Lecture 7 – WNT signalling cascade

MAP kinase signalling pathway is mitogenic. What does this mean?

It induces a cell to enter the cell cycle

• Can drive cells from G0 state to G1 state

What is the WNT receptor made of?

LRP5/6 and Frizzled form a complex.

• This is the WNT receptor

What occurs in the cell when the WNT receptor is in its inactive state (No WNT binding)?

• Beta-catenin levels are kept low due to its constant ubiquination

• This prevents transcription of WNT related genes

What occurs in the cell when the WNT receptor is in its active state (WNT binding)?

• WNT binds to its receptor which results in disassembly of the ubiquination complex.

• This prevents Beta-catenin destruction and allows intracellular levels to rise

• This allows transcription of WNT related genes 

WNT signaling is pleiotropic. What does this mean?

WNT signalling regulates several distinct cell functions

• Proliferation, differentiation, migration

WNT signalling has multiple different outcomes 

How does WNT signalling prevent Beta-catenin destruction?

• Binding of WNT to its receptor causes the ubiquination complex to be pulled apart and inactivated. 

• Cytoplasmic Beta-catenin levels can then rise.

What does Beta-catenin bind to in the nucleus to activate gene transcription? 

T-cell factor/lymphoid enhancer factor 1

• TCF/LEF1

WNT are displayed by signalling/effector cells and signal via cell-cell interactions with WNT receptors.

True or False?

False.

• Secreted by signalling/effector cells and are received by target cells both adjacent and distal to WNT releasing cell 

Of the 19 WNT proteins, which four are involved in canonical WNT signalling?

WNT-1, -3a, -8a and -8b

What is the role of Porcupine (PORCN) in WNT signalling?

WNT proteins are palmitoylated by Porcupine.

• This makes them extremely hydrophobic and is essential for WNT signalling 

What protein palmitoylates WNT?

Porcupine (PORCN)

What must Frizzled receptor complex with in order for WNT signalling?

Single pass-transmembrane protein of the LRP family.

• LRP5 or LRP6

Dishevelled associates with which domains of Fizzled/LRP5/6? 

The C-terminal domain.

What is the role of Dishevelled?

Following WNT binding, Dishevelled recruits the kinase GSK3 (Glycogen synthase kinase-3)

• GSK3 then phosphorylates LRP5/6

Which two proteins are involved in LRP5/6 phosphorylation? 

GSK3

• Protein kinase

CKI

• Membrane-anchored kinase 

Following LRP5/6 phosphorylation, what occurs which allows Beta-catenin signalling?

Phosphorylation of LRP5/6 causes high affinity interaction with Axin 

• Accumulation of Axin at the plasma membrane disrupts the ubiquination complex

• This allows for accumulation of Beta-catenin in the cytoplasm 

Which two proteins are part of the Beta-catenin uquination complex and are pulled away by the WNT receptor?

GSK3 and Axin

• Associate with Dishevelled and LRP5/6

How does increased Beta-catenin levels result in gene transcription?

• Translocates to the nucleus where it binds TCF/LEF1

• This displaces histone deacetylase (HDAC) proteins

• It also recruits the co-activator CBP which has histone acetyltransferase (HAT) activity 

• DNA is then acetylated which relaxes the chromatin and opens up the gene for transcription 

What two main ways does WNT regulate itself?

WNT signalling causes:

• Reduction in Frizzled receptor expression

• Increase in Naked (A dishevelled inhibitor)

These dampen WNT signalling 

What is the role of Naked?

It’s an inhibitor of the Dishevelled protein.

• Regulates WNT signalling 

What is the role of Dickkopf?

Inhibits LRP5/6

Beta-catenin signalling causes increased expression of RNF43. What does this cause?

Ring Finger Protein 43 (RNF43) drives ubiquination of Frizzled receptor.

• Causes destruction of the receptor and regulation of WNT signalling 

How can a mutation in APC result in deregulated WNT signalling?

APC is part of the Beta-catenin ubiquination complex

Mutated APC cannot maintain the function of the destruction complex 

• B-catenin is not destroyed in cells and will result in hyperactive WNT signalling 

What is a FAP adenoma?

Familial Adenomatous polyposis (FAP) adenoma

• Inherited disorder which causes thousands of precancerous colonic adenomas (polyps)

• Caused by APC gene mutation

How can FAP adenomas in the colon lead to colon cancer?

These FAP adenomas can acquire additional mutations in p53 and Ras which drives transformation 

What is the role of WNT in crypt stem cells?

Stem cells in the crypts of villi will give rise to progenitor cells 

• These then terminally differentiate as they migrate along the crypt villus  

• This is highly regulated by WNT to prevent abnormal cell growth

Inhibition of WNT in stem cell crypts causes…?

Causes a loss of the stem cell rich crypts 

• Suggests WNT is necessary for self-renewal of stem cells 

Hyperactivation of WNT in stem cell crypts causes…?

Causes large amplification of the stem cell niche

• Fewer cells are differentiated and large, stem cell-like growths occur

How do Porcupine inhibitors work?

Treatment which targets WNT pathway.

• By blocking Porcupine-mediated WNT palmitoylation, WNT cannot bind to its receptor and the cascade is stopped 

How do B-catenin CBP inhibitors work?

Treatment which targets WNT pathway.

• Interaction between B-catenin and CBP is crucial for activation of target genes 

• By blocking the interaction, WNT target genes cannot be transcribed which reduces cell proliferation