Adhesion molecules in the regulation of cell differentiation: Mainly about Wnt
FIG 14.3 The Groucho/TCF-1 repressor complex.
(a)TCF-1 (or LEF-1), Groucho and HDAC form a repressor complex that renders DNA inaccessible to the transcription machinery (RNA polymerases).
(b)Binding of -catenin to TCF-1 (or LEF-1) removes Groucho and with it the histone deacetylase. Acetylation proceeds, allowing the relaxation of the nucleosomes. Not all components of the deacetylation complex are shown.
Contribution of different species to the elucidation of the Wnt signal transduction pathway
We have limited the description of the discovery of Wnt and its downstream signalling components to the epidermal segment polarity of the larval cuticle in Drosophila. However, experiments using Xenopus laevis have also contributed to the elucidation of this pathway. The Xenopus model attracted much attention after it was found that injection of Wnt mRNA into developing embryos causes a new and complete dorsal–ventral axis, giving rise to two-headed tadpoles.43 The local production of Wnt creates an independent Spemann organizer. With respect to molecular mechanisms of Wnt signalling, Xenopus has helped to elucidate the mechanism of -catenin stabilization and subsequent nuclear localization, it has helped to define the Wnt coreceptors LRP5 and LRP6, the discovery that -catenin binds TCF and the Wnt antagonists such as Dkk, sFRP, and Cerberus. Finally, Xenopus has played an important role in the discovery of non-canonical ( -catenin-independent) pathways (for a review, see Moon
et al.;44 for a note on the Spemann organizer, see page 618).
The TCF family of vertebrate transcription factors consists of four members: TCF-1–4. They all contain an HMG box, which is a DNA-binding
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TCF, T cell factor, initially cloned as a lymphoid cell transcription
factor. Members of the TCF family are now recognized as activators and repressors of genes implicated in regulation of the cell cycle in a number of cell types. The name T cell factor should not be confused with the ternary complex factor p62TCF, a quite distinct entity, linked with the MAP kinases (see
page 342).
FIG 14.4 Groucho Marx.
domain, first recognized in the non-histone high mobility group (HMG) of proteins. TCF-1 is mainly expressed in cells of T cell lineage. In mice, its
inactivation results in failure to undergo T cell development. Lef-1 is mainly expressed during development in the neural crest, mesencephalon, tooth germs and whisker follicles. TCF-3 is expressed in stomach epithelium, hair follicles, and keratinocytes. TCF-4 appears much later in embryogenesis and is most highly expressed in the midbrain and in intestinal and mammary epithelium. In adult mice, expression of TCF-4 is essential for the maintenance of the progenitor compartment of the gut epithelium.45
Dual regulation by TCF/LEF. Through their interaction with Groucho these proteins prevent access of the transcriptional machinery to DNA, an action similar to that of the retinoblastoma protein (see page 308). Association with-catenin (or with plakoglobin) relieves the repression and turns TCF into a true transcription factor, initiating transcription of genes that affect cell fate, proliferation, adhesion, and extracellular matrix degradation.46
Groucho and chromatin condensation. Groucho is the name of a mutant that has an increased number of bristles around the eye, reminiscent of the bushybrowed Groucho Marx (Figure 14.4). It encodes a nuclear protein expressed ubiquitously in both embryos and imaginal discs, and acts as a transcriptional repressor of several genes in Drosophila development.47 Drosophila Groucho is the archetype of a conserved family of transcriptional corepressors collectively named Groucho/TLE. It participates in a protein complex, enhancer of split ((spl)m9/m10) comprising at least five gene products. Together they repress neurogenesis in proneural clusters of the ventral neuroectoderm (to avoid
an excess of brain formation). Sequence analysis indicates that one of the components contains WD domains (sequence terminating with Trp-Asp), also characteristic of the -subunits of G proteins. The human variants of Groucho are therefore known as transducin-like enhancer-of-split (TLE).
The murine and human genomes harbour four full-length homologues of Groucho, as well as a gene that encodes a truncated Groucho protein termed hAES (amino-terminal enhancer of split). In mice, the homologues are called Groucho-related genes, Grg (1–4). There is also a truncated version, Grg-5. Members of this family all share a N-terminal Q (glutamine-rich) domain, GP (glycine/proline-rich) domain, CCN domain (containing putative casein kinase 2/Cdc2 phosphorylation sites and nuclear localization signal), SP (serine/ proline-rich) domain, and four WD40 repeats (protein interaction domain). Numerous transcription factors interact with Groucho.45
Adenomatous polyposis coli (APC) and the localization of -catenin
Hints of the mechanism by which -catenin is localized were provided by an inherited disorder, adenomatous polyposis coli (APC). This is characterized by
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Adhesion molecules in the regulation of cell differentiation: Mainly about Wnt
early onset of numerous polyps in the colon, which progress to malignancy.48 The polyps are a consequence of germline and (subsequent) somatic mutations that cause truncation of a protein, also termed APC49 (for a review, see Polakis50) (Figure 14.5).
Intact APC forms part of a complex of proteins that interact with -catenin and bring about its destruction, while stimulation of the Wnt pathway protects the -catenin, allowing it to re-accumulate.51 In Drosophila, genetic ablation of APC causes up-regulation of -catenin signalling.52 In humans, a lack of APC and the ensuing nuclear translocation of -catenin are linked to cell transformation giving rise to colorectal polyps. When APC is over-expressed there is a reduction in the cellular content of -catenin due to enhanced degradation.53
The importance of APC has also been established in a transgenic mouse model allowing inducible ablation of the APC gene. Here, Wnt signalling is acutely activated in intestinal epithelial cells. There is copious nuclear accumulation of -catenin, and this coincides with transformation towards a ‘crypt progenitor-like’ phenotype.54 Finally, over-expression of -catenin
FIG 14.5 -Catenin’s choice.
Following synthesis on free ribosomes, -catenin binds to the cytosolic segment of cadherin at the surface of the rough endoplasmic reticulum (rer) (1). This binding is rendered effective through prior phosphorylation of cadherin. The complex transfers to the plasma membrane where it forms a component of the adherent junctions. Destabilization of the junctions liberates -catenin, which is then recognized by Axin and APC (2).
This leads to its destruction. Any -catenin that escapes enters the nucleus and binds transcription factors of the TCF/LEF family (3).
APC (adenomatous polyposis coli) is not to be confused with APC/C (anaphase promoting complex/cyclosome).
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through gene transfection gives rise to a transformed phenotype as found in colorectal cancer.55 Thus, Wnt signalling causes an elevated level of -catenin by disabling the destruction-promoting activity of APC. It is the accumulation of cytosolic free -catenin that favours its translocation into the nucleus.
Importantly, intact APC also forms a complex with glycogen synthase kinase- 3 (GSK3 ), the homologue of the Drosophila gene shaggy. This finding positions APC and GSK3 at the centre of the wingless signal transduction pathway.56
Take your partner: which way -catenin?
-Catenin, freshly released from the ribosome has three potential binding partners (see Figure 14.5). Two of these, the cadherins and Axin/APC, are located in the cytoplasm; the other, TCF/LEF, in the nucleus. The first choice of partner is cadherin and association occurs as the nascent cadherin appears on the rough endoplasmic reticulum. Binding of -catenin first requires
that this cytoplasmic tail is phosphorylated (by casein kinase 2 or GSK3 ). The attachment of -catenin then facilitates cadherin transport through the Golgi and prevents its degradation by masking the so-called PEST region (or destruction box) that would otherwise attract ubiquitylation57 (see page 467). On reaching the plasma membrane, the two proteins contribute to the establishment of cell–cell contacts.
A shortage of cadherins or destabilization of the interaction between the two proteins directs -catenin to its second cytoplasmic partner, the Axin/ APC complex.58 Here, it is first phosphorylated by casein kinase 1 (CK1 ) and then by GSK3 , after which it dissociates to undergo multiple rounds of ubiquitylation by SCFTcrp ubiquitin E3-ligase. It is then recognized by the
regulatory PA700 and destroyed in the proteasome (see Figure 14.6). Activation of the Wnt pathway allows a stay of execution, causing disruption of the axin/ APC/CK1 /GSK3 complex, so preventing phosphorylation. The free -catenin can now enter the nucleus to unite with its third partner, the transcription factors of the TCF/LEF family.
The ( -catenin-dependent) canonical Wnt pathway
Our description of the continuing sequence of events refers to Wnts 1, 3a, and 8. These bind to two receptors, both discovered in genetic screens. Frizzled-1 (Fz-1) is a 7TM receptor (see page 55) having an extracellular cysteine-rich portion (CRD or Fz domain) thought to bind direct to Wnt. This group of receptors forms a large family, 10 members in humans (Figure 3.14, page 61).27 The other receptor (or coreceptor) is LRP (low-density lipoprotein-related protein, the arrow gene in Drosophila), which has only a
single membrane-spanning segment. The LRPs also comprise a large family of
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Adhesion molecules in the regulation of cell differentiation: Mainly about Wnt
Interestingly, CK and GSK3 contribute both to the destruction of-catenin (through
its phosphorylation) and its protection (by phosphorylation of LRP6 in the presence of Wnt Figure 14.8), a subtle mechanism that genetic screening in Drosophila failed to illuminate.
FIG 14.6 Path to destruction.
Binding of -catenin to axin and APC brings it into the vicinity of CK1 and GSK3 . These phosphorylate the N-terminal at numerous serines and one threonine residue. When dissociated from the complex, phosphorylated-catenin is recognized by Tcrp1, the receptor component of an SCF E3-ubiquitin ligase complex (2). The polyubiquitylated protein is next recognized by the PA700 subunit of the proteasome (3). The protein is unfolded and split into small polypeptides.
which LRP5 and LRP6 are of particular relevance.59 It is clear that both types of receptor are required for proper signalling, but how they interact with Wnt and with each other remains to be determined. The domain architecture of the main components of the Wnt pathway are illustrated in Figure 14.7.
Binding of Wnt activates a membrane-bound CK1 (Drosophila homologue Gilgamesh, Gish), which with GSK3 causes phosphorylation of residues
in the cytoplasmic tail of the LRP6 receptor (see Figures 14.8 and 14.9).60,61 Phosphorylated LRP6 acts as a docking site for axin, which is normally associated with APC, CK1 , and GSK3 as part of the -catenin destruction complex. The docking destabilizes the complex, liberating -catenin and, in consequence, its cytosolic concentration gradually increases. Simultaneously, Dsh is recruited to the membrane by Fz where it becomes phosphorylated by (soluble) CK1 (Figure 14.8). Fz, acting conventionally as a G-protein-linked receptor, also activates Go. Although loss of Dsh and G o impairs Wntmediated nuclear localization of -catenin, no effectors have so far been clearly defined (though possible candidates are CKII and Daam1).59,62
A failure to destroy -catenin or the lack of cytoplasmic binding sites leads to its nuclear localization. Several modes of transport to the nucleus have been proposed, including free exchange, APC-mediated import, or Bcl-9-mediated import (also known as Legless).
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FIG 14.7 Domain architecture of components of the Wnt pathway.
In the nucleus, -catenin finds its third binding partner, the transcription factors of the TCF/LEF-1 family (Figure 14.10). Importantly, its binding to TCF/ LEF-1 displaces Groucho (see Figure 14.3), thereby removing the transcription repression complex. Effective transcriptional activation requires the presence of many other factors, including Bcl-9, Pygopus (Pygo), CREB-binding protein (CBP), and Brg1 (the catalytic subunit of a chromatin-remodelling complex.63–66
Casein kinases are constitutively active, though not insensitive to allosteric modifications. Access to substrate is controlled by compartmentalization and
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Adhesion molecules in the regulation of cell differentiation: Mainly about Wnt
FIG 14.8 The canonical Wnt pathway.
Binding of Wnt3A to Fz-1 and LRP6 (1) causes activation of membrane-bound CK1 and the cytoplasmic GSK3 (2). These phosphorylate the cytoplasmic segment of LRP6 at numerous serine and threonine residues, allowing Axin to bind at the position normally occupied by CK1 (3). As a consequence, the destruction complex dissipates and -catenin is liberated (4). Simultaneously (5), the occupied Fz-1 receptor recruits Dsh, which is then phosphorylated by CK1 . It also recruits and activates Go. All three components, -catenin, Dsh, and G o, play a role in the signalling process.
FIG 14.9 Numerous CK1 and GSK3 phosphorylation sites in the cytosolic sequence of LRP6.
LRP6 contains numerous phosphorylation sites. The axin binding site (in the motif PPPSPATER) is first phosphorylated on threonine by CK1 . This acts as a priming point for further phosphorylations by GSK3 . Downstream of the axin binding site, four GSK3 consensus sequences ((P)PPS/TP) are evident. CK1 phosphorylation site clusters are in red (only the bold Ts are confirmed). Potential GSK3 phosphorylation sites are in green (the bold S is confirmed).
association of regulatory subunits. Substrates are numerous and include cytoskeletal proteins, receptors, ribosomal proteins, translation and transcription factors, kinases, phosphatases, and metabolic enzymes. There are two main families, CK1 and CK2, and in addition, a related mammary gland enzyme.
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