Wnt Signaling Triggers Release of a Transcription Factor from a Cytosolic Protein Complex

The components of the Wnt and Hedgehog signaling pathways have been conserved throughout the evolution of metazoan organisms and were elucidated mainly through genetic analysis of developmental mutants in Drosophila. In vertebrates, mutations in these pathways are thought to trigger several types of cancers. In fact, the first vertebrate Wnt gene to be discovered, the mouse Wnt-1 gene, attracted notice because it was overexpressed in certain mammary cancers because of insertion of a mouse retroviral DNA, the mammary tumor virus (MMTV) genome, near the Wnt-1 gene; the retrovirus LTR promoter (see Figure 8-13) activated inappropriate expression of the Wnt-1 gene.

The word Wnt is an amalgamation of wingless, the corresponding fly gene, with int for the retrovirus integration site in mice. The human genome encodes 19 different Wnt proteins, and Wnt proteins are essential for numerous critical developmental events, such as brain development, limb patterning, and organogenesis. A major role for Wnt signaling in bone formation was revealed by the finding that inactivating mutations in Wnt pathway components affect bone density in humans. Wnt signaling is now known to control formation of osteoblasts (bone-forming cells). Additionally, Wnt signals are essential for proliferation of many types of stem cells (see Chapter 21) and in many other aspects of development.

Wnt proteins are secreted signaling molecules that are modified by linkage of a monounsaturated fatty acid, palmitoleic acid, to a serine in the middle of the protein. Like other growth factors, Wnt proteins interact with several cell-surface proteins and activate multiple downstream signal transduction pathways. The principal signaling receptor for Wnt proteins is Frizzled (Fz), which contains seven transmembrane α helices. Like the glucagon receptor (see Figure 15-13c), Fz has a large extracellular domain that is connected to the first membrane-spanning α helix and comprises the major ligand binding site, but Fz does not activate a G protein. The palmitate attached to the Wnt protein binds to a specific site on the Fz extracellular domain and stabilizes the Wnt-Fz complex. This lipid is central to receptor engagement by Wnt proteins, and is the only known example of post-translational modification by a lipid that mediates a ligand-receptor interaction.

At least three different signal transduction pathways are activated by the binding of different Wnt proteins to Fz. The most widespread, “canonical” Wnt signaling pathway uses a second transmembrane protein, LRP (called Arrow in Drosophila), that associates with Frizzled in a Wnt signal–dependent manner (Figure 16-30). Inactivating mutations in the genes encoding Wnt proteins, Frizzled, or LRP all have similar effects on the development of embryos, indicating that all three proteins are essential for Wnt signaling.

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FIGURE 16-30 “Canonical” Wnt signaling pathway. (a) In the absence of Wnt, the transcription factor TCF is bound to promoters or enhancers of target genes, but its association with transcriptional repressors such as Groucho (Gro) inhibits gene activation. β-catenin is bound in a complex with Axin (a scaffold protein), APC, and the kinases CK1 and GSK3, which sequentially phosphorylate β-catenin at multiple serine and threonine residues. In particular, Axin-mediated formation of this complex facilitates phosphorylation of β-catenin by GSK3 by an estimated factor of 20,000. The E3 TrCP ubiquitin ligase then binds to two phosphorylated β-catenin residues, leading to β-catenin ubiquitinylation and degradation in proteasomes. (b) Binding of Wnt to its receptor Frizzled (Fz) and to the LRP co-receptor triggers phosphorylation of LRP by GSK3 and CK1, allowing subsequent binding of the Dishevelled scaffold protein. Binding of Axin to the phosphorylated LRP protein and to Dishevelled disrupts the Axin–APC–CK1–GSK3–β-catenin complex, preventing phosphorylation of β-catenin by CK1 and GSK3 and leading to accumulation of β-catenin in the cell. After translocation to the nucleus, β-catenin binds to TCF to displace the Gro repressor and recruits co-activator proteins including Pygo, LGS, and others to activate gene expression. See R. van Amerongen and R. Nusse, 2009, Development 136:3205; E. Verheyen and C. Gottardi, 2010, Dev. Dynam. 239:34; and J. Holland et al., 2013, Curr. Opin. Cell Biol. 25:254. See also the Wnt Homepage, http://web.stanford.edu/group/nusselab/cgi-bin/wnt.

The central player in the “canonical” Wnt intracellular signal transduction pathway is called β-catenin in vertebrates and Armadillo in Drosophila. This multi-talented protein functions both as a transcriptional activator and as a membrane-cytoskeleton linker protein (see Figure 20-14). In the absence of a Wnt signal, the β-catenin molecules that are not attached to cell-adhesion molecules are bound in a cytosolic complex based on the scaffold protein Axin. The complex contains the adenomatous polyposis coli (APC) protein, so named because its loss may result in colorectal cancer. In the resting state, two kinases in the complex, casein kinase 1 (CK1) and GSK3, sequentially phosphorylate β-catenin on multiple serine and threonine residues. Some of these phosphorylated residues serve as binding sites for a ubiquitin-ligase protein named TrCP. β-Catenin is then ubiquitinylated and rapidly degraded by the 26S proteasome (Figure 16-30a; for more on ubiquitinylation, see Figures 3-31 and 3-36).

The complete pathway by which Wnt signaling blocks the degradation of β-catenin has not yet been identified. We do know that Wnt binding to the complex of Fz and LRP leads to the phosphorylation of the LRP cytosolic domain, probably by free GSK3 or CK1. This enables Axin to bind to the cytosolic domain of the LRP co-receptor. This shift in Axin localization disrupts the interactions that stabilize the cytosolic complex containing Axin, GSK3, CK1, and β-catenin and thus prevents phosphorylation of β-catenin by CK1 and GSK3. This change, in turn, prevents ubiquitinylation and subsequent degradation of β-catenin and stabilizes it in the cytosol (Figure 16-30b). This process requires the Dishevelled (Dsh) protein, which becomes bound to the cytosolic domain of the Frizzled receptor and stabilizes Axin binding to LRP. The freed β-catenin translocates to the nucleus, where it associates with a transcription factor (TCF) and functions as a co-activator to induce expression of particular target genes, often including those that promote cell proliferation. (The name is unfortunately confusing; this TCF is different from the TCF protein that functions in the MAP kinase pathway; see Figure 16-26.)

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Aberrant hyperactive Wnt signaling is implicated in the progression of many cancers; more than 90 percent of human colon cancers display hyperactivity of the Wnt signaling pathway in that the level of free β-catenin is abnormally high (see Chapter 24). This observation provided one of the earliest clues that β-catenin can activate many growth-promoting genes. Inactivating mutations in genes encoding APC and Axin are found in multiple types of human cancers, as are mutations in β-catenin phosphorylation sites for GSK3 or CK1; these mutations reduce formation of the cytosolic complex that inactivates β-catenin (see Figure 16-30a), reduce β-catenin degradation, and allow β-catenin to activate gene expression in the absence of the normal Wnt signal.

Among the Wnt target genes are many that also control Wnt signaling, indicating a high degree of feedback regulation. The importance of β-catenin stability and location means that Wnt signals affect a critical balance between the three pools of β-catenin in the cell: at the membrane-cytoskeleton interface, in the cytosol, and in the nucleus.

In order to signal, Wnt must also bind to cell-surface proteoglycans. Evidence for the participation of proteoglycans in Wnt signaling comes from Drosophila Sugarless (Sgl) mutants, which lack a key enzyme needed to synthesize the glycosaminoglycans heparan and chondroitin sulfate. These mutants have greatly depressed levels of Wingless (the fly Wnt protein) and exhibit other phenotypes associated with defects in Wnt signaling. How proteoglycans facilitate Wnt signaling is unknown, but perhaps binding of Wnt to specific glycosaminoglycan chains is required for it to bind to its receptor, Fz, or its co-receptor, LRP. This mechanism would be analogous to the binding of fibroblast growth factor to heparan sulfate, which enhances binding of FGF to its receptor tyrosine kinase (see Figure 16-15).