenin into the nucleus occurs by a novel pathway and can be inhibited by factors in the cytosol without affecting stability of the beta-catenin protein. Additionally, GSK3beta has been demonstrated to regulate the nuclear localization of NF-ATc and both the nuclear localization and the stability of cyclin D1. With regard to cyclin D1, the effect on nuclear localization appears to be an early regulatory step and destabilization of the protein happens later in the cell cycle. A similar biphasic form of regulation might occur during beta-catenin signaling and could be a way of allowing a quick response to a signal followed by long-term 'memory' of the response (Guger, 2000).

The adenomatous polyposis coli (APC) tumor-suppressor protein, together with Axin and GSK3, forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of ß-catenin by the proteasome. Siah-1, the human homolog of Drosophila Seven in absentia, is a p53-inducible mediator of cell cycle arrest, tumor suppression, and apoptosis. Siah-1 interacts with the carboxyl terminus of APC and promotes degradation of ß-catenin in mammalian cells. The ability of Siahß-1 to downregulate ß-catenin signaling was also demonstrated by hypodorsalization of Xenopus embryos. Unexpectedly, degradation of ß-catenin by Siah-1 is independent of GSK3ß-mediated phosphorylation and does not require the F box protein ß-TrCP. These results indicate that APC and Siahß-1 mediate a novel ß-catenin degradation pathway linking p53 activation to cell cycle control (Liu, 2001).

Destruction of ß-catenin is regulated through phosphorylation-dependent interactions with the F box protein ß-TrCP. A novel pathway for ß-catenin degradation was discovered involving mammalian homologs of Drosophila Sina (Siah), which bind ubiquitin ß-conjugating enzymes, and Ebi, an F box protein that binds ß-catenin independent of the phosphorylation sites recognized by ß-TrCP. A series of protein interactions were identified in which Siah is physically linked to Ebi by association with a novel Sgt1 homolog SIP that binds Skp1, a central component of Skp1-Cullin-F box complexes. Expression of Siah is induced by p53, revealing a way of linking genotoxic injury to destruction of ß-catenin, thus reducing activity of Tcf/LEF transcription factors and contributing to cell cycle arrest (Matsuzawa, 2001).

A pathway linking the RING protein Siah-1 to the F box protein Ebi has been mapped and it has been shown that Ebi can bind ß-catenin. Unlike ß-TrCP, however, which requires GSK3ß-mediated phosphorylation of ß-catenin on serine 33 and serine 37, Ebi interacts with ß-catenin independently of these phosphorylation sites. Also, the Siah binding protein SIP associates with complexes containing Ebi but not ß-TrCP, suggesting differences compared to previously characterized E3 ubiquitin ligase complexes, where E2 enzymes are supplied via Cullin-mediated interactions with RING-containing proteins such as Rbx-1/Roc-1. Recent identification of interactions between Siah-1 and the ß-catenin binding protein APC suggest that this scaffold protein represents a point of common intersection of the Wnt and Siah-1 pathways for ß-catenin degradation (Matsuzawa, 2001).

Two alternative pathways for regulation of ß-catenin levels are presented, involving different F box proteins (Ebi versus ß-TrCP). One pathway is initiated by increases in the expression of Siah-family proteins, which can be induced, for example, by p53 in response to DNA damage, and involves sequential protein interactions with SIP, Skp1, and Ebi. Ebi binds ß-catenin, thus recruiting it to the Siah-1-SIP-Skp1 complex for polyubiquitination and subsequent proteosome-mediated degradation. Siah-1 binds the E2 UbcH5. The other pathway is regulated by Wnt signals (Dsh) and possibly PI3K/Akt. This pathway is phosphorylation dependent and involves GSK3ß-induced phosphorylation of Ser-33 and Ser-37 on ß-catenin, allowing ß-TrCP binding, resulting in recruitment of ß-catenin to Skp1-Cullin-1- ß-TrCP complexes (SCF). Cullin-1, in collaboration with other proteins, supplies this SCF complex with E2s, such as UbcH3. APC is required for both pathways as a scaffold protein, binding ß-catenin via one domain and also binding Siah-1 and GSK3ß (Matsuzawa, 2001).

In the fly, Sina recruits E2s to Phyllopod/Tramtrack complexes, targeting Tramtrack for ubiquitination. The ebi-gene product also binds Tramtrack and promotes its degradation in vitro and when expressed in insect cells in culture. Loss-of-function mutations of ebi cause Tramtrack accumulation and prevent R7 cell differentiation. Similar to ß-TrCP, the ebi gene of Drosophila encodes an F box/WD-40-repeat protein with sequence homology to Cdc4 (yeast), Sel-10 (C. elegans), and Slimb (Drosophila), suggesting that it provides a functional connection between a Sina-regulated pathway and SCF complexes. How this linkage between Sina and SCF complexes is achieved, however, has been unclear (Matsuzawa, 2001).

The finding that SIP functions as a molecular bridge between the human homologs of Sina and the SCF-component Skp1 provides evidence of a physical linkage between components of these two ubiquitin ligase systems, thus corroborating the genetic evidence from Drosophila that these two pathways for targeted protein degradation interact. The Drosophila ortholog of SIP is also capable of bridging the fly Skp1 and Sina proteins in three-hybrid experiments. Thus, an evolutionarily conserved network of protein interactions exists in which Siah-1 (Sina) binds to SIP, which in turn binds to Skp1, which binds Ebi (Matsuzawa, 2001).

p53 can induce expression of Siah-family genes in mammals, establishing p53 as one factor capable of invoking Siah-dependent pathways for protein degradation. Siah-family proteins are normally maintained at a relatively low level through ubiquitination-dependent protein turnover, where human Siah-1 and Siah-2 promote their own degradation through interactions of their RING domains with E2s. This therefore suggests that activation of p53 leads to a burst of Siah-1 mRNA and protein production, triggering the Siah/SIP/Skp1/Ebi pathway for ß-catenin degradation. In contrast to Siah-family proteins, it seems unlikely that SIP, Skp1, or Ebi are limiting components of this pathway, since overexpression of them has little effect on ß-catenin levels (Matsuzawa, 2001).

Though p53-mediated degradation of ß-catenin correlates with cell cycle arrest, it remains to be established whether these events are functionally linked. Activation of Tcf/LEF-family transcription factors by ß-catenin is known to induce expression of cyclin D1, c-myc, and other genes important for cell proliferation, making it plausible that ß-catenin degradation is linked to p53-mediated cell cycle arrest. However, given the role established for the cyclin-dependent kinase inhibitor p21Waf1 in mediating G1 arrest induced by p53, it is unclear whether a parallel pathway for ß-catenin degradation would be required. Circumstances have been described where p53 fails to induce cell cycle arrest despite inducing p21Waf1 expression, raising the question of whether p21Waf1 is necessary but insufficient for p53-mediated G1 arrest. Recently, a genetic interaction between ebi and p21Waf1 has been identified using an assay in Drosophila where flies are engineered to ectopically express human p21Waf1 in the developing eye disc (Boulton, 2000). Specifically, mutant alleles of ebi abrogated inhibition of S phase entry by p21Waf1, implying a need for Ebi in p21-mediated cell cycle arrest. Flies with mutant ebi also display ectopic S phases and overproliferation phenotypes (Boulton, 2000), further implying a role for ebi in growth suppression. Defects in cell cycle arrest in ebi mutants, however, do not necessarily implicate ß-catenin/Armadillo. For example, p53 can induce degradation of c-Myb through a proteosome-dependent mechanism partly mediated by Siah (Tanikawa, 2000). Thus, Ebi may have other targets in addition to ß-catenin that are relevant to mechanisms of p53-mediated cell cycle arrest. Future experiments should explore whether the fly homolog of p53 is linked to an ebi-dependent pathway for cell cycle arrest entailing degradation of Armadillo. In the M1 cell model, p53 induces both G1 arrest and apoptosis. Though Ebi(DeltaF)-expressing M1 cells may exhibit some delay in p53-induced apoptosis, this could result indirectly because of failed G1 arrest. Moreover, Siah-1 often fails to induce apoptosis when overexpressed in cells. However, links of Siah to apoptosis can occur under some circumstances, as demonstrated by the observation that coexpression of Siah-1 with a Siah binding protein Pw1/Peg3 causes apoptosis, whereas neither Siah-1 nor Pw1/Peg3 alone are sufficient. Mutations affecting components of the Wnt-signaling pathway are commonly observed in human cancers, resulting in aberrant accumulation of ß-catenin and activation of Tcf/LEF-target genes. Wnt-family ligands, frizzled-family receptors, and the signaling proteins downstream of these define one mechanism for regulating ß-catenin levels. However, additional inputs into pathways controlling ß-catenin turnover have recently been identified, including a mitogen-activated protein kinase pathway involving a Tak1 homolog and Nemo-like kinases in C. elegans and a cell adhesion-dependent pathway involving integrin-linked kinase. The findings reported here reveal yet another pathway for regulating ß-catenin levels that is linked at least in part to p53-dependent responses to genotoxic injury. It is speculated that loss of p53 or components of the Siah/SIP/Skp/Ebi pathway for ß-catenin destruction may contribute to aberrant ß-catenin accumulation in cancers (Matsuzawa, 2001).

The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the >70 human F box proteins, allowing the recognition of a wide range of substrates. The F box protein β-TrCP1 recognizes the doubly phosphorylated DpSGφXpS destruction motif, present in β-catenin and IκB, and directs the SCFβ-TrCP1 to ubiquitinate these proteins at specific lysines. The 3.0 Å structure of a β-TrCP1-Skp1-β-catenin complex reveals the basis of substrate recognition by the β-TrCP1 WD40 domain. The structure, together with the previous SCFSkp2 structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant β-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection (Wu, 2003).

The identities of the ubiquitin-ligases active during myogenesis are largely unknown. A RING-type E3 ligase complex is described that is specified by the adaptor protein, Ozz, a novel SOCS protein that is developmentally regulated and expressed exclusively in striated muscle. The encoded protein is 285 amino acids long and includes three domains: two N-terminal Neuralized homology repeats (NHR1 and NHR2) which are found in the Neuralized protein family, and a C-terminal SOCS box. In mice, the absence of Ozz results in overt maturation defects of the sarcomeric apparatus. ß-catenin as one of the target substrates of the Ozz-E3 in vivo. In the differentiating myofibers, Ozz-E3 regulates the levels of sarcolemma-associated ß-catenin by mediating its degradation via the proteasome. Expression of ß-catenin mutants that reduce the binding of Ozz to endogenous ß-catenin leads to membrane-bound ß-catenin accumulation and myofibrillogenesis defects similar to those observed in Ozz null myocytes. These findings reveal a novel mechanism of regulation of membrane-bound ß-catenin and the role of this pool of the protein in myofibrillogenesis, and implicate the Ozz-E3 ligase in the process of myofiber differentiation (Nastasi, 2004).

Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly, the Wnt signaling pathway controls HSC self-renewal and bone marrow repopulation. This study shows that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation, demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocks wnt-induced alterations in HSC formation. PGE2 modifies the wnt signaling cascade at the level of beta-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulates murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt is also conserved during regeneration of other organ systems. This work provides in vivo evidence that Wnt activation in stem cells requires PGE2, and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery (Goessling, 2009).

Protein interactions of Armadillo homologs: Interactions with Bcl9-2, a Legless homolog

ß-Catenin controls both cadherin-mediated cell adhesion and activation of Wnt target genes. The ß-catenin-binding protein BCL9-2, a homolog of the human proto-oncogene product BCL9, induces epithelial-mesenchymal transitions of nontransformed cells and increases ß-catenin-dependent transcription. RNA interference of BCL9-2 in carcinoma cells induces an epithelial phenotype and translocates ß-catenin from the nucleus to the cell membrane. The switch between ß-catenin's adhesive and transcriptional functions is modulated by phosphorylation of Tyr 142 of ß-catenin, which favors BCL9-2 binding and precludes interaction with alpha-catenin. During zebrafish embryogenesis, BCL9-2 acts in the Wnt8-signaling pathway and regulates mesoderm patterning (Brembeck, 2004).

Vertebrate BCL9-2 proteins show an overall amino acid sequence identity of 60%, and 35% identity to the human proto-oncogene product BCL9. Up to 90% sequence identity was found in seven short clusters of 20-30 amino acids, which are also conserved in Legless from Drosophila: the ß-catenin-binding domain (ß-catBD), the Pygopus (PyBD)-binding domain, a domain that contains a classical nuclear localization signal (NLS, KRRK motif), three C-terminal homology domains (C-HD1 to C-HD3), and a novel N-terminal homology domain (N-HD) that contains a lysine-rich potential sumoylation motif (K*K*KXE/D; in amino acid residues 108-137) and a sequence similar to classical nuclear localization signals (PRSKRRC; in amino acids 138-173) (Brembeck, 2004).

BCL9-2 was expressed in epithelial MDCK cells and cell clones were established that had stably incorporated the expression vector. Control cells formed round compact colonies at subconfluency, and exhibited the typical cobblestone morphology of epithelial cells. In contrast, BCL9-2 expressing colonies were flattened and irregularly shaped, and contained scattered cells. Moreover, BCL9-2 expressing colonies were completely scattered in the presence of suboptimal dosages of Hepatocyte Growth Factor (HGF), which activates the receptor tyrosine kinase Met. Control clones were not scattered at these low concentrations of HGF. BCL9-2-expressing cells also exhibited a threefold increased cell migration. These results suggest that BCL9-2 induces epithelial-mesenchymal transitions, and that tyrosine phosphorylation by Met collaborates with BCL9-2 action (Brembeck, 2004).

Epithelial-mesenchymal transitions correlated with complex formation of BCL9-2 and ß-catenin in the nucleus of the scattered cells; BCL9-2 was located in the nucleus (the nucleoli were excluded) and translocated ß-catenin to the nuclear compartment, whereas ß-catenin located at the plasma membrane, but not in the nucleus in control cells. A fragment of BCL9-2 that contains only the N-terminal domain (amino acids 1-175) also located to the nucleus. In contrast, BCL9-2 with a deletion of the nuclear localization signal of the N-terminal domain (Delta amino acids 138-173) was located in the cytoplasm. Mutations of the putative sumoylation motif in the N-terminal domain of BCL9-2 (e.g., the K110R mutation) favored accumulation in nuclear bodies. The BCL9/Legless homologs, which lack the nuclear localization signal in the N-terminal domain, did not localize to the nucleus (Brembeck, 2004).

Tyrosine phosphorylation of ß-catenin also contributed to nuclear translocation. A deletion fragment of BCL9-2 that contained only the ß-catenin-binding domain (amino acids 387-530) located to the cytoplasm. Upon HGF treatment, ß-catenin was partially released from the plasma membrane, translocated to the nucleus, where it colocalized with the BCL9-2 fragment (Brembeck, 2004).

Colon cancer cell lines were used that exhibit activated Wnt/ß-catenin signaling by mutation of the tumor suppressor gene APC. SW480 cells are fibroblast-like and show high levels of nuclear ß-catenin, and they express high levels of BCL9-2. Transfection with specific siRNAs against BCL9-2 reduced endogenous BCL9-2 (but not BCL9) RNA levels almost completely within 24 h, and abolished protein expression of transfected BCL9-2 in HEK293 cells. Remarkably, the morphology of the BCL9-2 siRNAs-treated SW480 cells became epithelial-like, and ß-catenin was translocated from the nucleus to the cell membrane. Moreover, cell migration of the treated SW480 cells was drastically reduced. BCL9-2 siRNA's treatment of DLD-1 colon cancer cells also induced loss of nuclear ß-catenin, and the cells showed reduced colony formation in soft agar (Brembeck, 2004).

BCL9-2 was initially identified as an interaction partner of tyrosine-phosphorylated ß-catenin in a yeast two-hybrid screen. BCL9-2 interacted efficiently with ß-catenin in yeast when the armadillo domains were fused to kinase-active, but not to kinase-defective Met. Armadillo repeats 1 and 2 of ß-catenin are required to bind BCL9-2, which contains only one tyrosine residue at position 142. Mutation of Y142 to alanine indeed abrogates BCL9-2 binding, whereas mutation to glutamic acid (Y142E), which can mimic tyrosine phosphorylation, increases binding efficiency (Brembeck, 2004).

The presence of a nonphosphorylated Tyr 142 in ß-catenin is required for alpha-catenin binding. The mutations Y142A and Y142E both abrogated binding to alpha-catenin. Interaction of BCL9-2 with tyrosine-phosphorylated ß-catenin was confirmed by coimmunoprecipitation from tissue culture cells. HGF treatment of cells induced tyrosine phosphorylation of ß-catenin and promoted interaction with BCL9-2, as did the Y142E mutation of ß-catenin in GST pull-down experiments. In contrast, a strongly reduced binding between ß-catenin and alpha-catenin was observed when the Y142A and Y142E mutations were present in ß-catenin (Brembeck, 2004).

HEK293 cells, which contain low levels of endogenous BCL9-2 and ß-catenin, were used for testing the transcriptional activity of BCL9-2. Full-size BCL9-2 enhanced ß-catenin-dependent transcription threefold. BCL9-2 had no effect in the absence of ß-catenin. Moreover, mutation of the conserved lysines in the potential sumoylation motif of the N-terminal domain of BCL9-2 (K108R, K110R, or K112R) activated ß-catenin-dependent transcription up to ninefold. Remarkably, expression of the BCL9-2 fragment that contains the ß-catenin-binding domain only (amino acids 387-530) and does not affect binding of ß-catenin to Lef/Tcfs abolished ß-catenin-dependent transcription. Deletion of the nuclear localization signal in the N-terminal domain of BCL9-2 (Delta amino acids 138-173) abolished transcriptional activation. Internal deletion of the previously identified Pygopus-binding domain of BCL9 proteins showed that this domain is not required in BCL9-2 to potentiate ß-catenin's transcriptional activity. BCL9/Legless stimulated transcription to a similar degree, but this activation was dependent on the Pygopus-binding domain. BCL9-2 expression increased ß-catenin-dependent transcription also in other assays, as assessed for the expression levels of the Wnt target gene conductin. BCL9-2 siRNA treatment of SW480 and DLD-1 cells significantly reduced transcriptional activity of ß-catenin. The coactivator function of BCL9-2 depended on Y142 of ß-catenin; in the presence of ß-catenin that contains a Y142A mutation, BCL9-2 had a reduced effect on transcription . alpha-Catenin blocked ß-catenin's transcriptional activity. This inhibition was overcome by cotransfected BCL9-2, but only in the presence of wild-type and not of Y142A mutant ß-catenin. Tyrosine phosphorylation of ß-catenin was forced by HGF treatment of cells and by cotransfection of the receptor tyrosine kinase trk-Met. BCL9-2 cofactor function was increased, but only in the case of wild-type, and not Y142A mutant ß-catenin (Brembeck, 2004).

In zebrafish embryos, BCL9-2 and BCL9 mRNAs are contributed maternally and are highly expressed during gastrulation. Injection of antisense BCL9-2 morpholinos (MOs) into zebrafish embryos resulted in severe defects of trunk and tail developmental. BCL9-2 MOs directed against the start codon were highly effective at minimal dosages. The mutant phenotype was observed in 80% of the cases. In contrast, different MOs against the homolog BCL9/Legless had no effect. Moreover, coinjection of BCL9-2 and BCL9 MOs did not alterate the developmental defects induced by BCL9-2 MOs alone. The loss-of-function of BCL9-2 suggested a deficit in Wnt8 signaling. At 70% epiboly of wild-type embryos, floating head was expressed in the dorsal axial mesoderm, and tbx6 in the ventro-lateral mesoderm. After injections of BCL9-2 MOs, the expression of floating head was broadened, and tbx6 expression was lost. Moreover, injection of low concentrations of MOs against BCL9-2 and Wnt8 synergized to suppress tbx6 expression in the ventro-lateral mesoderm. No change of expression of pax2.1 or otx2 in the neuroectoderm was observed. Loss of tbx6 expression induced by BCL9-2 MOs was completely rescued by mouse BCL9-2 mRNA, indicating that the MOs specifically target zebrafish BCL9-2. Injection of mouse BCL9-2 mRNA alone expanded tbx6 expression. Injection of BCL9-2 mRNA that encodes a fragment lacking the N-terminal domain did not expand tbx6 expression, nor did such injection rescue the phenotypes of BCL9-2 MOs. The epistasic relationship between Wnt8, Diversin (a negative regulator of ß-catenin signaling and BCL9-2 was examined in zebrafish embryos. Injection of Wnt8 DNA or stabilization of ß-catenin by injection of Diversin MOs lead to an expansions of tbx6 expression. This was completely blocked by coinjection of BCL9-2 MOs (Brembeck, 2004).

Epithelial-mesenchymal transitions occur during critical phases of embryonic development. Such transitions are also observed late in the progression of carcinomas and provide a possible metastatic mechanism. Several signaling systems can induce epithelial-mesenchymal transitions, such as Wnt/ß-catenin, TGFß/BMPs, or tyrosine kinases. Epithelial-mesenchymal transitions are initiated by a breakdown of the E-cadherin/ß-catenin/alpha-catenin complex at the plasma membrane and a dissociation of this adhesive complex from the cytoskeleton, which can be induced by tyrosine phosphorylation of ß-catenin. This study demonstrates that BCL9-2 forms a complex with ß-catenin that is phosphorylated at Tyr 142, which precludes interaction with alpha-catenin. Phosphorylation of Tyr 142 of ß-catenin and interaction with BCL9-2 allows location of the complex in the nucleus and increases transcription of Wnt/ß-catenin target genes. Thus, BCL9-2 appears to contribute to oncogenicity by two mechanisms: (1) interfering with cadherins, which act as tumor suppressor genes, and (2) by increased signaling of family members of the Wnt pathway, which contains many oncogenes and tumor suppressor genes (Brembeck, 2004).

During vertebrate embryogenesis, Wnt/ß-catenin signals control formation of the dorso-anterior axis and patterning of the mesoderm at early stages, and organ specification at later stages of development. In zebrafish embryos, Wnt8/ß-catenin signaling is required to pattern ventro-lateral mesoderm and to posteriorize neural ectoderm. This study has shown that BCL9-2, but not the homolog BCL9/Legless, is essential for mesoderm patterning in early zebrafish embryogenesis, and that BCL9-2 acts downstream of Wnt8/ß-catenin. However, BCL9-2 does not appear to be required for other early functions of the canonical Wnt pathway, such as formation of the dorsal organizer or posteriorization of anterior neuroectoderm. These data therefore suggest that BCL9-2 acts as a specific modulator of canonical Wnt signaling at particular developmental stages, rather than as a general component of Wnt signaling (Brembeck, 2004).

The autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) results from low levels of survival motor neuron (SMN) protein; however, it is unclear how reduced SMN promotes SMA development. This study determined that ubiquitin-dependent pathways regulate neuromuscular pathology in SMA. Using mouse models of SMA, widespread perturbations were observed in ubiquitin homeostasis, including reduced levels of ubiquitin-like modifier activating enzyme 1 (Uba1). SMN physically interacts with UBA1 in neurons, and disruption of Uba1 mRNA splicing was observed in the spinal cords of SMA mice exhibiting disease symptoms. Pharmacological or genetic suppression of UBA1 was sufficient to recapitulate an SMA-like neuromuscular pathology in zebrafish, suggesting that UBA1 directly contributes to disease pathogenesis. Dysregulation of UBA1 and subsequent ubiquitination pathways lead to beta-catenin accumulation, and pharmacological inhibition of beta-catenin robustly ameliorates neuromuscular pathology in zebrafish, Drosophila, and mouse models of SMA. UBA1-associated disruption of beta-catenin is restricted to the neuromuscular system in SMA mice; therefore, pharmacological inhibition of beta-catenin in these animals failed to prevent systemic pathology in peripheral tissues and organs, indicating fundamental molecular differences between neuromuscular and systemic SMA pathology. These data indicate that SMA-associated reduction of UBA1 contributes to neuromuscular pathogenesis through disruption of ubiquitin homeostasis and subsequent beta-catenin signaling, highlighting ubiquitin homeostasis and beta-catenin as potential therapeutic targets for SMA (Wishart, 2104).

Abnormal activation of Wnt/β-catenin-mediated transcription is associated with a variety of human cancers. This study reports that LATS2 inhibits oncogenic Wnt/β-catenin-mediated transcription by disrupting the β-catenin/BCL9 interaction. LATS2 directly interacts with β-catenin and is present on Wnt target gene promoters. Mechanistically, LATS2 inhibits the interaction between BCL9 and β-catenin and subsequent recruitment of BCL9, independent of LATS2 kinase activity. LATS2 is downregulated and inversely correlated with the levels of Wnt target genes in human colorectal cancers. Moreover, nocodazole, an antimicrotubule drug, potently induces LATS2 to suppress tumor growth in vivo by targeting β-catenin/BCL9. These results suggest that LATS2 is not only a key tumor suppressor in human cancer but may also be an important target for anticancer therapy (Li, 2013).

Protein interactions of Armadillo homologs: Reptin and WNT signaling

Pontin and Reptin (see Drosophila Reptin) are nuclear beta-catenin interaction partners that antagonistically modulate beta-catenin transcriptional activity. Hint1/PKCI, a member of the evolutionary conserved family of histidine triad proteins, was characterised as a new interaction partner of Pontin and Reptin. Pull-down assays and co-immunoprecipitation experiments show that Hint1/PKCI directly binds to Pontin and Reptin. The Hint1/PKCI-binding site was mapped to amino acids 214-295 and 218-289 in Pontin and Reptin, respectively. Conversely, Pontin and Reptin bind to the N-terminus of Hint1/PKCI. Moreover, by its interaction with Pontin and Reptin, Hint1/PKCI is associated with the LEF-1/TCF-beta-catenin transcription complex. In this context, Hint1/PKCI acts as a negative regulator of TCF-beta-catenin transcriptional activity in Wnt-transfected cells and in SW480 colon carcinoma cells as shown in reporter gene assays. Consistent with these observations, Hint1/PKCI represses expression of the endogenous target genes cyclin D1 and axin2 whereas knockdown of Hint1/PKCI by RNA interference increases their expression. Disruption of the Pontin/Reptin complex appears to mediate this modulatory effect of Hint1/PKCI on TCF-beta-catenin-mediated transcription. These data now provide a molecular mechanism to explain the tumor suppressor function of Hint1/PKCI recently suggested from the analysis of Hint1/PKCI knockout mice (Weiske, 2005).

While the biological roles of canonical Wnt/beta-catenin signaling in development and disease are well documented, understanding the molecular logic underlying the functionally distinct nuclear transcriptional programs mediating the diverse functions of beta-catenin remains a major challenge. This study reports an unexpected strategy for beta-catenin-dependent regulation of cell-lineage determination based on interactions between beta-catenin and a specific homeodomain factor, Prop1, rather than Lef/Tcfs. beta-catenin acts as a binary switch to simultaneously activate expression of the critical lineage-determining transcription factor, Pit1, and to repress the gene encoding the lineage-inhibiting transcription factor, Hesx1, acting via TLE/Reptin/HDAC1 corepressor complexes. The strategy of functionally distinct actions of a homeodomain factor in response to Wnt signaling is suggested to be prototypic of a widely used mechanism for generating diverse cell types from pluripotent precursor cells in response to common signaling pathways during organogenesis (Olson, 2006).

AP/TAZ incorporation in the beta-Catenin destruction complex orchestrates the Wnt response

The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. This study provides biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the β-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for β-TrCP recruitment to the complex and beta-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/beta-catenin signaling. In line, the beta-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression (Azzolin, 2014).

Table of contents

armadillo continued: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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