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Cell Signaling Pathways – A Case Study Approach L. Emtage, L. Bradbury, N. Coleman, D. Davenport, A. Dunning and J. Grew Assignment: The Wnt/ β-catenin pathway Wnt Wnt Fzd Plasma membrane LRP LRP Fzd DSH GSK3 A x i n Ubiqui n-mediated degrada on β-catenin APC DSH GSK3 GBP A x i n APC β-catenin Ub Ub Ub Ub β-catenin β-catenin TCF TCF Note: GSK3, APC and Axin together are called the β-catenin Destruction Complex. DSH is the abbreviation for Dishevelled. The human genome encodes many Fzd receptors, many Wnts, several Dsh proteins, one GSK3, and one β-catenin. Wnts are a family of protein ligands that activate members of the Frizzled (Fzd) family of receptors. Fzd receptors often associate with co-receptors, such as the lipoprotein related-receptor protein-5/6, or LRP, shown here. There are several signaling pathways that may be activated by the binding of a Wnt to a Fzd, but the canonical pathway, activated by Fzd-LRP, modulates transcription through βcatenin. β-catenin is constitutively expressed, but in the absence of Wnt signaling, it is constitutively destroyed in the cytoplasm through the action of the β-catenin Destruction Complex. The β-catenin Destruction Complex targets β-catenin for ubiquitination. Once ubiqutinated, β-catenin is broken down by the proteasome. Upon activation of the Fzd-LRP receptor complex by Wnts, the receptor complex binds to the β-catenin Destruction Complex via the adaptor Dishevelled (Dsh). The Destruction Complex is sequestered and prevented from ubiquitinating β-catenin. The inhibition of the Destruction Complex stabilizes β-catenin, allowing cytoplasmic and nuclear levels to rise. Although β-catenin does not have a DNA- Cell Signaling Pathways – A Case Study Approach L. Emtage, L. Bradbury, N. Coleman, D. Davenport, A. Dunning and J. Grew binding domain, it is a transcriptional co-activator that associates with the TCF/LEF family of transcription factors to modulate transcription. For a recent review of the factors and molecular mechanisms that mediated canonical Wnt/ β-catenin signaling, see The way Wnt works: Components and mechanism; Saito-Diaz, et al., Growth Factors, 2013. Freely available at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697919/ Questions 1. What are the components of the receptor complex? What is the ligand? The receptor complex is composed of LRP and Fzd. The ligand for Fzd is Wnt. 2. What is the molecular function of β-catenin itself? Apart from its role in adherens junctions, β-catenin is a transcriptional coactivator. β-catenin has a transactivation domain, but does not have a DNA binding domain, and so it activates transcription through binding to transcription factors such as TCF. 3. Where is β-catenin found within the cell? Does its subcellular location ever change? Low levels of β-catenin are found in the cytoplasm until inhibition of the β-catenin Destruction Complex allows levels of β-catenin to rise. It is subsequently found in the cytoplasm and nucleusd. 4. What are the molecular functions of GSK3, APC, and Axin? They work together to ubiquitinate β-catenin, targeting it for destruction by the proteasome. GSK3 is a kinase, Axin is a scaffolding protein, the function of APC is unclear, although it binds to both Axin and β-catenin. 5. Define proteolysis. The break-down of polypeptides to amino acids. 6. Describe the ultimate outcome of Wnt pathway activation. Changes in gene expression when β-catenin moves into the nucleus. 7. Give two examples of negative regulation found here. Contrast their molecular mechanisms. β-catenin is ubiquitinated and removed by proteolysis, ie. it is destroyed. The β-catenin Destruction Complex is sequestered by the receptor complex upon receptor activation. In contrast to β-catenin itself, sequestration does not result in the proteolysis of the destruction complex, its only held and prevented from acting on β-catenin. 8. Describe the effect of loss-of-function mutations in Frizzled, DSH, GSK3, and β-catenin separately on the transcription of target genes. You can assume that mutations in any part of the β-catenin Destruction Complex will cause the loss of function of the entire complex. The loss of Fz, DSH or β-catenin will prevent the pathway from being activated. The loss of GSK3 will Cell Signaling Pathways – A Case Study Approach L. Emtage, L. Bradbury, N. Coleman, D. Davenport, A. Dunning and J. Grew constitutively activate the pathway. However, because there are many Fz genes, the loss of any particular Fz is less severe than the loss of β-catenin. 9. What would be the result of a double knock-out of DSH and APC? Of GSK3 and β-catenin? DSH acts upstream of APC, so a DKO of both will result in constitutive activation of the pathway. Any KO of β-catenin will inactivate the pathway regardless of the identity of the other gene.