Lecture 17 - Wnt Network Flashcards
Paracrine cell signalling
Cells that are near one another communicate through the release of chemical messengers (ligands that can diffuse through the space between the cells)
This type of signalling, in which cells communicate over relatively short distances, is known as paracrine signalling
Int experiments - Roel Nusse 1982
Retroviral insertion of wnt randomly into genome of mice
Leads to downstream gene activation
Identification of Int1 (insertion 1)
Proto oncogene
A normal gene that could become an oncogene due to mutations or increased expression
Proto-oncogenes code for proteins that help to regulate the cell growth and differentiation
Oncogene
A gene that has the potential to cause cancer
Wingless experiments - Volhard and Weischaus 1980
Knock out wing genes in fruit flies
Gene identified as ‘wingless’
Wingless and Int = orthologs
Wingless + Int
Wnt
The Wnt pathway summary
Wnt -> Dvl -> GSK3 -> b-catenin -> TCF
Wnt pathway functions
Embryogenesis (wing development in drosophila)
Disease (Oncogene in mice)
The Wnt/b-catenin pathway is
Highly conserved
Wnt regulates
Cell proliferation
Cell fate
Apoptosis
Wnt can influence the development of
An entire organism
mutations at certain stage of development can induce a second head in xenopus
Wnt ‘network’ in 2017
many pathways
Embryogenesis
Regeneration
Disease
Wnt embryogenesis
Body axis formation Patterning of nervous system (anterior posterior) Neural crest induction Hair follicle growth Limb polarity Muscle development
Wnt regeneration
Limb bud
Heart
Brain
Wnt diseases
Colon carcinoma Mamma carcinoma Melanoma Alzheimers Diabetes
3 pathways in the Wnt signalling network
- Planar cell polarity signalling (cell polarity and migration)
- b-catenin signalling (cell differentiation and proliferation)
- Wnt Calcium signalling (cell fate and migration)
Pathways interact
- Planar cell polarity pathway
Tells cells where to go
Uses Frizzled family GTpases
e.g. neural crest cells
Frizzled
A family of G protein-coupled receptor proteins that serves as receptors in the Wnt signaling pathway
When activated, Frizzled leads to activation of Dishevelled in the cytosol
Dishevelled
Dsh (Dvl in mammals) is a cytoplasmic phosphoprotein that acts directly downstream of frizzled receptors
It takes its name from its initial discovery in flies, where a mutation in the dishevelled gene was observed to cause improper orientation of body and wing hairs
Dishevelled plays important roles in both the embryo and the adult, ranging from cellular differentiation and cell polarity to social behavior
- b-catenin signalling pathway
Wnt molecule binds to a receptor
Deactivates a protein complex
b-catenin is translocated to the nucleus
Activates gene transcription
The 3 Wnt pathways
Interact with each other
Pathways 1 and 2 repress each other
Wnt ligand structure
350 a.a
22 cysteines (disulfide bridges important for form of protein)
Serine 187 (palmitoleic acid)
Asparagin 87 + 298 (glycosylation)
Wnt ligand numbers
19 ligands
10 Frizzled receptors
5 co receptors
Two post translational modifications on Wnt ligands
Palmitoylation
Glycosylation
Palmitoylation occurs at
Serine 187
Glycosylation occurs at
Asparagine 87 and 298
The Wnt protein look like a
Hand
Palmitoleic acid at thumb
Glycosylations in the palm
The presence of the hydrophobic palmitoleic acid
Makes it hard to diffuse into the cell
Production of the Wnt ligand
- Wnt is lipid modified by the ER by O-acetyltransferase (Porcupine)
- Palmitolated Wnt is transported in vesicles to the Golgi by p24
Porcupine
Enzyme that modifies Wnt (O acetyltransferase)
Allows transport, secretion and activity
Without porcupine Wnt cannot be
Palmitoylated
Secretion of the Wnt ligand
Transported from the Golgi by Evi/Wntless
Wnt loaded onto cytonemes (signalling filopodia)
Wnt is packaged in exosomes (mutli vesicular bodies)
The released
Evi/Wntless
Chaperone type protein that binds Wnt
An important checkpoint
Mutli vesicular bodies
A specialised subset of endosomes that contain membrane-bound vesicles
These vesicles form by budding into the lumen of the MVB
The content of MVBs can be degraded, via fusion with lysosomes, or released into the extracellular space, via fusion with the plasma membrane
Once released, Wnt exosomes can travel
Through the entire body
Interaction of Wnt and Frizzled
Wnt binds to the CRD on Frizzled as a monomer
Thumb and finger of Wnt bind to the CRD
Frizzled has a
7 TM domain and a CRD (cysteine rich domain)
The ‘thumb’ of Wnt (palmitoleic site) is projected into a
Groove in the Frizzled CRD
Specific Wnt ligands have
Very specific functions
e.g. Wnt 4 mutants have non functional kidneys
Wnt 1 mutants have no mid brain
Specific Wnt ligands bind to
Specific Frizzled receptors
When Wnt is NOT bound to Frizzled
- Fzd Receptors continually endocytosed at the membrane (recycled)
- The protein b-catenin is bound by a ‘destruction complex’ with protein kinases CK1y and GSK3-b
- The protein kinases phosphorylate b-catenin, targeting for degradation in the proteosome (no free b-catenin in nucleus)
- In the absence of b-catenin, transcriptional co repressors bind to TCF transcription factors to prevent expression of certain genes
The beta catenin destruction complex
APC + Dvl + Axin1 + GSK3b
All structural proteins
hen Wnt IS bound to Frizzled
- Signal transmitted by Fzd and LRP
- Activation of Fzd and LRP causes CK1y and GSK3-b to associate with membrane
- Protein kinases phosphorylate the tail of of LRP
- Dishevelled and Axin associate with cytoplasmic tails of Fzd and LRP
- This prevents the formation of the destruction complex
- Beta catenin increases in the cytoplasm
- Beta catenin moves into the nucleus
- Catenin binds to TCF, displacing repressors
Target genes expressed
beta catenin is the
Key effector of Wnt signalling
Beta catenin can
- interact with scaffold proteins Axin1 and APC
- be phosphorylated by GSK3b
- be ubiquitinated by bTRCP
- can translocate to the nucleus to regulate transcription
- further role in cell adhesion with cadherin
Ubiquitination
Addition of ubiquitin to a substrate protein
Ubiquitination affects proteins in many ways: it can mark them for degradation via the proteasome, alter their cellular location, affect their activity, and promote or prevent protein interactions
Phosphorylation
Activity of proteins can be altered after they are formed
A phosphate group ( PO3−4 ) is added to a protein by specific enzymes called kinases
Cytoneme
Thin, cellular projections that are specialized for exchange of signaling proteins between cells
Cytonemes emanate from cells that make signaling proteins, extending directly to cells that receive signaling proteins
Advantages of a ‘handbreak on’ system
Keeping all components in place with the ‘lock on’
System ready to go
Wnt ligand receptor releases the ‘hand break’
Very fast responses
Wnt signalling is
Very fast
Activates transcription within minutes
Wnt special features
The negative feedback loop of Axin2
Wnt signalling activates the transcription of Axin
Axin is an inhibitor of Wnt/b-catenin signalling
Does this via negative feedback loop
Ribosyltransferase Tankyrase mediates poly-ADP ribosylation of Axin = degradation
Chemical inhibitors of Tnks block Wnt signalling (drug therapy using Axin)
Axin is an
Inhibitor of wnt/catenin pathway
Wnt is difficult to synthesise for regenerative medical applications so
Wnt surrogates are made instead
all you have to do is cause Fzd to associate with LRP
Wnt catenin signalling in diseases
10 billion cells are lost from gut villi daily
Wnt signalling controls regeneration of stem cells into Goblets and enterocytes
Wnt ligands are expressed in intestinal
Paneth cells and stroma cells
The APC protein is a
Tumour suppressor gene
When the APC or beta catenin genes are mutated
Colon cancer occurs
In cancer, APC can no longer
Keep beta catenin out of the nucleus
Inside the nucleus beta catenin
Causes uncontrolled cell proliferation
