2012 Group 5 Project

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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)[9] and TCF (T-cell factor)[10] 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).[11]

1993: Through nucleotide sequence analysis and peptide mapping, APC was found to associated with β-catenin[12][13]

1994: Mutations in Dishevelled affected phenotype and gene expression, indicating its crucial role in the Wnt signalling pathway[14]

1995: APC is involved in the degradation of β-catenin and hence regulates its cytoplasmic levels[15]

1996: The interaction of β-catenin with the transcription factors LEF and TCF was discovered[16]

1996: Bhanot et al identifies Frizzled as a receptor for Wnt ligands[17]

1996: A transmembrane protein identified as Porcupine was found to be involved in the processing of Wnt ligands[18]

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[19]

1997: Huber et al investigates the three dimensional structure of β-catenin[20]

1997: Phosphorylation of β-catenin by GSK3β (in the absence of Wnt) signals degradation via ubiquitin/proteasome[21]

1997: Identification of the homeotic gene Ubx as the first Wnt target gene [22]

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 [23][24]

1999: Casein kinase 1 (CK1) was found to regulate β-catenin function [25]

1999: Protein phosphatase 2A (PP2A) interacts with the β-catenin destruction complex and modulates GSK3β (Glycogen synthase kinase 3β) function [26][27]

2000: The three-dimensional structure of the β-catenin–TCF complex was determined [28]

2000: Arrow, LRP5 and LRP6 were identified as coreceptors of Frizzled [29]

2001: LRP5 was found to transduce Wnt signals by recruitment of axin to the plasma membrane [30]

2003: The tyrosine kinase receptor Derailed in Drosophila (RYK in mammals) was identified as an alternative Wnt receptor [31]

2006: LEF1 mutations were associated with sebaceous gland tumours in humans, showing that Wnt–β-catenin signalling is inhibited in these tumours [32]

2007: Dishevelled was found to polymerize at the plasma membrane and to recruit axin upon Wnt stimulation [33]

Mechanism of action

  • β-catenin commonly exists as a subunit making up the cadherin protein complex, where it links E-cadherin to α-catenin [34]
  • Cadherin proteins play an integral role in the formation of adhesion junctions between cells [34]
  • β-catenin also acts to help anchor the actin cytoskeleton within the cell [34]
  • 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.[35][36][37]

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:
    • Intracellular Axin, GSK-3β (glycogen synthase kinase β) and APC (Adenomatous polyposis coli – coded for by the APC tumour suppressor gene)[38]
  • 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 [39]
  • β-catenin is recognised by E3 ligase β-Trcp, ubiquinated and subsequently degraded by the 26S proteosome [40][41]
  • 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
    • TCF/LEF proteins act as transcriptional repressors, binding to proteins of the TLE /Groucho family [42]
    • HDAC represses transcription via chromatin remodelling in the form of histone deacetylation [43]

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" [44][45]
  • 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 [46]
  • 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 [38]
  • β-catenin directly competes with Groucho/TLEs for TCF binding [47]
  • 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 [48]
    • MMP-7 and uPA, associated with metastasis and the invasion of cancer cells [49]

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 [50]
      • 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 [51]


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

Diseases associated with Wnt/β-catenin signalling

The Wnt pathways is heavily involved in the fate of stem cells and plays a role in the regulation of stem cell choice to proliferate or self renew. Thus there is a strong correlation between mutations of genes involved in the Wnt signalling pathway and the onset of cancer. The table below outlines some diseases associated with mutations in the Wnt pathway.


File:Table 1. Human Diseases Associated with Mutations of the Wnt Signaling Components.png
Table 1. Human Diseases Associated with Mutations of the Wnt Signaling Components[52]
File:Figure. Schematic representation of a colon crypt and proposed model for polyp formation.png
Figure 3. Schematic representation of a colon crypt and proposed model for polyp formation[53]
Disease Gene affected Mutation Normal function Effects of mutation
Familial Adenomatous Polyposis (FAP) APC Loss of function (LOF) mutation. One defective APC allele is inherited. [54][55] Facilitates B-catenin degradation and acts as a tumour suppressor Large numbers of colon adenomas or polyps (benign out growths of epithelial cells) are developed. Inevitably, some of these polyps progress into malignant adenocarnicoma.
Colorectal cancer APC LOF in both APC alleles. [56] Facilitates B-catenin degradation and acts as a tumour suppressor Mutational inactivation of APC leads to the inappropriate stabilization of B-catenin [57]. Through the action of constitutive B-catenin/Tcf4 complexes, tcf reporter constructs are inappropriately transcribed [58]. In intestinal epithelial cells the constitutive B-catenin/Tcf4 complex leads to an unrestrained proliferation of crypt progenitors, resulting in cancer. (picture)
Hepatocellular carcinomas (liver cancer) Axin2 Loss of function mutation Facilitates B-catenin degradation and acts as a tumour suppressor Mutations cause familial tooth agenesis and are predisposed to colorectal cancer [59].
A wide variety of solid tumours (eg. hair follicle tumours) B-catenin Oncogenic B-catenin GOF mutations Primary Wnt effector and acts as an oncogene Activating point mutations in B-catenin remove its N-terminal Ser/Thr destruction motif (Morin et al., 1997), resulting in a constitutively active oncogenic B-catenin transgene.This leads to excessive stem cell renewal and proliferation, predisposing the cells to the formation of tumours [60].

Treatment Options

  1. Non-steroidal anti-inflammatory drugs (NSAIDs) have proven promising for the prevention of colorectal cancers. NSAIDs interfere with B-catenin/TFC-dependent transcription by inhibiting prostaglandin production, thus preventing the proliferation of cancer. [61]
  2. Frizzled-related proteins can be used as natural antagonists to manage the Wnt pathway.
  3. Recombinant adenovirus (Ad-CBR) that constitutively expresses the B-catenin binding domain of APC would prevent the accumulation of nuclear B-catenin [62].
  4. Monoclonal antibodies against Wnt proteins induce apoptosis in cancer cells [63].

Key players in Wnt/β-catenin Signalling

Protein (Gene) Diagram Structure Function Inhibitors
Wnt (WNT) 250px Evolutionarily conserved protein across species[64]. 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[65] Initiates Wnt signalling when it binds as a ligand to the Fz receptor with LRP[66] and destabilises the β-catenin degradation complex, dephosphorylating β-catenin and subsequently enabling its migration and accumulation in the cell nucleus for gene transcription [67]
  • The FRP family resembles the Wnt-interacting domain of Fz receptors, thus competitvely sequesters Wnt from the Fz receptor [68].
  • WIF-1 has been shown to competitively antagonise Wnt binding to the Fz receptor [69]
  • Cerberus is a secreted protein that directly binds to the Wnt protein and inhibits signal transduction [70]
  • 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 [71]
  • 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 [72]
  • Experiments suggest that axin indirectly blocks Wnt signalling, in a manner that renders further increases in Wnt levels ineffective [73]
Fz (FZD) 150px Fz houses a CRD on the N-terminus, seven transmembrane domains, and a PDZ domain on the C-terminus[74] Binds the Wnt ligand at the extracellular N-terminus for the initiation of the Wnt/β-catenin signalling pathway[75]
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 [76] Co-receptor to the Wnt ligand in its interaction with the Fz receptor in signal transduction[77]. Causes translocation and association of axin to its intracellular tail, thus destabilising the β-catenin binding activity of axin[78]
  • Dkk antagonises LRP-5/6 binding to Wnt through competitive binding[79] and complexes with the transmembrane protein Kremen2 for intracellular removal cell through endocytosis[80]
Dsh (DVL) 250px Prevents phosphorylatory activity of GSK-3β upon hyperphosphorylation by the Wnt/LRP-5/6 complex binding to the Fz receptor[81], thus preventing β-catenin ubiquitination and degradation, to promote translocation into the nucleus.
  • Nkd blocks feedback signalling, thus inhibiting Dsh[82]
  • Stbm binds to and jointly immunoprecipitates Dsh[83]
  • Dapper antagonises Dsh in complex with Axin, GSK-3β, CK-I and β-catenin, leading to degradation of β-catenin and subsequent reduced signalling[84]
  • PKC, a serine/threonine-specific protein kinase product of the non-canonical Wnt/Ca2+ pathway, inhibits Dsh through phosphorylation, preventing β-catenin nuclear translocation[85]
  • Experiments suggest that axin indirectly blocks Dsh function, in a manner that renders further increases in Dsh levels ineffective [86]
Axin (AXIN) 250px In the absence of Wnt signalling, it constitutes the β-catenin ubiquitination complex along with APC and GSK-3β [87] and its role as a scaffolding protein enhances GSK-3β phosphorylation of β-catenin[88]
  • Wnt downregulates axin through increased Dsh expression, hindering axin phosphorylation by GSK-3b, leading to decreased stability and ultimately a shorter half-life[89]
GSK-3β (GSK3β) 250px Constitutes the β-catenin ubiquitination complex along with axin and APC[90]; 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[91] after priming by CK-1[92], for subsequent ubiquitination and degradation by the proteasome. This activity is enhanced by GSK-3β phosphorylation of axin[93] and APC[94] 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[95]
  • PKC inhibits GSK-3β phosphorylatory activity[96]
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[97]
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[98]. In the Wnt/β-catenin signalling pathway, APC constitutes the β-catenin ubiquitination complex along with axin and GSK-3β[99]. It binds to axin via SAMP elements[100], and to β-catenin via three 15-amino acid repeats and seven 20-amino acid repeats[101]
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[102]. The C-terminus houses a glycine-rich transactivation domain[103] 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[104]
  • The axin/APC/GSK-β complex phosphorylates b-catenin and marks it for ubiquitination and degradation by the proteasome[105]
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[106] Mediates DNA binding when in complex with β-catenin; the N-terminus of TCF/LEF associates with the β-catenin C-terminus[107]
  • CamKII, a serine/threonine-specific protein kinase product of the non-canonical Wnt/Ca2+ pathway, inhibits LEF-1 downstream of β-catenin, acting at the transcription factor complex level[108]

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.[109] 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.[110] For example, Oct4 has the effect of repressing Wnt/β-catenin signaling in self renewing hESCs and is depressed during hESC differentiation.[111] Hence, this suggests that Wnt/β-catenin signaling is involved in differentiation rather than self renewal.

Future directions

  • What is the mode of translocation of β-catenin into the nucleus upon dissociating with axin?[112]
  • Elucidate the mechanism for the hyperphosphorylation of Dsh by Wnt/Fz/LRP-5/6 binding concurrent to, but mutually exclusive from, axin destabilisation[113]
  • 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[114] - 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

  • 7TM: seven transmembrane receptor
  • 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

  • C-terminus: the end of the polypeptide chain which features a free carboxyl group (-COOH)
  • canonical: standard and well accepted
  • differentiation: the process by which cells become mature and specialised in structure and function
  • glycolipoprotein: a protein with attached lipid and carbohydrate groups
  • homeotic gene: genes involved in embryonic development, specifically controlled the anterior-posterior axis
  • 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
  • self renewal: the capability to undergo numerous cell divisions and maintain undifferentiation
  • ubiquitylation: the process of adding ubiquitin

External links

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