Catenin

ß-catenin is a 92 kDa multifunctional protein that belongs to the armadillo family of proteins, characterized by a central domain of 12 repeats of about 40 amino acids called arm repeats (Figure 2). The arm domain was originally described in armadillo, which is the Drosophila homologue of ß-catenin (Kodama et al., 1999). ß-catenin serves as a link between cadherins and the actin cytoskeleton. ß-catenin also binds to numerous other proteins in cadherin-independent complexes (Behrens, 2002) such as APC, lymphoid enhancer factor and T-cell factor (LEF/TCF) transcription factors, RGS domain proteins axin/conductin (Ki-kuchi, 1999; Kikuchi, 2000; Von Kries et al., 2000; Akiyama, 2000) and prontin 52 (Bauer et al, 1998). ß-catenin also associates with fascin, an actin-binding protein, in a cadherin independent manner (Tao et al., 1996).

ß-catenin

ARMADILLO 12

E-cadherin

Figure 2. Diagram of the twelve armadillo repeats of p-catenin. The (3-catenin protein consists of 12 armadillo repeats designated as 1-12. (-Catenin associates with specific proteins within the indicated region of the 12 repeats, in a mutually exclusive manner. Armadillo repeats 1-7 is designated as the LEF binding region; E-cadherin binds to repeats 4-12; APC binds to repeats 1-10; Tcf binds to repeats 3-8 of the (3-catenin protein. Armadillo protein 12 has been shown to be involved in transactivation of Wnt-responsive genes. N, N-terminus; C, Carboxy-terminus; LEF, lymphoid enhancer-binding factor ; APC, adenomatous polyposis coli;TCF,T-cellTranscription factor.

In addition to its role in cell-adhesion, p-catenin is associated with Wnt signal transduction pathway (Figure 3). This pathway is important in regulating embryonic development, and generation of cell polarity. Wnt proteins are differentially expressed in tissues during mammalian development (Cadigan and Nusse, 1997). These proteins are particularly important in regulating tissue differentiation and organogenesis (Behrens, 2002; Parr and McMahon, 1994; Willert and Nusse, 1998; Brown and Moon, 1998; Bullions and Levine, 1998). When Wnt proteins are aberrantly activated, tumor formation ensues (Moon and Kimelman, 1998; Zeng et al., 1997; Wodarz and Nusse, 1998; Peifer and Polakis, 2000; Bienz and Clevers, 2000; Barker and Clevers, 2000). Wnt has also been demonstrated to play a role in cancer development by transmitting a signal via its cytoplasmic component, p-catenin protein (Lejeune et al., 1995; Shimizu et al., 1997; Polakis, 2001; Polakis, 2000; Polakis 1999; Eastman and Gros-schedl, 1999; Cadigan and Nusse, 1997). Recent studies have suggested that Wnt proteins may have a role in tumor-induced osteoblastic activity, which is characterized by increased bone production as a result of prostate caner metastasis to the bone (Hall et al., 2006). Wnt proteins bind to cell surface receptors termed Frizzled (Fz). This interaction results in the activation of the cytoplasmic phosphoprotein disheveled (Dvl). Activated Dvl inhibits activation of axin and conductin proteins in the Wnt signaling cascade. Axin and its homolog, conductin (Axin2/Axil) form a multiprotein complex with APC and GSK3p; this activated complex catalyzes the phoshphorylation of p-catenin at specific residues in its N-terminal domain (Behrens, 2002; Ikeda et al., 1998). Axin and conductin act as scaffold proteins that directly bind several components of the Wnt signaling pathway, promoting the phosphory-lation of p-catenin by GSK-3p (Jho et al., 2002; Ikeda et al., 1998; Fagotto et al., 1999; Itoh et al., 1998; Hsu et al., 1999; Julius et al., 2000). Four ser/thr residues in the N-terminal region of p-catenin are targets for GSK-3p phosphorylation. In the absence of a Wnt signal, GSK3p phosphorylates p-catenin, which is then targeted for ubiquitination and subsequently degraded by proteasomes. Interestingly, recent studies show that additional proteins are involved in priming p-catenin for phosphorylation by GSK3p. Casein kinase I, Casein kinase II and GSK3p act together in marking p-catenin for phosphorylation (Polakis, 2002; Amit et al., 2002; Liu et al., 2002; Yanagawa e al., 2002; Zhang et al., 2002).

Figure 3. Diagram of Wnt signaling pathway. This schematic represents the Wnt-mediated signaling pathway that functions to stabilize cytoplasmic p-catenin. In the absence of Wnt signaling, p-catenin is degraded by the activity of glycogen synthase kinase 3p (GSK3P) in a complex with APC, axin, axin2 (conductin/Axil), and p-TrCP. The binding of Wnt proteins to its receptor, Frizzled (Fz) at the cell surface leads to the activation of Disheveled (Dvl) in the cytoplasm. Subsequently, GSK3P complex is inactivated and p-catenin accumulates in the cytoplasm, then enters the nucleus to interact with LEF/TCF proteins. p-Catenin-Tcf transcription factor activates the expression of Wnt responsive genes.

Figure 3. Diagram of Wnt signaling pathway. This schematic represents the Wnt-mediated signaling pathway that functions to stabilize cytoplasmic p-catenin. In the absence of Wnt signaling, p-catenin is degraded by the activity of glycogen synthase kinase 3p (GSK3P) in a complex with APC, axin, axin2 (conductin/Axil), and p-TrCP. The binding of Wnt proteins to its receptor, Frizzled (Fz) at the cell surface leads to the activation of Disheveled (Dvl) in the cytoplasm. Subsequently, GSK3P complex is inactivated and p-catenin accumulates in the cytoplasm, then enters the nucleus to interact with LEF/TCF proteins. p-Catenin-Tcf transcription factor activates the expression of Wnt responsive genes.

Regulation of p-catenin degradation is pivotal in downstream signaling. Several gene mutations have been reported in human cancers that render p-catenin resistant to GSK-3p mediated degradation. First, mutations in APC, a suppressor in human cancers, are associated with aberrant expression of p-catenin in colon cancers (Kawahara et al., 2000; Bienz and Clevers, 2000; Polakis 2000; Bright-Thomas and Hargest, 2002; Kawasaki et al., 2003). Second, oncogenic mutations have been identified in p-catenin at putative GSK-3p phosphorylation sites, which stabilize p-catenin in colorectal cancer and melanoma (Van Noort et al., 2002, Morin et al., 1997 and Korinek et al., 1997). Third, a mutation in human AX-IN1 has been found to be associated with hepatocellular carcinoma (Satoh et al., 2000), while a mutation in AXIN2 (also called conductin) is found in colorectal and liver cancers

(Liu et al., 2000; Lustig et al., 2002). Conversely, constitutive Wnt signaling negatively regulates the ubiquitination and degradation of cytosolic p-catenin leading to its stabilization. In summary, stabilization of p-catenin in the cytosol is altered by three independent mechanisms: 1) gene mutation of any one of the degradation complex components: APC, axin, axin2 or GSK-3p, 2) gene mutation of p-catenin, or 3) constitutive Wnt signaling. As a result, the level of cytosolic p-catenin increases, and p-catenin translocates to the nucleus where it interacts with transcription factors of the LEF/TCF family. Several negative feedback loops could limit the duration or intensity of a Wnt-initiated signal. First, the F-box protein p-TrCP is an ubiquitin-ligase complex that has been shown to be involved in the proteasome mediated degradation of phosphorylated p-catenin (Chen et al., 1997; Behrens, 2002; Winston et al., 1999, Hart et al., 1999; Latres et al., 1999; Kitagawa et al., 1999). p-TrCP is post-transcriptionally induced by p-catenin/TCF signaling. As a result of this signal, p-catenin degradation is accelerated. Second, Tcf4/p-catenin signaling regulates transcription of the Tcfl gene in epithelial cells. While TCF1 does not bind p-catenin, TCF1 binds to transcriptional repressors such as groucho, which would allow TCF1 to serve as a feedback repressor of p-catenin/Tcf4 target genes (Roose et al., 1999; Polakis 2002). Third, axin2 (conductin) appears to downregulate p-catenin to normal levels after a Wnt signal in a negative feedback loop mechanism (Jho et al., 2002; Leung et al., 2002). This would suggest that, without precise regulation of Wnt-initiated signaling, p-catenin is aberrantly expressed. As a result, downstream target genes that might contribute to tu-morigenesis are either up- or downregulated.

Increased concentration of p-catenin in the cytoplasm promotes its binding to LEF/TCF family of DNA-binding proteins. As a result, p-catenin translocates to the nucleus where it transcriptionally activates specific target genes. Although the exact mechanism of nuclear translocation of p-catenin has not been elucidated, association of p-catenin with several nuclear transport proteins, including importin/karyopherin and Ran (Wiechens and Fagotto, 2001; Fagotto et al., 1998), is not responsible. p-catenin lacks a classical nuclear localization sequence, but the armadillo repeats at the C-terminus are essential for nuclear translocation (Figure 2; Giannini et al., 2000; Funayama et al., 1995). Recent studies have suggested that, in prostate cancer cells, p-catenin can translocate into the nucleus as part of a complex with androgen receptor, AR, (Mulholland et al., 2002). This association of p-catenin with the androgen receptor is abrogated in the absence of armadillo repeat 6, further supporting the association of certain armadillo repeats with specific p-catenin functions. Armadillo repeats 4-12 are required for p-catenin to bind to E-cadherin (Hulsk-en et al., 1994; Orsulic 1996; Piedra et al., 2001). The expression of cadherin proteins could thus sequester p-catenin to the plasma membrane, preventing its nuclear translocation (Heasman et al., 1994; Fagotto et al., 1996; Weng et al., 2002). In the absence of sequestering proteins, p-catenin co-localizes with LEF/TCF in the nucleus to transactivate specific genes that contain LEF/TCF binding sites.

LEF-1 and TCF1-4 were first identified in immune cells (Clevers and van De Wetering, 1997). LEF-1 is a sequence-specific DNA-binding protein that is expressed in pre-B and pre-T lymphocytes of adult mice as well as in the neural crest, mesencephalon, tooth germs and whisker follicles (Van Genderen et al., 1994). In addition to its role in organogenesis and embryogenesis, constitutive LEF/TCF/ß-catenin transactivation is associated with oncogenesis in human colon carcinomas and melanomas (Korinek et al., 1997; Morin et al., 1997; Rubinfeld et al., 1997; Aoki et al., 1999). Although LEF/TCFs can bind directly to DNA through their HMG or DNA-binding domain, they are incapable of independently activating gene transcription (Polakis 2000; Polakis 2002, Behrens, 2002; Jiang and Struhl,1998; Kiatagawa et al., 1999; Hecht et al., 1999; Eastman and Grosschedl, 1999; Roose et al., 1999). Specific regions of ß-catenin are required to interact with either LEF or TCF proteins. Armadillo repeats 1-7 of ß-catenin interact with LEF while armadillo repeats 3-8 interact with TCF (Fig 1-3; Piedra et al., 2001; Sadot 1998; Behrens et al., 1996; Van de Wetering, 1997). ß-catenin forms a complex with LEF/TCF proteins, depending on the amount of free ß-catenin available. In this complex, LEF/TCF provides the DNA binding domain while ß-catenin provides the transactivation domain. ß-catenin binds specifically to sequences 1-51 of Tcf-4 (Miravet et al., 2002). Activation of this transcriptional complex between ß-catenin and Tcf induces the expression of specific target genes (Miz-ushima et al., 2002; Behrens, 2002; Polakis 2002). Examples of these genes include ultrabi-thorax in Drosophila, nodal related 3 (McKendry et al., 1997), and siamois in Xenopus (Brannon et al., 1997), and c-myc (He 1998; Kolligs et al., 2000) and cyclin D1 (Tetsu and McCormick, 1999; Shtutman et al., 1999) in mammals. The list of target genes also include genes that regulate cellular functions other than stimulating cell growth, such as cyclooxy-genase-2 (Howe et al., 2001); multi-drug resistance gene (Yamada et al., 2000); AF17 (Lin et al., 2001); metalloproteinase 7 (MMP-7) (Crawford et al., 1999; Brabletz et al., 1999); per-oxisome proliferator-activated receptor 5 (He 1999); laminin-5 y2 (Hlubek 2001); c-jun/ fra-1 (Mann et al., 1999) TCF-1 (Roose et al., 1999); axin2 (Jho et al., 2002; Leung et al., 2002); ITF-2 (Kolligs et al., 2002); E-cadherin (Huber et al., 1998; Novak et al., 1998); and mesenchymal genes (Huber et al., 1996; Miller and Moon, 1996; Novak and Dedhar, 1999).

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