Difference between revisions of "2012 Group 5 Project"

From CellBiology
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| align="center" | '''Wnt''' (WNT)
 
| align="center" | '''Wnt''' (WNT)
 
| align="center" width="150px" | [[File:Genomic_structure_and_localization_of_WNT-4.JPG|250px]]
 
| align="center" width="150px" | [[File:Genomic_structure_and_localization_of_WNT-4.JPG|250px]]
| align="center" | Evolutionarily conserved protein across species<ref><pubmed>9407023</pubmed></ref>. Most Wnt proteins figure in the region of 40 kDa, each possessing a characteristic 23 or 24 cysteine residues highly conserved in their spacing. This suggests that the formation of ‘‘‘[[#GLOSSARY |disulfide bonds]]’’’ plays a significant role in determining tertiary protein structure<ref><pubmed>11806834</pubmed></ref>  
+
| align="center" | Evolutionarily conserved protein across species.<ref name="PMID9407023"><pubmed>9407023</pubmed></ref> Most Wnt proteins figure in the region of 40 kDa, each possessing a characteristic 23 or 24 cysteine residues highly conserved in their spacing. This suggests that the formation of ‘‘‘[[#GLOSSARY |disulfide bonds]]’’’ plays a significant role in determining tertiary protein structure<ref name="PMID11806834"><pubmed>11806834</pubmed></ref>
| align="center" | Initiates Wnt signalling when it binds as a ‘‘‘[[#GLOSSARY |ligand]]’’’ to the ‘‘‘[[#GLOSSARY |Fz]]’’’  receptor with ‘‘‘[[#GLOSSARY |LRP]]’’’ <ref name="PMID9312064"/> and destabilises the β-catenin degradation complex, dephosphorylating β-catenin and subsequently enabling its migration and accumulation in the cell nucleus for gene transcription <ref><pubmed>12781368</pubmed></ref>  
+
| align="center" | Initiates Wnt signalling when it binds as a ‘‘‘[[#GLOSSARY |ligand]]’’’ to the ‘‘‘[[#GLOSSARY |Fz]]’’’  receptor with ‘‘‘[[#GLOSSARY |LRP]]’’’ <ref name="PMID9312064"/> and destabilises the β-catenin degradation complex, dephosphorylating β-catenin and subsequently enabling its migration and accumulation in the cell nucleus for gene transcription <ref name="PMID12781368"><pubmed>12781368</pubmed></ref>
 
|  
 
|  
 
*The FRP family resembles the Wnt-interacting domain of ‘‘‘[[#GLOSSARY |Fz]]’’’  receptors, thus competitvely ‘‘‘[[#GLOSSARY |sequester]]’’’s Wnt from the ‘‘‘[[#GLOSSARY |Fz]]’’’  receptor <ref><pubmed>9118212</pubmed></ref>.
 
*The FRP family resembles the Wnt-interacting domain of ‘‘‘[[#GLOSSARY |Fz]]’’’  receptors, thus competitvely ‘‘‘[[#GLOSSARY |sequester]]’’’s Wnt from the ‘‘‘[[#GLOSSARY |Fz]]’’’  receptor <ref><pubmed>9118212</pubmed></ref>.
 
*WIF-1 has been shown to competitively antagonise Wnt binding to the ‘‘‘[[#GLOSSARY |Fz]]’’’  receptor <ref><pubmed>10201374</pubmed></ref>
 
*WIF-1 has been shown to competitively antagonise Wnt binding to the ‘‘‘[[#GLOSSARY |Fz]]’’’  receptor <ref><pubmed>10201374</pubmed></ref>
 
*Cerberus is a secreted protein that directly binds to the Wnt protein and inhibits signal transduction <ref><pubmed>10067895</pubmed></ref>
 
*Cerberus is a secreted protein that directly binds to the Wnt protein and inhibits signal transduction <ref><pubmed>10067895</pubmed></ref>
*Dkk indirectly inhibits Wnt protein signalling by binding to the co-receptor ‘‘‘[[#GLOSSARY |LRP]]-6’’’, thus making it unable to bind to Wnt for ‘‘‘[[#GLOSSARY |Fz]]’’’  signal transduction <ref><pubmed>11357136</pubmed></ref>  
+
*Dkk indirectly inhibits Wnt protein signalling by binding to the co-receptor ‘‘‘[[#GLOSSARY |LRP]]-6’’’, thus making it unable to bind to Wnt for ‘‘‘[[#GLOSSARY |Fz]]’’’  signal transduction <ref name="PMID11357136"><pubmed>11357136</pubmed></ref>
 
* The extracellular sFRP family of proteins possess a consensus ‘‘‘[[#GLOSSARY |CRD]]’’’  sequence on their ‘‘‘[[#GLOSSARY |N-terminus]]’’’, with which they directly bind to Wnt, and prevent its association with ‘‘‘[[#GLOSSARY |Fz]]’’’  receptors, thus antagonising the signal conduction <ref><pubmed>9096311</pubmed></ref>  
 
* The extracellular sFRP family of proteins possess a consensus ‘‘‘[[#GLOSSARY |CRD]]’’’  sequence on their ‘‘‘[[#GLOSSARY |N-terminus]]’’’, with which they directly bind to Wnt, and prevent its association with ‘‘‘[[#GLOSSARY |Fz]]’’’  receptors, thus antagonising the signal conduction <ref><pubmed>9096311</pubmed></ref>  
*Experiments suggest that axin indirectly blocks Wnt signalling, in a manner that renders further increases in Wnt levels ineffective <ref><pubmed>9230313</pubmed></ref>   
+
*Experiments suggest that axin indirectly blocks Wnt signalling, in a manner that renders further increases in Wnt levels ineffective <ref name="PMID9230313"><pubmed>9230313</pubmed></ref>   
 
|-
 
|-
 
| align="center" | ''‘[[#GLOSSARY |Fz]] ''' (FZD)
 
| align="center" | ''‘[[#GLOSSARY |Fz]] ''' (FZD)
 
| align="center" width="150px" | [[File:Frizzled.JPG|150px]]
 
| align="center" width="150px" | [[File:Frizzled.JPG|150px]]
| align="center" | ‘‘‘[[#GLOSSARY |Fz]]’’’  houses a ‘‘‘[[#GLOSSARY |CRD]]’’’  on the ‘‘‘[[#GLOSSARY |N-terminus]]’’’, seven transmembrane domains, and a ‘‘‘[[#GLOSSARY |PDZ domain]]’’’ on the ‘‘‘[[#GLOSSARY |C-terminus]]’’’ <ref><pubmed>11806834</pubmed></ref>  
+
| align="center" | ‘‘‘[[#GLOSSARY |Fz]]’’’  houses a ‘‘‘[[#GLOSSARY |CRD]]’’’  on the ‘‘‘[[#GLOSSARY |N-terminus]]’’’, seven transmembrane domains, and a ‘‘‘[[#GLOSSARY |PDZ domain]]’’’ on the ‘‘‘[[#GLOSSARY |C-terminus]]’’’ <ref name="PMID11806834"/>
| align="center" | Binds the Wnt ‘‘‘[[#GLOSSARY |ligand]]’’’ at the extracellular ‘‘‘[[#GLOSSARY |N-terminus]]’’’ for the initiation of the Wnt/β-catenin signalling pathway<ref><pubmed>11806834</pubmed></ref>  
+
| align="center" | Binds the Wnt ‘‘‘[[#GLOSSARY |ligand]]’’’ at the extracellular ‘‘‘[[#GLOSSARY |N-terminus]]’’’ for the initiation of the Wnt/β-catenin signalling pathway<ref name="PMID11806834"/>
 
|  
 
|  
 
|-
 
|-
 
| align="center" | ''‘[[#GLOSSARY |LRP]]-5/6''' (LRP)
 
| align="center" | ''‘[[#GLOSSARY |LRP]]-5/6''' (LRP)
 
| align="center" width="150px" | [[File:LRP6_active_sites.JPG|250px]]
 
| align="center" width="150px" | [[File:LRP6_active_sites.JPG|250px]]
| align="center" | A protein that spans multiple domains. The extracellular component is comprised of four EGF-like repeats on the ‘‘‘[[#GLOSSARY |N-terminus]]’’’ <ref><pubmed>11357136</pubmed></ref>
+
| align="center" | A protein that spans multiple domains. The extracellular component is comprised of four EGF-like repeats on the ‘‘‘[[#GLOSSARY |N-terminus]]’’’ <ref name="PMID11357136"/>
 
| align="center" | Co-receptor to the Wnt ‘‘‘[[#GLOSSARY |ligand]]’’’ in its interaction with the ‘‘‘[[#GLOSSARY |Fz]]’’’  receptor in signal transduction<ref><pubmed>11029008</pubmed></ref>. Causes translocation and association of axin to its intracellular tail, thus destabilising the β-catenin binding activity of axin<ref name=PMID11336703/>
 
| align="center" | Co-receptor to the Wnt ‘‘‘[[#GLOSSARY |ligand]]’’’ in its interaction with the ‘‘‘[[#GLOSSARY |Fz]]’’’  receptor in signal transduction<ref><pubmed>11029008</pubmed></ref>. Causes translocation and association of axin to its intracellular tail, thus destabilising the β-catenin binding activity of axin<ref name=PMID11336703/>
 
|  
 
|  
* Dkk antagonises ‘‘‘[[#GLOSSARY |LRP]]-5/6’’’ binding to Wnt through competitive binding<ref><pubmed>11357136</pubmed></ref> and complexes with the transmembrane protein Kremen2 for intracellular removal cell through endocytosis<ref><pubmed>12050670</pubmed></ref>
+
* Dkk antagonises ‘‘‘[[#GLOSSARY |LRP]]-5/6’’’ binding to Wnt through competitive binding<ref name="PMID11357136"/> and complexes with the transmembrane protein Kremen2 for intracellular removal cell through endocytosis<ref><pubmed>12050670</pubmed></ref>
 
|-
 
|-
 
| align="center" | ''‘[[#GLOSSARY |Dsh]] ''' (DVL)
 
| align="center" | ''‘[[#GLOSSARY |Dsh]] ''' (DVL)
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* Dapper antagonises ‘‘‘[[#GLOSSARY |Dsh]]’’’  in complex with Axin, ‘‘‘[[#GLOSSARY |GSK-3β]]’’’ , CK-I and β-catenin, leading to degradation of β-catenin and subsequent reduced signalling<ref><pubmed>11970895</pubmed></ref>  
 
* Dapper antagonises ‘‘‘[[#GLOSSARY |Dsh]]’’’  in complex with Axin, ‘‘‘[[#GLOSSARY |GSK-3β]]’’’ , CK-I and β-catenin, leading to degradation of β-catenin and subsequent reduced signalling<ref><pubmed>11970895</pubmed></ref>  
 
* ‘‘‘[[#GLOSSARY |PKC]]’’’ , a serine/threonine-specific protein kinase product of the non-canonical Wnt/Ca2+ pathway, inhibits ‘‘‘[[#GLOSSARY |Dsh]]’’’  through phosphorylation, preventing β-catenin nuclear translocation<ref><pubmed>11472835</pubmed></ref>  
 
* ‘‘‘[[#GLOSSARY |PKC]]’’’ , a serine/threonine-specific protein kinase product of the non-canonical Wnt/Ca2+ pathway, inhibits ‘‘‘[[#GLOSSARY |Dsh]]’’’  through phosphorylation, preventing β-catenin nuclear translocation<ref><pubmed>11472835</pubmed></ref>  
*Experiments suggest that axin indirectly blocks ‘‘‘[[#GLOSSARY |Dsh]]’’’  function, in a manner that renders further increases in ‘‘‘[[#GLOSSARY |Dsh]]’’’  levels ineffective <ref><pubmed>9230313</pubmed></ref>
+
*Experiments suggest that axin indirectly blocks ‘‘‘[[#GLOSSARY |Dsh]]’’’  function, in a manner that renders further increases in ‘‘‘[[#GLOSSARY |Dsh]]’’’  levels ineffective <ref name="PMID9230313"/>
 
|-
 
|-
 
| align="center" | Axin (AXIN)
 
| align="center" | Axin (AXIN)
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| align="center" | Involved in mediating both ‘‘‘[[#GLOSSARY |morphogenesis]]’’’ and maintenance of tissue integrity in the endothelium, bound to a-catenin that subsequently binds to the actin cytoskeleton. Gene expression is mediated in conjunction with ‘‘‘[[#GLOSSARY |TCF]] /[[#GLOSSARY |LEF]]’’’, upon translocation from the cytosol into the nucleus<ref><pubmed>10966653</pubmed></ref>  
 
| align="center" | Involved in mediating both ‘‘‘[[#GLOSSARY |morphogenesis]]’’’ and maintenance of tissue integrity in the endothelium, bound to a-catenin that subsequently binds to the actin cytoskeleton. Gene expression is mediated in conjunction with ‘‘‘[[#GLOSSARY |TCF]] /[[#GLOSSARY |LEF]]’’’, upon translocation from the cytosol into the nucleus<ref><pubmed>10966653</pubmed></ref>  
 
|  
 
|  
* The axin/’‘‘[[#GLOSSARY |APC]]’’’ /GSK-β complex phosphorylates b-catenin and marks it for ubiquitination and degradation by the ‘‘‘[[#GLOSSARY |’’’proteasome]]’’’<ref><pubmed>12781368</pubmed></ref>  
+
* The axin/’‘‘[[#GLOSSARY |APC]]’’’ /GSK-β complex phosphorylates b-catenin and marks it for ubiquitination and degradation by the ‘‘‘[[#GLOSSARY |’’’proteasome]]’’’<ref name="PMID12781368"/>
 
|-
 
|-
 
| align="center" | '''[[#GLOSSARY |TCF]] /[[#GLOSSARY |LEF]] ''' (TCF7/LEF)
 
| align="center" | '''[[#GLOSSARY |TCF]] /[[#GLOSSARY |LEF]] ''' (TCF7/LEF)
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! scope="col" width="150" | Normal function
 
! scope="col" width="150" | Normal function
 
! scope="col" width="290" | Effects of mutation
 
! scope="col" width="290" | Effects of mutation
! scope="col" width="150" | Associated cancers <ref name="PMID12781368"><pubmed>12781368</pubmed></ref>
+
! scope="col" width="150" | Associated cancers <ref name="PMID12781368"/>
 
|-  
 
|-  
 
| style="background: #FFDAB9; color: Black"|'''β-catenin'''
 
| style="background: #FFDAB9; color: Black"|'''β-catenin'''
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There is a large focus on finding more effective means of treating and preventing cancer. Current researchers are looking at inhibiting various components of the Wnt/β-catenin pathway to prevent the proliferation of cancer. Such treatments include:
 
There is a large focus on finding more effective means of treating and preventing cancer. Current researchers are looking at inhibiting various components of the Wnt/β-catenin pathway to prevent the proliferation of cancer. Such treatments include:
 
# Small molecule inhibitors can be used to block the interaction between β-catenin and TCF, thus preventing constitutive transcriptional activities that lead to the proliferation of cancer.  <ref name="PMID19619488"/>
 
# Small molecule inhibitors can be used to block the interaction between β-catenin and TCF, thus preventing constitutive transcriptional activities that lead to the proliferation of cancer.  <ref name="PMID19619488"/>
# Non-steroidal anti-inflammatory drugs (NSAIDs) function by interfering with β-catenin/TFC-dependent transcription, and have proven promising for the treatment and prevention of colorectal cancers. <ref name="PMID19619488"/> Examples of NSAIDs include exisulind, sulindac and aspirin.<ref><pubmed>15372092</pubmed></ref> The success of NSAIDs was measured, indicating a "40-50% reduction in mortality due to CRC in indicidulas taking NSAIDs".<ref name="PMID19619488"/> Furthermore, studies are being conducted into the use of NSAIDs to inhibit coclooxygenase-2 (COX-2) as an effective method of treatment and prevention of cancers. <ref name="PMID19619488"/>
+
# Non-steroidal anti-inflammatory drugs (NSAIDs) function by interfering with β-catenin/TFC-dependent transcription, and have proven promising for the treatment and prevention of colorectal cancers. <ref name="PMID19619488"/> Examples of NSAIDs include exisulind, sulindac and aspirin.<ref name="PMID15372092"/> The success of NSAIDs was measured, indicating a "40-50% reduction in mortality due to CRC in indicidulas taking NSAIDs".<ref name="PMID19619488"/> Furthermore, studies are being conducted into the use of NSAIDs to inhibit coclooxygenase-2 (COX-2) as an effective method of treatment and prevention of cancers. <ref name="PMID19619488"/>
 
# Frizzled-related proteins can be used as natural antagonists to manage the Wnt pathway.
 
# Frizzled-related proteins can be used as natural antagonists to manage the Wnt pathway.
 
# A “recombinant adenovirus (Ad-CBR) that constitutively expresses the β-catenin binding domain of APC” <ref name="PMID15578921"><pubmed>15578921</pubmed></ref> was developed, enabling APC to maintain its function of β-catenin degradation.
 
# A “recombinant adenovirus (Ad-CBR) that constitutively expresses the β-catenin binding domain of APC” <ref name="PMID15578921"><pubmed>15578921</pubmed></ref> was developed, enabling APC to maintain its function of β-catenin degradation.
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* The biochemistry of the activities of the destruction complex
 
* The biochemistry of the activities of the destruction complex
  
Taken from ''Wnt signaling: a common theme in animal development''<ref><pubmed>9407023</pubmed></ref> - the research outlined will be explored in the current literature
+
Taken from ''Wnt signaling: a common theme in animal development''<ref name="PMID9407023"/> - the research outlined will be explored in the current literature
 
* How do the ‘‘‘[[#GLOSSARY |Fz]]’’’  receptors work?
 
* How do the ‘‘‘[[#GLOSSARY |Fz]]’’’  receptors work?
 
* What is ‘‘‘[[#GLOSSARY |Dsh]]’’’  doing to transduce the signal?
 
* What is ‘‘‘[[#GLOSSARY |Dsh]]’’’  doing to transduce the signal?

Revision as of 10:10, 19 May 2012


2012 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7 | Group 8 | Group 9
Figure 1. 30 Years of Wnt Signalling Conference

Wnt/β-catenin Signalling Pathway

Introduction

2012 marks the 30th anniversary of the identification of Wnt-1, the first component of the Wnt signaling pathway to be discovered. Since this breakthrough, researchers from around the globe have extensively studied and pieced together the multiple components that form the Wnt signaling pathway as it is known and accepted today.

The classical pathway of Wnt signalling that is best understood in current research is the Wnt/β-catenin signalling pathway which features heavily in development, regeneration, stem cell regulation and cellular processes such as proliferation and migration[1].

Wnt proteins encompass a network of secreted glycolipoproteins[1], forming the basis of this highly conserved pathway. The various elements, agonists and antagonists of this pathway will be discussed, with key emphasis on the structure and function.

Furthermore, mutations in components of this pathway have been associated with cancers, developmental defects and other diseases which will be discussed in further detail.

It is important to note that the Wnt/β-catenin signalling pathway is becoming increasingly complex with the addition of new molecules being discovered in recent years. Areas of current research and future directions will also be outlined.

History

Figure 2. Overall structure of the β-catenin/XAxin-CBD complex

1973: A mutant of Drosophila melanogaster which had no wings was characterised as Wingless (Wg) [2]

1982: Roel Nusse and Harold Varmus found that mouse mammary tumour virus (MMTV) stimulated tumour formation when the Int1 (integration 1) gene was activated.[3]

1987: Rijsewijk et al identifies the mammalian homolog of the Drosophila Wingless gene as Int1.[4]

1989: Screening of zygotic lethal mutations in Drosophila identified new Wnt signalling molecules e.g. Dishevelled and Shaggy.[5]

1990: Riggleman et al reports on how the accumulation of Armadillo (mammalian homolog is beta-catenin) in Drosophila is posttranscriptionally controlled by the Wingless gene.[6]

1991: The nomenclature of Wingless and Int-1 was combined to form the mnemonic, Wnt.[7]

1991: Molecular cloning of β-catenin[8], LEF (lymphoid enhancer factor)[6] and TCF (T-cell factor)[9] transcription factors was conducted.

1992: Wingless signaling was discovered to be regulated by zeste-white 3 (also called Shaggy), which is the Drosophila homolog of mammalian Glycogen Synthase Kinase-3 (GSK-3).[10]

1993: Through nucleotide sequence analysis and peptide mapping, APC was found to associated with β-catenin[11][12]

1994: Mutations in Dishevelled affected phenotype and gene expression, indicating its crucial role in the Wnt signalling pathway[13]

1995: APC is involved in the degradation of β-catenin and hence regulates its cytoplasmic levels[14]

1996: The interaction of β-catenin with the transcription factors LEF and TCF was discovered[15]

1996: Bhanot et al identifies Frizzled as a receptor for Wnt ligands[16]

1996: A transmembrane protein identified as Porcupine was found to be involved in the processing of Wnt ligands[17]

1996: β-catenin levels were reported to be three times higher in tumour tissues than in normal specimens, indicating the link between β-catenin accumulation and colorectal cancers[18]

1997: Huber et al investigates the three dimensional structure of β-catenin[19]

1997: Phosphorylation of β-catenin by GSK3β (in the absence of Wnt) signals degradation via ubiquitin/proteasome[20]

1997: Identification of the homeotic gene Ubx as the first Wnt target gene [21]

1998: Axin 1 and axin 2 were found to interact with β-catenin, GSK3β and APC and to promote GSK3β - dependent phosphorylation and degradation of β-catenin [22] [23]

1999: Casein kinase 1 (CK1) was found to regulate β-catenin function [24]

1999: Protein phosphatase 2A (PP2A) interacts with the β-catenin destruction complex and modulates GSK3β (Glycogen synthase kinase 3β) function [25][26]

2000: The three-dimensional structure of the β-catenin–TCF complex was determined [27]

2000: Arrow, LRP-5 and LRP-6 were identified as coreceptors of Frizzled [28]

2001: LRP-5 was found to transduce Wnt signals by recruitment of axin to the plasma membrane [29]

2003: The tyrosine kinase receptor Derailed in Drosophila (RYK in mammals) was identified as an alternative Wnt receptor [30]

2006: LEF mutations were associated with sebaceous gland tumours in humans, showing that Wnt–β-catenin signalling is inhibited in these tumours [31]

2007: Dishevelled was found to polymerize at the plasma membrane and to recruit axin upon Wnt stimulation [32]

Normal function

Role of Wnt/β-catenin signalling in embryonic development

Figure 5. Neural crest stem cells that were cultured with bone morphogenic protein (BMP) and Wnt were shown to maintain stem cell markers (indicated in red and green)

The Wnt/β-catenin signaling pathway has been implicated as an important pathway in human fetal development. Through immunohistochemical staining, Eberhart and Argani (2001) localised nuclear beta catenin in fetal lung, placenta, kidney, cartilage, capillaries, adrenal glands and skin. This indicates that Wnt signaling regulates the development of specific set of organs and tissues.[33] For example, Wnt genes such as Wnt4 regulate the conversion of mesenchyme to epithelial cells in kidney morphogenesis.

In addition, Wnt/β-catenin signaling is also involved in maintaining the pluripotency of human embryonic stem cells (hESCs). Wnt3a promotes the reprogramming of somatic cells to pluripotency in conjunction with the classical transcription factors, Oct4, Sox2 and Nanog.[34] For example, Oct4 has the effect of repressing Wnt/β-catenin signaling in self renewing hESCs and is depressed during hESC differentiation[35]. Hence, this suggests that Wnt/β-catenin signaling is involved in differentiation rather than self renewal.

Key players in Wnt/β-catenin signalling

Protein (Gene) Diagram Structure Function Inhibitors
Wnt (WNT) 250px Evolutionarily conserved protein across species.[36] Most Wnt proteins figure in the region of 40 kDa, each possessing a characteristic 23 or 24 cysteine residues highly conserved in their spacing. This suggests that the formation of ‘‘‘disulfide bonds’’’ plays a significant role in determining tertiary protein structure[37] Initiates Wnt signalling when it binds as a ‘‘‘ligand’’’ to the ‘‘‘Fz’’’ receptor with ‘‘‘LRP’’’ [38] and destabilises the β-catenin degradation complex, dephosphorylating β-catenin and subsequently enabling its migration and accumulation in the cell nucleus for gene transcription [39]
  • The FRP family resembles the Wnt-interacting domain of ‘‘‘Fz’’’ receptors, thus competitvely ‘‘‘sequester’’’s Wnt from the ‘‘‘Fz’’’ receptor [40].
  • WIF-1 has been shown to competitively antagonise Wnt binding to the ‘‘‘Fz’’’ receptor [41]
  • Cerberus is a secreted protein that directly binds to the Wnt protein and inhibits signal transduction [42]
  • Dkk indirectly inhibits Wnt protein signalling by binding to the co-receptor ‘‘‘LRP-6’’’, thus making it unable to bind to Wnt for ‘‘‘Fz’’’ signal transduction [43]
  • The extracellular sFRP family of proteins possess a consensus ‘‘‘CRD’’’ sequence on their ‘‘‘N-terminus’’’, with which they directly bind to Wnt, and prevent its association with ‘‘‘Fz’’’ receptors, thus antagonising the signal conduction [44]
  • Experiments suggest that axin indirectly blocks Wnt signalling, in a manner that renders further increases in Wnt levels ineffective [45]
Fz ' (FZD) 150px ‘‘‘Fz’’’ houses a ‘‘‘CRD’’’ on the ‘‘‘N-terminus’’’, seven transmembrane domains, and a ‘‘‘PDZ domain’’’ on the ‘‘‘C-terminus’’’ [37] Binds the Wnt ‘‘‘ligand’’’ at the extracellular ‘‘‘N-terminus’’’ for the initiation of the Wnt/β-catenin signalling pathway[37]
LRP-5/6' (LRP) 250px A protein that spans multiple domains. The extracellular component is comprised of four EGF-like repeats on the ‘‘‘N-terminus’’’ [43] Co-receptor to the Wnt ‘‘‘ligand’’’ in its interaction with the ‘‘‘Fz’’’ receptor in signal transduction[46]. Causes translocation and association of axin to its intracellular tail, thus destabilising the β-catenin binding activity of axin[29]
  • Dkk antagonises ‘‘‘LRP-5/6’’’ binding to Wnt through competitive binding[43] and complexes with the transmembrane protein Kremen2 for intracellular removal cell through endocytosis[47]
Dsh ' (DVL) 250px Prevents phosphorylatory activity of ‘‘‘GSK-3β’’’ upon hyperphosphorylation by the Wnt/’‘‘LRP-5/6’’’ complex binding to the ‘‘‘Fz’’’ receptor[48], thus preventing β-catenin ubiquitination and degradation, to promote translocation into the nucleus.
  • ‘‘‘Nkd’’’ blocks feedback signalling, thus inhibiting ‘‘‘Dsh’’’ [49]
  • ‘‘‘Stbm’’’ binds to and jointly immunoprecipitates ‘‘‘Dsh’’’ [50]
  • Dapper antagonises ‘‘‘Dsh’’’ in complex with Axin, ‘‘‘GSK-3β’’’ , CK-I and β-catenin, leading to degradation of β-catenin and subsequent reduced signalling[51]
  • ‘‘‘PKC’’’ , a serine/threonine-specific protein kinase product of the non-canonical Wnt/Ca2+ pathway, inhibits ‘‘‘Dsh’’’ through phosphorylation, preventing β-catenin nuclear translocation[52]
  • Experiments suggest that axin indirectly blocks ‘‘‘Dsh’’’ function, in a manner that renders further increases in ‘‘‘Dsh’’’ levels ineffective [45]
Axin (AXIN) 250px In the absence of Wnt signalling, it constitutes the β-catenin ubiquitination complex along with ‘‘‘APC’’’ and ‘‘‘GSK-3β’’’ [38] and its role as a ‘‘‘scaffolding protein’’’ enhances ‘‘‘GSK-3β’’’ phosphorylation of β-catenin[53]
  • Wnt downregulates axin through increased ‘‘‘Dsh’’’ expression, hindering axin phosphorylation by GSK-3b, leading to decreased stability and ultimately a shorter half-life[54]
GSK-3β ' (GSK3β) 250px Constitutes the β-catenin ubiquitination complex along with axin and ‘‘‘APC’’’ [38]; its primary role in the Wnt/β-catenin signalling pathway is inhibition of β-catenin nuclear translocation through phosphorylation of three amino acids at the ‘‘‘N-terminus’’’[55] after priming by ‘‘‘CK-I’’’ [56], for subsequent ubiquitination and degradation by the ‘‘‘’’’proteasome’’’. This activity is enhanced by ‘‘‘GSK-3β’’’ phosphorylation of axin[53] and ‘‘‘APC’’’ [57] which appears to promote phosphorylation of β-catenin by ‘‘‘GSK-3β’’’ in the complex.
  • Frat1, which appears to be recruited by ‘‘‘Dsh’’’ in complex, tightly binds GSK-3b even after complex dissociation and b-catenin degradation[58]
  • ‘‘‘PKC’’’ inhibits ‘‘‘GSK-3β’’’ phosphorylatory activity[59]
Diversin Diversin.JPG Primes b-catenin for degradation by recruiting CK-I to phosphorylate serine 45 on the ‘‘‘N-terminus’’’ before subsequent phosphorylation of threonine 41, serine 37 and serine 33 by GSK-b and degradation by E3 ubiquitin ligase β-TrCP[60]
APC ' (APC) 150px A large 312 kDa protein Possesses a numerous and varied set of roles ranging from cell migration and adhesion, cell cycle regulation and chromosome stability[61]. In the Wnt/β-catenin signalling pathway, ‘‘‘APC’’’ constitutes the β-catenin ubiquitination complex along with axin and ‘‘‘GSK-3β’’’ [38]. It binds to axin via ‘‘‘SAMP’’’ elements[22], and to β-catenin via three 15-amino acid repeats and seven 20-amino acid repeats[62]
β-catenin (CTNNB1) Murine bcat.jpg The ‘‘‘N-terminus’’’ accommodates a critical sequence of thirteen armadillo repeats that competitively bind E-cadherin, ‘‘‘LEF-1’’’ and ‘‘‘APC’’’ [63]. The ‘‘‘C-terminus’’’ houses a glycine-rich transactivation domain[64] Involved in mediating both ‘‘‘morphogenesis’’’ and maintenance of tissue integrity in the endothelium, bound to a-catenin that subsequently binds to the actin cytoskeleton. Gene expression is mediated in conjunction with ‘‘‘TCF /LEF’’’, upon translocation from the cytosol into the nucleus[65]
  • The axin/’‘‘APC’’’ /GSK-β complex phosphorylates b-catenin and marks it for ubiquitination and degradation by the ‘‘‘’’’proteasome’’’[39]
TCF /LEF (TCF7/LEF) Protein structure LEF1 TCF4 TCF3.JPG The ‘‘‘N-terminus’’’ constitutes the β-catenin interaction domain; ‘‘‘TCF /LEF’’’ also possesses a ‘‘‘HMG’’’ box DNA-binding domain[66] Mediates DNA binding when in complex with β-catenin; the ‘‘‘N-terminus’’’ of ‘‘‘TCF /LEF’’’ associates with the β-catenin ‘‘‘C-terminus’’’ [67]
  • ’‘‘CAMKII’’’ , a serine/threonine-specific protein kinase product of the non-canonical Wnt/Ca2+ pathway, inhibits ‘‘‘LEF’’’ downstream of β-catenin, acting at the transcription factor complex level[68]

Mechanism of action

  • β-catenin commonly exists as a subunit making up the cadherin protein complex, where it links E-cadherin to α-catenin [69]
  • Cadherin proteins play an integral role in the formation of adhesion junctions between cells [69]
  • β-catenin also acts to help anchor the actin cytoskeleton within the cell [69]
  • The β-catenin gene can be said to function as an oncogene given that the promotion of transcription of Wnt target genes by β-catenin has been shown to be involved in the development of an array of cancers including basal cell carcinoma, colorectal cancer and breast cancer.[70][71][72]

In the absence of Wnt (“off state”)

Figure 3. "Off" and "On" States of the Wnt/Beta-catenin Signalling Pathway
  • No extracellular "Wnt signal"
  • β-catenin targeted for proteosomal degradation by its incorporation into a “destruction complex” comprising:
  • APC and axin assemble into a structural scaffold, allowing the phosphorylation of the N-terminal of β-catenin by the coordinated action of CK1 and GSK-3β[74]
  • β-catenin is recognised by E3 ligase β-Trcp, ubiquinated and subsequently degraded by the 26S proteosome [75][38]
  • Level of β-catenin in the cytosol is low, and therefore does not reach the nucleus at a level sufficient enough to affect transcription
  • No transcription of Wnt target genes

In the presence of Wnt (“on state”)

  • Extracellular “Wnt signal” binds to a cell surface G-protein coupled receptor of the “Frizzled” (FRZ) family
  • Low-density lipoprotein receptor-related protein (LRP) family functions as a transmembrane co-receptor for FRZ
    • Recruitment of axin to the membrane where it is degraded upon initiation of the Wnt signalling cascade
    • Dissociation of β-catenin into the cytoplasm without degradation by the “destruction complex" [78][38]
  • Activation (phosphorylation and poly-ubiquination) of proteins of the “Dishevelled” (Dsh) family
    • Implicated in the inactivation of the “destruction complex” by the recruitment of axin and GSK-3β away from the complex [32]
  • With the complex now interrupted, β-catenin resists ubiquination and reaches the nucleus in increased levels where it functions as a transcriptional enhancer alongside TCF[73]
  • β-catenin directly competes with Groucho/TLEs for TCF binding [79]
  • TCF promotes binding of RNA polymerase to DNA template strand
  • Induction of the downstream transcription of Wnt target genes, for example:
    • oncogenes such as c-Myc and cyclin D1 involved in cell cycle control [80]
    • MMP-7 and uPA, associated with metastasis and the invasion of cancer cells [71]

Tumour Cells

  • In a tumour cell, the Wnt siganalling pathway is activated abberantly either:
    • Innappropriately by an extracellular Wnt signal
    • In the absence of an extracellular Wnt signal
      • Usually results from a mutation in one of the subunits that comprise the “destruction complex”, resulting in an inability to degrade cytosolic β-catenin [80]
      • Mutant APC proteins characteristic of those associated with cancer, have been shown to upregulate transcription of Wnt target genes due to an inability to regulate levels of cytosolic and nuclear β-catenin [14]

MOVIE: A detailed explanation of Wnt/β-catenin signalling

Diseases associated with Wnt/β-catenin signalling

As described above, the Wnt/β-catenin signalling pathway plays a critical role in development, stem cell regulation and cellular processes such as proliferation and migration. Thus mutations in components of this pathway have been associated with cancers, hereditary disorders, developmental defects and other diseases.

Wnt/B-Catenin Pathway and the Onset of Cancer

The high number of repressor genes involved in the Wnt/β-catenin pathway indicates that it is imperative for this pathway to be tightly regulated. [81] These repressor genes include APC, Axin 1 and Axin 2. Given the pathways involvements in the regulation of stem cell choice to proliferate or self renew, there is a strong correlation between mutations in these genes and the onset of cancer. The table below describes in further detail some mutations of the Wnt signalling pathway and associated cancers.

Mutations of the Wnt signalling pathway and associated cancers

Figure 4. The colon of a patient with Familial Adenomatous Polyposis (FAP)
Gene Normal function Effects of mutation Associated cancers [39]
β-catenin Primary Wnt effector. Acts as an oncogene. In the nucleus, β-catenin functions as a cofactor for TCF transcription factors which specify a subset of genes, such as cyclin D1 and c-MYC, which are responsible for determining cell fate and regulation of proliferation. [39] Any mutations that inhibit its destruction motif would cause constitutively active β-catenin signalling, leading to excessive stem cell renewal and proliferation thus predisposing the cells to the formation of tumours. [81]
  • Colorectal cancer
  • Prostate cancer
  • Uterine endometrial cancer
  • Melanoma
  • Hepatoblastoma (liver cancer)
  • Medulloblastoma (brain cancer)
  • Pancreatoblastoma (pancreatic cancer)
  • Ovarian carcinoma
  • Thyroid carcinoma
  • Pancreatic carcinoma
  • Hepatocellular carcinoma
  • Lung adenocarcinomas
  • Esophageal adenocarcinomas
  • Synovial sarcoma
APC Facilitates β-catenin degradation; acts as a tumour suppressor Mutational inactivation of APC inhibits degradation of β-catenin, leading to the over-stabilisation and accumulation of β-catenin in the nucleus of the cell. [82] (See β-catenin above).


Colorectal Cancer (CRC)

  • APC mutations in intestinal epithelial cells lead constitutive β-catenin/Tcf4 complex activation, causing unrestrained production of crypt stem cells, resulting in cancer. [83] Once the cancer has spread widely through the body, it is often incurable and management focuses on chemotherapy and improving quality of life.
  • Activating mutations in the canonical Wnt pathway is responsible for approximately 90% of all colorectal cancer cases. [73] Of these, 85% are caused by loss of function mutations in APC. [39]
  • Colorectal cancer is the fourth most commonly diagnosed cancer in the world.


  • Colorectal cancer
  • Prostate cancer
  • Melanoma
  • Hepatoblastoma
  • Medulloblastoma
  • Ovarian carcinoma
  • Pancreatic non-ductal acinar cell carcinomas
  • Synovial carcinoma
  • Desmoid tumor
  • Gastric adenoma
  • Breast fibromatoses
  • Familial Adenomatous Polyposis (FAP) [Figure 4]
Axin 1 & Axin 2 Serve as scaffolding components for the β-catenin degradation complex. Acts as a tumour suppressor. Mutational inactivation of Axin 1 or Axin 2 inhibits degradation of β-catenin, leading to the over-stabilisation and accumulation of β-catenin in the nucleus of the cell.
  • Ovarian carcinoma
  • Hepatocellular carcinoma
  • Medulloblastoma
  • Predisposition to colon cancer [84]


For a comprehensive list of human diseases associated with mutations of the Wnt signalling components, please refer to Table 1 in the following article: Wnt/β-catenin signaling: components, mechanisms, and diseases[73]

Treatment:

There is a large focus on finding more effective means of treating and preventing cancer. Current researchers are looking at inhibiting various components of the Wnt/β-catenin pathway to prevent the proliferation of cancer. Such treatments include:

  1. Small molecule inhibitors can be used to block the interaction between β-catenin and TCF, thus preventing constitutive transcriptional activities that lead to the proliferation of cancer. [73]
  2. Non-steroidal anti-inflammatory drugs (NSAIDs) function by interfering with β-catenin/TFC-dependent transcription, and have proven promising for the treatment and prevention of colorectal cancers. [73] Examples of NSAIDs include exisulind, sulindac and aspirin.[82] The success of NSAIDs was measured, indicating a "40-50% reduction in mortality due to CRC in indicidulas taking NSAIDs".[73] Furthermore, studies are being conducted into the use of NSAIDs to inhibit coclooxygenase-2 (COX-2) as an effective method of treatment and prevention of cancers. [73]
  3. Frizzled-related proteins can be used as natural antagonists to manage the Wnt pathway.
  4. A “recombinant adenovirus (Ad-CBR) that constitutively expresses the β-catenin binding domain of APC” [85] was developed, enabling APC to maintain its function of β-catenin degradation.
  5. Monoclonal antibodies are being used against Wnt proteins by inducing apoptosis in cancer cells [85].

Current research

Future directions

  • What is the mode of translocation of β-catenin into the nucleus upon dissociating with axin?[29]
  • Elucidate the mechanism for the hyperphosphorylation of ‘‘‘Dsh’’’ by Wnt/’‘‘Fz /LRP-5/6 binding concurrent to, but mutually exclusive from, axin destabilisation[86]
  • The biochemical events connecting the components of the canonical Wnt pathway
  • Production of purified Wnt proteins to assist with research
  • Production of effective Wnt antibodies
  • Structure of the Wnt1 protein
  • Complete understanding of the routing and the coincident posttranslational modifications of Wnt proteins in the secreting cell
  • The rules that dictate the movement of Wnt proteins between cells
  • The biochemistry of the activities of the destruction complex

Taken from Wnt signaling: a common theme in animal development[36] - the research outlined will be explored in the current literature

  • How do the ‘‘‘Fz’’’ receptors work?
  • What is ‘‘‘Dsh’’’ doing to transduce the signal?
  • What is the relationship between ‘‘‘APC’’’ and Wnt signaling?
  • How does Arm/β-catenin get into the nucleus?

Glossary

List of Abbreviations Used

  • APC: adenomatous polyposis coli
  • CAMKII: Ca2+/calmodulin-dependent protein kinase II
  • CK-I: casein kinase I
  • CRD: cysteine rich domain
  • Dsh: Dishevelled
  • Fz: Frizzled
  • GSK-3β: glycogen synthase kinase-3β
  • HMG: high mobility group
  • LEF: lymphoid enhancer-binding factor
  • LRP: lipoprotein receptor–related protein
  • Nkd: naked cuticle
  • PKC: protein kinase C
  • SAMP: Ser-Ala-Met-Pro repeated amino acid segment
  • Stbm: strabismus
  • TCF: T cell factor

List of Terminology

  • agenesis: the inability to form an organ during embryogenesis due to the lack of primordial tissues
  • agonist: a molecule which binds to a receptor to produce a physiological response
  • antagonist: a molecule which inhibits the physiological function of another substance
  • apoptosis: programmed cell death
  • C-terminus: the end of the polypeptide chain which features a free carboxyl group (-COOH)
  • canonical: standard and well accepted
  • destruction motif: short sequences which provide stability of other proteins when deleted or mutated
  • differentiation: the process by which cells become mature and specialised in structure and function
  • disulfide bonds: a covalent bond between sulfur atoms
  • gain of function mutation: mutation produces a protein which has an enhanced or abnormal function
  • glycolipoprotein: a protein with attached lipid and carbohydrate groups
  • homeotic gene: genes involved in embryonic development, specifically controlled the anterior-posterior axis
  • histone deacetylation: the process by which histone deacetylase removes acetyl groups on the histones to increase affinity and condense DNA, thus preventing transcription
  • homolog: gene which is related to another gene by common descent
  • immunohistochemistry: detecting antigens on a tissue using labelled antibodies
  • immunoprecipitation: the process of isolating a protein antigen using an antibody that binds to it to form an insoluble complex
  • lethal mutation: a mutation which can result in the death of the organism
  • ligand: a molecule which binds to another entity
  • loss of function mutation: mutation produces a protein which has decreased or no function
  • mnemonic: a tool which assists memory
  • monoclonal antibody: an antibody engineered from a single clone of cells
  • morphogenesis: the differentiation and growth of tissues and organs during embryo development
  • N-terminus: the start of the polypeptide chain which features a free amine group (-NH2)
  • oncogene: genes which transform normal cells into cancerous cells
  • pluripotency: the ability of stem cells to differentiate into ectoderm, mesoderm and endoderm
  • PDZ domain: a common structural domain of 80-90 amino acids of signalling proteins, whose name is derived from the first three letters of the first three proteins to be discovered to share the domain: post synaptic density protein, Drosophila disc large tumor suppressor, zonula occludens-1 protein
  • proteasome: a large intracellular particle which degrades proteins
  • repressor: a protein which binds to the operator and prevents transcription
  • self renewal: the capability to undergo numerous cell divisions and maintain undifferentiation
  • sequester: to isolate or separate
  • scaffolding protein: proteins which regulate signal transduction by organising and assembling signalling complexes
  • tumour suppressor gene: produces proteins which control cell growth to prevent uncontrolled cell proliferation
  • ubiquitylation: the process of adding ubiquitin

External links

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