1. The Wnt Signaling Pathway Jennifer Slade B.Sc. (Hon) M.Sc. Candidate

2. Outline Introduction –Overview of Wnt signaling Components of Wnt signaling –Wnt proteins Palmitoylation Transport –Wnt receptors Interactions with extracellular proteins How they signal –Cytoplasmic Signaling cascade –Nuclear Signaling Cascade –Target genes Non-Wnt pathway genes Feedback loop Mutant Wnt Pathway Phenotypes –Wnt Redundancy Wnt and Human Disease Summary

3. Introduction Important in multiple developmental events –Mutated: leads to disease Canonical pathway: –Wnt signaling through Frizzled to β-catenin Main intracellular proteins involved: –Dishevelled (Dsh)- Glycogen synthase kinase 3 –Axin(GSK-3) –β –catenin- Adenomatous Polyposis Coli (APC) Wnt signaling inhibits degradation of β -catenin β -catenin interacts with Tf lymphoid enhancer- binding factor (TCF)

4. Overview of Pathway Dsh APC GSK3 Axin Β-c TCF Β-c TCF Axin Β-c WNT protein Frizzled LRP5/6 DNA

5. First Component: Wnt Proteins

6. Wnt Proteins Large family of secreted molecules –350 to 400 amino acids –Signal sequence –Invariant pattern of 23-24 conserved cysteines Name derived from first 2 members discovered: –Drosophila Wingless –Mouse int-1 Involved in intercellular signaling during development –Early mesodermal patterning of embryo –Morphogenesis of brain and kidneys

7. Wnt Proteins Secreted but insoluble (hydrophobic) –Palmitoylated –Enzyme responsible: porcupine (por) in Drosophila or mom-1 in C. elegans –Essential for function and signaling Mutation of cysteine Removal of palmitate Drosophila homologue – Wingless –Loses hydrophobicity and activity when por eliminated –Por is necessary for lipidation/membrane targeting Inactive WNT Protein

8. Palmitoylation WNT H-C-CH 2 -S-H O=C-O - HO-C-(CH 2 ) 14 CH 3 O WNT H-C-CH 2 -S O=C-O - C-(CH 2 ) 14 CH 3 O Cysteine Residue Palmitic Acid Palmitoylated WNT protein pormom-1

9. Function of Palmitoylation Remains unclear Experiments in: –Drosophila Loss of por Excess of Wingless Circumvents the loss of por –Vertebrates Excess expression of mutant Wnt Still some Wnt signaling Presence of lipid moiety targets Wnt to the membrane Absence of lipid is overcome by high concentration of Wnt protein

10. Transport of Wnt Proteins Secreted from cells Experiment in Drosophila –Antibody to Wingless Significant spread in imaginal discs Concentration-dependent long-range morphogenetic signals acting on distant neighbours Flies have vessicles in imaginal discs –Argosomes –Might carry Wingless as cargo

11. Extracellular Binding Partners Extracellular enhancer: –HSPG – Heparin-sulfated forms of proteoglycans Co-receptor on target cells Drosophila’s Dally –Lost or mutated: similar phenotype to wingless mutants Extracellular inhibitors: –SFRP – Secreted Frizzled-related protein Resembles ligand-binding domain of Frizzled –WIF – Wnt inhibitory factor Secreted molecules resembling extracellular portion of receptor Might promote signaling through protection of Wnts from degradation

12. Extracellular Binding of Wnts Por/mom-1 SFRP WIF

13. Second Component: WNT Receptors

14. Wnt Receptors Frizzled (Fz) proteins –Seven transmembrane receptors –Long N-terminal extension Cysteine rich domain (CRD) Overexpression of Fz: –No Wnt signal –Co-overexpression of Wingless: Signaling –Fz activation is ligand dependent Fz forms receptor complex with another single- pass transmembrane protein –LRP (Low density receptor related protein) –Arrow in Drosophila

15. WNT Receptors Derailed –Distinct from Frizzled –Transmembrane tyrosine kinase Belongs to RYK subfamily –Contains WIF domain In Drosophila: –Binds Dwnt-5 Regulator of axon guidance in CNS –Cytoplasmic kinase domain dispensable In Vertebrates: –Wnt4 and Wnt5 implicated in axon guidance Wnt4 binds to Fz Wnt5 receptor remains undetermined

16. Non-Wnt Proteins that Interact with Wnt receptors Dickkopf – Dkk1 –Encodes cysteine-rich secreted protein –Binds Wnt coreceptor LRP6 If Fz has Wnt bound, can still bind to Dkk1 and LRP5/6 to induce canonical signaling pathway –Also binds transmembrane protein Kremen –Endocytosed, depleting LRP6 Norrin –Ligand that binds to Fz –No sequence similarity to Wnt –Can induce canonical signaling pathway

17. Wnt Receptors and Non-Wnt Proteins Dkk N N Canonical Signaling

18. How Wnt Receptors Signal Frizzled –Binds to Dishevelled (Dsh) Ubiquitously expressed –C-terminal cytoplasmic Lys-Thr-X-X-X-Trp motif Required for Fz signaling LRP –Binds to Axin –Cytoplasmic tail has several Pro-Pro-Pro-(SerTrp)Pro motifs Phosphorylated upon Wnt binding Axin and Dsh: DIX domains –Can heterodimerize –LRP and Fz may promote interaction between Dsh and Axin

19. Third Component: Cytoplasmic Cascade Dsh APC Axin Β-c GSK3

20. Canonical Signaling Absence of Wnt Signaling: –β-catenin phosphorylated by serine/threonine kinase Casein Kinase or GSK-3 –Facilitated by scaffolding proteins APC and Axin –Degradation complex –Recognized by β -TrCP Ubiquitinates for degradation via proteosome Activation of Wnt signaling: –β -catenin levels accumulate –Enter the nucleus to induce transcription of target genes Mutant β-catenin (no phosphorylation sites) –Wnt unresponsive –β–catenin active in entering the nucleus –Constitutive Wnt signaling common in neoplasms

21. Presence of Wnt Signaling Three ways of β-catenin accumulation –Disruption of degradation complex 1.Recruitment of Axin to LRP or Fz/Dsh –Amount of Axin in cell much lower than other complex proteins Limiting factor 2.Protein phosphatases –PP2A Binds to Axin, dephosphorylates GSK-3 3.GBP/Frat –GSK-3 binding protein –Removes GSK-3 from degradation complex

22. Regulation of Cytoplasmic Cascade Dsh APC Axin Β-c GSK3 PP2A GBP/Frat Β-c

23. Fourth Component: Signaling in the Nucleus Β-c TCF DNA Β-c

24. Signaling in the Nucleus In presence of Wnt binding only –β-catenin enters nucleus –Binds to TCF DNA-binding proteins No Wnt: TCF represses gene transcription –Forms complex with Groucho Interacts with histone deacetylases β-catenin converts TCF to activator –Displaces Groucho –Recruits histone acetylase (CBP – cyclic AMP response element binding protein) Co-activator

25. Other controls of nuclear signaling Protein partners –Chibby Nuclear antagonist Binds C-terminus of β-catenin –ICAT Blocks binding of β-catenin to TCF Disassociates TCF/ β-catenin-CBP complex –Mitogen-activated protein kinase(MAPK)-related protein kinase NLK/Nemo Phosphorylates TCF, sending it to the cytoplasm –Sequestered by 14-3-3 binding protein Regulated itself by MAPK kinase TAK1

26. Groucho Regulation of Nuclear Signaling Β-c TCF Groucho Chibby ICAT TCF NLK TCF 14-3-3 P

27. Fifth Component: Target Genes Β-c TCF DNA Β-c

28. Target Genes: Non-Wnt Pathway Many transcription factors and signaling proteins –Including: Members of the homeobox family –Engrailed (en) –Ultrabithorax (Ubx) Genes expressed in development of the embryo –Siamois (organizing center) –Achaete (ac – proneural gene) Differential control dependent on cellular context Ac activated in wing imaginal disc, but repressed in eye imaginal disc Cellular proliferation genes –Cell cycle regulators

29. Target Genes: Wnt Pathway Components Feedback control Receptor components: –Frizzled family of receptors DFz2 in Drosophila down-regulated by wingless –Number of LRP receptors controlled by Wg signaling Cytoplasmic negative regulators –Naked cuticle (naked) Encodes protein that binds to Dsh and inhibits Wnt signaling –Axin2 gene Β-c TCF Β-c TCF Dsh naked

30. Target GeneInteracts WithEffect on Target Gene Expression Effect on Wnt Pathway FzWntDownInactivate Dfz2WntDownInactivate Dfz3WntUpActivate Fz7WntUp-- Arrow/LRPWnt and AxinDownInactivate DallyWntDown-- Wingful/notumHSPGUpInactivate NakedDshUpInactivate Axin2β-cateninUpInactivate β-TRCPβ-cateninUpInactivate TCF1TCFUpInactivate LEF1β-cateninDownActivate Nemoβ-catenin and LEF/TCF UpInactivate (Drosophila) Activate (Zebrafish)

31. Mutant Wnt Pathway Phenotypes Study knock-outs –Gene expression pattern correlates with mutant phenotype Demonstrates Wnt requirement in developmental process Wnt3 –Expressed in primitive streak in mouse embryo –Wnt3 mutants – gastrulation defects Frizzled4 –Cerebellar, auditory and esophageal defects TCF1 –Defects in limb bud development –Mammory and gut tumours Many more

32. Wnt Redundancy Knockout both Wnt1 and Wnt3: –Larger area of CNS disturbed (compared to single knockouts of either) Frizzled mutants do not reveal specific Wnt/Fz pairs –Single Fz activated by many Wnts –Single Wnt may bind many Fzs

33. Wnt Signaling and Human Disease GeneDisease Wnt3Tetra-amelia LRP5Bone density defects Fzd4Familial Exudative Vitreoretinopathy (FEVR) Axin2Tooth agenesis Predisposition to Colorectal Cancer APCFamilial adenomatous polyposis (FAP) Colon Cancer Extracellular Wnt ProteinTarget Cell Membrane ProteinIntracellular Protein

34. Wnt3 and Tetra-amelia Rare human genetic disorder Absence of of all four limbs Mutated extracellular Wnt Protein –Loss of function Wnt3 mutations

35. LRP and Bone density Target cell membrane protein Mutation of single amino-acid –Substitution –LRP insensitive to Dkk-mediated Wnt inhibition –Increased bone density of the jaw and palate Mutation causing frameshift –Loss of function LRP –Decreased bone density Wnt Signaling mediated by LRP –Important in maintenance of normal bone density

36. Fz4 and FEVR Rare eye disease affecting: –The retina –The vitreous –Progressive genetic disease Congenital and bilateral Target cell membrane proteins Mutations in both Fz4 and LRP –Frizzled mutated in seventh transmembrane domain –LRP proteins prematurely terminated –Loss of Fz4/LRP signaling

37. Axin2 and Tooth Agenesis Intracellular protein Nonsense mutation in Axin2 Oligodontia –Condition where multiple permanent teeth are missing Mutation in Axin2 also results in pre- disposition to colon cancer

38. APC and FAP Intracellular protein Truncations in APC –Aberrant activation of Wnt pathway –Increased cell proliferation and adenomatous lesions Autosomal dominantly inherited disease Hundreds or thousands of polyps in the colon and rectum Mutations in APC also found in: –Sporadic colon cancer –Several types of tumours –Hepatocellular carcinoma

39. Summary Wnt signaling includes: –Wnt proteins Palmitoylated –Receptors Frizzled and LRP, Derailed –Cytoplasm proteins Dsh, Degradation complex: Axin, APC and GSK-3 β-catenin –Nuclear proteins TCF, Inhibitors: Groucho, Chibby, ICAT and NLK –Target genes include non-wnt developmental genes or wnt pathway components Feedback Pathway involved in many human diseases

40. References Logan, C.Y. and Nusse, R. 2004. The Wnt Signaling Pathway in Development and Disease. Annu. Rev. Cell. Dev. Biol. 20: 781-810 Wodarz, A. and Nusse, R. 1998. Mechanisms of Wnt Signaling in Development. Annu. Rev. Cell Dev. Biol. 14:59–88