WNT Signalling Pathway

1. THE WNT
SIGNALING PATHWAY
ADITYA BASAK
14BCB0010
VIT UNIVERSITY

2. OUTLINE
• Introduction
Overview of Wnt
signaling
• Components of Wnt
signaling
Wnt proteins
Palmitoylation
Transport
Wnt receptors
Interactions with
extracellular

3. 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

4. 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)

5. OVERVIEW OF PATHWAY

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. 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

9. 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

10. 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

11. SECOND COMPONENT:
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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. Target Gene Interacts With Effect on Target
Gene Expression
Effect on Wnt
Pathway
Fz Wnt Down Inactivate
Dfz2 Wnt Down Inactivate
Dfz3 Wnt Up Activate
Fz7 Wnt Up --
Arrow/LRP Wnt and Axin Down Inactivate
Dally Wnt Down --
Wingful/notum HSPG Up Inactivate
Naked Dsh Up Inactivate
Axin2 β-catenin Up Inactivate
β-TRCP β-catenin Up Inactivate
TCF1 TCF Up Inactivate
LEF1 β-catenin Down Activate
Nemo β-catenin and
LEF/TCF
Up Inactivate
(Drosophila)
Activate (Zebrafish)

21. 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

22. 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

23. WNT SIGNALING AND HUMAN DISEASE
Gene Disease
Wnt3 Tetra-amelia
LRP5 Bone density defects
Fzd4 Familial Exudative Vitreoretinopathy
(FEVR)
Axin2 Tooth agenesis
Predisposition to Colorectal Cancer
APC Familial adenomatous polyposis (FAP)
Colon Cancer
Extracellular Wnt Protein Target Cell Membrane Protein Intracellular Protein

24. 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

25. 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

26. 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

27. 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

28. REFERENCES
• https://en.wikipedia.org/wiki/Wnt_signaling_pathway
• https://www.ncbi.nlm.nih.gov/pubmed/19279717
• “An Introduction to Systems Biology”-Uri Alon