Transcript WNT Signaling

The Wnt
Signaling Pathway
Jennifer Slade B.Sc. (Hon)
M.Sc. Candidate
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
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)
– Axin
– β –catenin
- Glycogen synthase kinase 3
(GSK-3)
- Adenomatous Polyposis Coli
(APC)
• Wnt signaling inhibits degradation of β -catenin
• β -catenin interacts with Tf lymphoid enhancerbinding factor (TCF)
Overview of Pathway
WNT
protein
LRP5/6
Frizzled
Dsh Axin
Β-c
GSK3
APC
Axin
Β-c
Β-c
Dsh
Β-c
TCF
Β-c TCF
DNA
First Component: Wnt Proteins
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
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
Inactive WNT
Protein
• Drosophila homologue – Wingless
– Loses hydrophobicity and activity when por eliminated
– Por is necessary for lipidation/membrane targeting
Palmitoylation
O=C-O-
O
H-C-CH2-S-H
WNT
HO-C-(CH2)14CH3
Cysteine
Residue
por
Palmitic
Acid
mom-1
O=C-O-
O
H-C-CH2-S C-(CH2)14CH3
WNT
Palmitoylated
WNT protein
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
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
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
Extracellular Binding of Wnts
SFRP
Por/mom-1
WIF
Second Component: WNT Receptors
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 singlepass transmembrane protein
– LRP (Low density receptor related protein)
– Arrow in Drosophila
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
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
Wnt Receptors and Non-Wnt Proteins
Dkk
Dkk
N
Canonical
Signaling
N
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
Third Component: Cytoplasmic Cascade
Dsh Axin
GSK3
APC
Axin
Β-c
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
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
Regulation of Cytoplasmic Cascade
Β-c
Dsh Axin
GBP/Frat
GSK3
APC
Axin
PP2A
Β-c
Β-c
Β-c
Β-c
Fourth Component: Signaling in the Nucleus
Β-c
Β-c TCF
DNA
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
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
Regulation of Nuclear Signaling
Β-c
Groucho
Groucho
Β-c TCF
Β-c
TCF NLK
Chibby
Β-c
ICAT
P
14-3-3 TCF
Fifth Component: Target Genes
Β-c
Β-c TCF
DNA
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
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
Dsh
Β-c
TCF
naked
Dsh
naked
Β-c
TCF
naked
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)
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
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
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
Wnt3 and Tetra-amelia
• Rare human genetic disorder
• Absence of of all four limbs
• Mutated extracellular
Wnt Protein
– Loss of function
Wnt3 mutations
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
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
Axin2 and Tooth Agenesis
• Intracellular protein
• Nonsense mutation in Axin2
• Oligodontia
– Condition where multiple permanent teeth are
missing
• Mutation in Axin2 also results in predisposition to colon cancer
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
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
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