Beta-catenin

Catenin beta-1 also known as β-catenin is a is_associated_with::protein that in humans is encoded by the CTNNB1 is_associated_with::gene.

β-catenin is a dual function is_associated_with::protein, regulating the coordination of is_associated_with::cell–cell adhesion and is_associated_with::gene transcription. In humans, the CTNNB1 protein is encoded by the CTNNB1 is_associated_with::gene. In is_associated_with::Drosophila, the homologous protein is called armadillo. β-catenin is a subunit of the is_associated_with::cadherin protein complex and acts as an intracellular signal transducer in the is_associated_with::Wnt signaling pathway. It is a member of the is_associated_with::catenin protein family and homologous to is_associated_with::γ-catenin.

Mutations and overexpression of β-catenin are associated with many cancers, including is_associated_with::hepatocellular carcinoma, colorectal carcinoma, is_associated_with::lung cancer, malignant breast tumors, ovarian and is_associated_with::endometrial cancer. β-catenin is regulated and destroyed by the beta-catenin destruction complex, and in particular by the is_associated_with::adenomatous polyposis coli (APC) protein, encoded by the tumour-suppressing is_associated_with::APC gene. Therefore is_associated_with::genetic mutation of the APC gene is also strongly linked to cancers, and in particular is_associated_with::colorectal cancer resulting from is_associated_with::familial adenomatous polyposis (FAP).

Discovery
Beta-catenin was initially discovered in the early 1990s as a component of a mammalian is_associated_with::cell adhesion complex: a protein responsible for cytoplasmatic anchoring of is_associated_with::cadherins. But very soon, it was realized that the Drosophila protein armadillo - implicated in mediating the morphogenic effects of Wingless/Wnt - is homologous to the mammalian β-catenin, not just in structure but also in function. Thus beta-catenin became one of the very first examples of moonlighting: a protein performing more than one radically different cellular function.

Protein structure
The core of beta-catenin consists of several very characteristic repeats, each approximately 40 amino acids long. Termed armadillo repeats, all these elements fold together into a single, rigid is_associated_with::protein domain with an elongated shape - called armadillo (ARM) domain. An average armadillo repeat is composed of three is_associated_with::alpha helixes. The first repeat of β-catenin (near the N-terminus) is slightly different from the others - as it has an elongated helix with a kink, formed by the fusion of helices 1 and 2. Due to the complex shape of individual repeats, the whole ARM domain is not a straight rod: it possesses a slight curvature, so that an outer (convex) and an inner (concave) surface is formed. This inner surface serves as a ligand-binding site for the various interaction partners of the ARM domains.



The segments N-terminal and far C-terminal to the ARM domain do not adopt any structure in solution by themselves. Yet these intrinsically disordered regions play a crucial role in beta-catenin function. The N-terminal disordered region contains a conserved is_associated_with::short linear motif responsible for binding of TrCP1 (also known as β-TrCP) E3 ubiquitin ligase - but only when it is phosphorylated. Degradation of β-catenin is thus mediated by this N-terminal segment. The C-terminal region, on the other hand, is a strong transactivator when recruited onto is_associated_with::DNA. This segment is not fully disordered: part of the C-terminal extension forms a stable helix that packs against the ARM domain, but may also engage separate binding partners. This small structural element (HelixC) caps the C-terminal end of the ARM domain, shielding its hydrophobic residues. HelixC is not necessary for beta-catenin to function in cell-cell adhesion. On the other hand, it is required for Wnt signaling: possibly to recruit various coactivators. Yet its exact partners among the general transcription complexes are still unknown. Notably, the C-terminal segment of β-catenin can mimic the effects of the entire Wnt pathway if artificially fused to the DNA binding domain of LEF1 transcription factor.

is_associated_with::Plakoglobin (also called gamma-catenin) has a strikingly similar architecture to that of beta-catenin. Not only their ARM domains resemble each other in both architecture and ligand binding capacity, but the N-terminal β-TrCP-binding motif is also conserved in plakoglobin, implying common ancestry and shared regulation with β-catenin. However, plakoglobin is a very weak transactivator when bound to DNA - this is probably caused by the divergence of their C-terminal sequences (plakoglobin appears to lack the transactivator motifs, and thus inhibits the Wnt pathway target genes instead of activating them).

Partners binding to the armadillo domain


As sketched above, the ARM domain of beta-catenin acts as a platform to which specific linear motifs may bind. Located in structurally diverse partners, the β-catenin binding motifs are typically disordered on their own, and typically adopt a rigid structure upon ARM domain engagement - as seen for is_associated_with::short linear motifs. However, β-catenin interacting motifs also have a number of peculiar characteristics. First, they might reach or even surpass the length of 30 is_associated_with::amino acids in length, and contact the ARM domain on an excessively large surface area. Another unusual feature of these motifs is their frequently high degree of phosphorylation. Such Ser/Thr phosphorylation events greatly enhance the binding of many β-catenin associating motifs to the ARM domain.

The structure of beta-catenin in complex with the catenin binding domain of the transcriptional transactivation partner TCF provided the initial structural roadmap of how many binding partners of beta-catenin may form interactions. This structure demonstrated how the otherwise disordered N-terminus of TCF adapted what appeared to be a rigid conformation, with the binding motif spanning many beta-catenin repeats. Relatively strong charged interaction "hot spots" were defined (predicted, and later verified, to be conserved for the beta-catenin/E-cadherin interaction), as well as hydrophobic regions deemed important in the overall mode of binding and as potential therapeutic small molecule inhibitor targets against certain cancer forms. Furthermore, following studies demonstrated another peculiar characteristic, plasticity in the binding of the TCF N-terminus to beta-catenin.

Similarly, we find the familiar E-cadherin, whose cytoplasmatic tail contacts the ARM domain in the same canonical fashion. The is_associated_with::scaffold protein axin (two closely related paralogs, axin 1 and axin 2) contains a similar interaction motif on its long, disordered middle segment. Although one molecule of axin only contains a single β-catenin recruitment motif, its partner the is_associated_with::Adenomatous Polyposis Coli (APC) protein contains 11 such motifs in tandem arrangement per protomer, thus capable to interact with several β-catenin molecules at once. Since the surface of the ARM domain can typically accommodate only one peptide motif at any given time, all these proteins compete for the same cellular pool of β-catenin molecules. This competition is the key to understand how the is_associated_with::Wnt signaling pathway works.

However, this "main" binding site on the ARM domain β-catenin is by no means the only one. The first helices of the ARM domain form an additional, special protein-protein interaction pocket: This can accommodate a helix-forming linear motif found in the coactivator is_associated_with::BCL9 (or the closely related is_associated_with::BCL9L) - an important protein involved in Wnt signaling. Although the precise details are much less clear, it appears that the same site is used by alpha-catenin when beta-catenin is localized to the adherens junctions. Because this pocket is distinct from the ARM domain's "main" binding site, there is no competition between alpha-catenin and E-cadherin or between TCF1 and BCL9, respectively. On the other hand, BCL9 and BCL9L must compete with α-catenin to access β-catenin molecules.

Regulation of degradation through phosphorylation
The cellular level of beta-catenin is mostly controlled by its is_associated_with::ubiquitination and proteosomal degradation. The E3 ubiquitin ligase TrCP1 (also known as β-TrCP) can recognize β-catenin as its substrate through a is_associated_with::short linear motif on the disordered N-terminus. However, this motif (Asp-Ser-Gly-Ile-His-Ser) of β-catenin needs to be phosphorylated on the two is_associated_with::serines in order to be capable to bind β-TrCP. Phosphorylation of the motif is performed by Glycogen Synthase Kinase 3 alpha and beta (GSK3α and GSK3β). GSK3s are constitutively active enzymes implicated in several important regulatory processes. There is one requirement, though: substrates of GSK3 need to be pre-phosphorylated four amino acids downstream (C-terminally) of the actual target site. Thus it also requires a "priming kinase" for its activities. In the case of beta-catenin, the most important priming kinase is Casein Kinase I (CKI). Once a serin-threonine rich substrate has been "primed", GSK3 can "walk" across it from C-terminal to N-terminal direction, phosphorylating every 4th is_associated_with::serine or is_associated_with::threonine residues in a row. This process will result in dual phosphorylation of the aforementioned β-TrCP recognition motif as well.

The beta-catenin destruction complex
For GSK3 to be a highly effective kinase on a substrate, pre-phosphorylation is not enough. There is one additional requirement: Similar to the is_associated_with::mitogen-activated protein kinases (MAPKs), substrates need to associate with this enzyme through high-affinity docking motifs. Beta-catenin contains no such motifs, but a special protein does: axin. What is more, its GSK3 docking motif is directly adjacent to a β-catenin binding motif. This way, axin acts as a true is_associated_with::scaffold protein, bringing an enzyme (GSK3) together with its substrate (β-catenin) into close physical proximity.



But even axin does not act alone. Through its N-terminal regulator of G-protein signaling (RGS) domain, it recruits the is_associated_with::adenomatous polyposis coli (APC) protein. APC is like a huge "Christmas tree": with a multitude of β-catenin binding motifs (one APC molecule alone possesses 11 such motifs ), it may collect as many β-catenin molecules as possible. APC can interact with multiple axin molecules at the same time as it has three SAMP motifs (Ser-Ala-Met-Pro) to bind the RGS domains found in axin. In addition, axin also has the potential to oligomerize through its C-terminal DIX domain. The result is a huge, multimeric protein assembly dedicated to β-catenin phosphorylation. This complex is usually called the beta-catenin destruction complex, although it is distinct from the is_associated_with::proteosome machinery actually responsible for β-catenin degradation. It only marks β-catenin molecules for subsequent destruction.

Wnt signaling and the regulation of destruction
In resting cells, axin molecules oligomerize with each other through their C-terminal DIX domains, which have two binding interfaces. Thus they can build linear oligomers or even polymers inside the cytoplasm of cells. DIX domains are unique: the only other protein known to have a DIX domain is is_associated_with::Dishevelled. (The single Dsh protein of is_associated_with::Drosophila corresponds to three paralogous genes, Dvl1, Dvl2 and Dvl3 in is_associated_with::mammals.) Dsh associates with the cytoplasmatic regions of is_associated_with::Frizzled receptors with its PDZ and DEP domains. When a is_associated_with::Wnt molecule binds to Frizzled, it induces a poorly known cascade of events, that result in the exposure of dishevelled's DIX domain and the creation of a perfect binding site for axin. Axin is then titrated away from its oligomeric assemblies - the β-catenin destruction complex - by Dsh. Once bound to the receptor complex, axin will be rendered incompetent for β-catenin binding and GSK3 activity. Importantly, the cytoplasmatic segments of the Frizzled-associated is_associated_with::LRP5 and is_associated_with::LRP6 proteins contain GSK3 pseudo-substrate sequences (Pro-Pro-Pro-Ser-Pro-x-Ser), appropriately "primed" (pre-phosphorylated) by CKI, as if it were a true substrate of GSK3. These false target sites greatly inhibit GSK3 activity in a comptetitive manner. This way receptor-bound axin will abolish mediating the phosphorylation of β-catenin. Since beta-catenin is no longer marked for destruction, but continues to be produced, its concentration will increase. Once β-catenin levels rise high enough to saturate all binding sites in the cytoplasm, it will also translocate into the nucleus. Upon engaging the transcription factors LEF1, is_associated_with::TCF1, is_associated_with::TCF2 or is_associated_with::TCF3, β-catenin forces them to disengage their previous partners: Groucho proteins. Unlike Groucho, that recruit transcriptional repressors (e.g. histone-lysine methyltransferases), beta-catenin will bind transcriptional activators, switching on target genes.

Role in cell-cell adhesion


Cell–cell adhesion complexes are essential for the formation of complex animal tissues. β-catenin is part of a protein complex that form the so-called is_associated_with::adherens junctions. These cell-cell adhesion complexes are necessary for the creation and maintenance of epithelial cell layers and barriers. As a component of the complex, β-catenin can regulate cell growth and adhesion between cells. It may also be responsible for transmitting the contact inhibition signal that causes cells to stop dividing once the epithelial sheet is complete. The E-cadherin – β-catenin – α-catenin complex is weakly associated to actin filaments. Adherent junctions thus form a dynamic, rather than a stable link to the actin cytoskeleton.

The heart of the adherent junctions are the is_associated_with::cadherin proteins. Cadherins form the cell-cell junctional structures known as is_associated_with::adherens junctions as well as the is_associated_with::desmosomes. Cadherins are capable of homophilic interactions through their extracellular cadherin repeat domains, in a Ca2+-dependent manner: this can hold adjacent epithelial cells together. While in the adherens junction, cadherins recruit β-catenin molecules onto their intracellular regions. β-catenin, in turn, associates with another important protein, α-catenin that directly binds to the actin filaments. This is possible because α-catenin and cadherins bind at distinct sites to β-catenin. The β-catenin - α-catenin complex can thus physically bridge cadherins with the actin cytoskeleton. Organization of the cadherin–catenin complex is additionally regulated through phosphorylation and is_associated_with::endocytosis of its components.

Roles in development
Beta-catenin has a central role in directing several developmental processes, as it can directly bind is_associated_with::transcription factors and be regulated by a diffusible extracellular substance: Wnt. It acts upon early embryos to induce entire body regions, as well as individual cells in later stages of development. It also regulates physiological regeneration processes.

Early embryonic patterning
Wnt signaling and beta-catenin dependent gene expression plays a critical role during the formation of different body regions in the early embryo. Experimentally modified embryos that do not express this protein will fail to develop is_associated_with::mesoderm and initiate is_associated_with::gastrulation. During the blastula and gastrula stages, Wnt as well as BMP and FGF pathways will induce the antero-posterior axis formation, regulate the precise placement of the primitive streak (gastrulation and mesoderm formation) as well as the process of neurulation (central nervous system development).

In is_associated_with::Xenopus oocytes, β-catenin is initially equally localized to all regions of the egg, but it is targeted for ubiquitination and degradation by the β-catenin destruction complex. is_associated_with::Fertilization of the egg causes a rotation of the outer cortical layers, moving clusters of the Frizzled and Dsh proteins closer to the equatorial region. β-catenin will be enriched locally under the influence of is_associated_with::Wnt signaling pathway in the cells that inherit this portion of the cytoplasm. It will eventually translocate to the nucleus to bind is_associated_with::TCF3 in order to activate several genes that induce dorsal cell characteristics. This signaling results in a region of cells known as the grey crescent, which is a classical organizer of embryonic development. If this region is surgically removed from the embryo, gastrulation does not occur at all. β-Catenin also plays a crucial role in the induction of the is_associated_with::blastopore lip, which in turn initiates is_associated_with::gastrulation. Inhibition of GSK-3 translation by injection of antisense mRNA may cause a second blastopore and a superfluous body axis to form. A similar effect can result from the overexpression of β-catenin.

Asymmetric cell division
Beta-catenin has also been implicated in regulation of cell fates through is_associated_with::asymmetric cell division in the model organism C. elegans. Similarly to the Xenopus oocytes, this is essentially the result of non-equal distribution of Dsh, Frizzled, axin and APC in the cytoplasm of the mother cell.

Stem cell renewal
One of the most important results of Wnt signaling and the elevated level of beta-catenin in certain cell types is the maintenance of is_associated_with::pluripotency. In other cell types and developmental stages, β-catenin may promote differentiation, especially towards is_associated_with::mesodermal cell lineages.

Epithelial-to-mesenchymal transition
Beta-catenin also acts as a morphogen in later stages of embryonic development. Together with TGF-β, an important role of β-catenin is to induce a morphogenic change in epithelial cells. It induces them to abandon their tight adhesion and assume a more mobile and loosely associated mesenchymal phenotype. During this process, epithelial cells lose expression of proteins like E-cadherin, Zonula occludens 1 (ZO1), and is_associated_with::cytokeratin. At the same time they turn on the expression of is_associated_with::vimentin, alpha smooth muscle actin (ACTA2), and fibroblast-specific protein 1 (FSP1). They also produce extracellular matrix components, such as type I collagen and is_associated_with::fibronectin. Aberrant activation of the Wnt pathway has been implicated in pathological processes such as fibrosis and cancer.

Involvement in cancer


Beta-catenin is a proto-oncogene. Mutations of this gene are commonly found in a variety of cancers: in primary hepatocellular carcinoma, is_associated_with::colorectal cancer, ovarial carcinoma, is_associated_with::breast cancer, is_associated_with::lung cancer and is_associated_with::glioblastoma. It has been estimated that approximately 10% of all tissue samples sequenced from all cancers display mutations in the CTNNB1 gene. Most of these mutations cluster on a tiny area of the N-terminal segment of β-catenin: the β-TrCP binding motif. Loss-of-function mutations of this motif essentially make ubiquitinylation and degradation of β-catenin impossible. It will cause β-catenin to translocate to the nucleus without any external stimulus and continuously drive transcription of its target genes. Increased nuclear β-catenin levels have also been noted in is_associated_with::basal cell carcinoma (BCC), head and neck squamous cell carcinoma (HNSCC), is_associated_with::prostate cancer (CaP), is_associated_with::pilomatrixoma (PTR) and is_associated_with::medulloblastoma (MDB) These observations may or may not implicate a mutation in the β-catenin gene: other Wnt pathway components can also be faulty.

Similar mutations are also frequently seen in the β-catenin recruiting motifs of APC. Hereditary loss-of-function mutations of APC cause a condition known as is_associated_with::Familial Adenomatous Polyposis. Affected individuals develop hundreds of polyps in their large intestine. Most of these polyps are benign in nature, but they have the potential to transform into deadly cancer as time progresses. Somatic mutations of APC in colorectal cancer are also not uncommon. Beta-catenin and APC are among the key genes (together with others, like K-Ras and is_associated_with::SMAD4) involved in colorectal cancer development. The potential of β-catenin to change the previously epithelial phenotype of affected cells into an invasive, mesenchyme-like type contributes greatly to metastasis formation.

As a therapeutic target
Due to its involvement in cancer development, inhibition of beta-catenin continues to receive significant attention. But the targeting of the binding site on its armadillo domain is not the simplest task, due to its extensive and relatively flat surface. However, for an efficient inhibition, binding to smaller "hotspots" of this surface is sufficient. This way, a "stapled" helical peptide derived from the natural β-catenin binding motif found in LEF1 was sufficient for the complete inhibition of β-catenin dependent transcription. Recently, several small-molecule compounds have also been developed to target the same, highly positively charged area of the ARM domain (CGP049090, PKF118-310, PKF115-584 and ZTM000990). In addition, β-catenin levels can also be influenced by targeting upstream components of the Wnt pathway as well as the β-catenin destruction complex. The additional N-terminal binding pocket is also important for Wnt target gene activation (required for BCL9 recruitment). This site of the ARM domain can be pharmacologically targeted by is_associated_with::carnosic acid, for example. That "auxiliary" site is another attractive target for drug development. Despite intensive preclinical research, no β-catenin inhibitors are available as therapeutic agents yet.

β-catenin destabilization by ethanol is one of two known pathways whereby alcohol exposure induces fetal alcohol syndrome (the other is ethanol-induced folate deficiency). Ethanol leads to β-catenin destabilization via a G-protein-dependent pathway, wherein activated Phospholipase Cβ hydrolyzes phosphatidylinositol-(4,5)-bisphosphate to diacylglycerol and inositol-(1,4,5)-trisphosphate. Soluble inositol-(1,4,5)-trisphosphate triggers calcium to be released from the endoplasmic reticulum. This sudden increase in cytoplasmic calcium activates Ca2+/calmodulin-dependent protein kinase (CaMKII). Activated CaMKII destabilizes β-catenin via a poorly characterized mechanism, but which likely involves β-catenin phosphorylation by CaMKII. The β-catenin transcriptional program (which is required for normal neural crest cell development) is thereby suppressed, resulting in premature neural crest cell apoptosis (cell death).

Binding partners
Beta-catenin has been shown to interact with:


 * APC,
 * is_associated_with::AXIN1,
 * is_associated_with::Androgen receptor,
 * is_associated_with::CBY1,
 * CDH1,
 * is_associated_with::CDH2,
 * CDH3,
 * is_associated_with::CDK5R1,
 * is_associated_with::CHUK,
 * is_associated_with::CTNND1,
 * CTNNA1,
 * EGFR,
 * is_associated_with::FHL2,
 * is_associated_with::GSK3B,
 * is_associated_with::HER2/neu,
 * HNF4A,
 * is_associated_with::IKK2,
 * LEF1,
 * is_associated_with::MAGI1,
 * is_associated_with::MUC1,
 * NR5A1,
 * is_associated_with::PCAF,
 * is_associated_with::PHF17,
 * is_associated_with::Plakoglobin,
 * is_associated_with::PTPN14,
 * is_associated_with::PTPRF,
 * is_associated_with::PTPRK (PTPkappa),
 * is_associated_with::PTPRT (PTPrho),
 * is_associated_with::PTPRU (PCP-2),
 * is_associated_with::PSEN1,
 * is_associated_with::PTK7
 * is_associated_with::RuvB-like 1,
 * SMAD7,
 * is_associated_with::SMARCA4
 * SLC9A3R1,
 * is_associated_with::USP9X, and
 * is_associated_with::VE-cadherin.