Signal Transduction
The name Casein kinases (CK) is misleading because the majority of them have little or nothing to do with phosphorylation of casein (a highly phosphorylated protein present in milk). CK2 was
discovered in a search for protein phosphorylating enzymes present in mitochondria.67 In
order to test the ability of mitochondrial enzyme systems to phosphorylate proteins other than those native to the organelle, various purified proteins were added to mitochondrial suspensions. Of the proteins tried, only casein showed a large and reproducible phosphorylation. One of its substrates, present in the inner mitochondrial membrane, is glycerol-3-phosphate acyltransferase, involved in de novo glycerolipid biosynthesis.68
FIG 14.10 -Catenin/TCF transcription complex.
Destabilization of the cadherin core complex results in liberation of -catenin (1). In the presence of Wnt, binding to Axin/APC is prohibited and the -catenin enters the nucleus, there to bind TCF/LEF-1 transcription factors (2). Numerous proteins associate with the complex, of which Bcl-9, Brg1, Pygo, and c-Jun are indicated. The DNA-bound complex interacts with CREB binding protein (CBP), which in turn plays an important role
in positioning RNA polymerase at the transcription initiation site. It does this by interaction with general transcription factors such as TATA-binding proteins (TBP) and the TBP-associated factors (TAF).
The CK1 family are further subdivided into , , 1, 2, 3, , and , of which the and forms exhibit a number of splice variants. The CK1 members are potentially membrane bound because they contain a consensus palmitoylation attachment site.69
CK2 is heterotetrameric with two different catalytic subunits and associated with two regulatory -subunits. It is not surprising that CK2 catalytic subunits are found in mitochondria, given their highly positively charged arginine-rich N-terminal region which serves as a ‘destination signal’ for soluble mitochondrial proteins. CK2 is involved in a number of processes, amongst which the regulation of cell polarity and morphology have attracted much attention.70 Casein is an excellent substrate.
Wnt target genes with a TCF-binding element
Many genes are directly affected by Wnt signalling. Here we only consider those having TCF binding sites and those listed below should not be considered universal Wnt targets, because common transcriptional outputs are unlikely to exist. It is the cell and its context, more than the signal itself, which determines
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Adhesion molecules in the regulation of cell differentiation: Mainly about Wnt
FIG 14.11 Parallels between the Wnt and Hedgehog pathways.
(a) Wnt and (b) Hh signalling pathways share components including GSK3 and members of the CK1 and Lrp families. They also have similar mechanisms. In the absence of ligands, transcription factors are phosphorylated (red arrows) and destroyed or partially degraded, resulting in repression of transcription. In the presence of their ligands, the receptors are phosphorylated (green arrows) and this prevents phosphorylation and destruction of the transcription factors, which now enter the nucleus to activate transcription (Smo, Smoothened).
the nature of the response. However, there are some themes that can be discerned in the types of target genes that are induced by Wnt:
•Genes that code for positive or negative regulators of the pathway. Amongst these are genes encoding TCF-1, LEF1, Axin-2, frizzled 7, Wnt-3a (mouse), Dickkopf
•Genes involved in proliferation and cell de-differentiation. Among these are Engrailed-2, Snail1, Snail2 (Slug), PPAR , c-Jun, Fra-1, ITF-2, FGF4, FGF18, VEGF, Dpp (Drosophila, homologue of BMP), or c-Myc binding protein
•Genes involved in adhesion, E-cadherin
•Genes coding for components of the extracellular matrix and their modifying enzymes, fibronectin and MMP7.
For further details, see the Wnt homepage: http://www.stanford.edu/~rnusse/ wntwindow.html
Wnt and Hedgehog
Hedgehog (Hh) is another secreted signalling protein that directs cell growth and patterning. The signalling pathways of Wnt and Hh have striking similarities (Figure 14.11). When Hh is absent from its receptor (Smoothened, panel b), PKA, CK1, and GSK3 sequentially phosphorylate the transcription
Extracellular inhibitors of Wnt and its receptors. A number
of proteins regulate Wnt signalling negatively. They do this by binding either to Wnt itself or
to the Wnt receptors.27 Dickkopf (Dkk), one of the best characterized, down-regulates the LRP5/6 receptor. Dickkopf are secreted proteins, four members in human and mouse. Binding
to LRP5/6 requires the presence of a member of the Kremen family of cell surface proteins (kringlecontaining protein marking the eye and the nose). Thus Dkk2 binds to LRP6 and Krm2 in order to inhibit Wnt signalling in human fibroblasts and in X. laevis embryos, to prevent dorsalization.71 Another inhibitor is Wise, that binds LRP6 and prevents Wnt8 binding.72
There are also soluble inhibitors that bind Wnts, preventing their binding to cellular receptors. Examples are WIF1, highly expressed in the unsegmented paraxial presomitic mesoderm in X. laevis embryos.73 Also, there are the secreted Frizzled receptor-related proteins (SFRP), five members in human, that sequester Wnt.74
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Signal Transduction
factor Ci-155 (red arrows). This causes it to be converted into a transcriptional repressor (Ci-75). In the presence of Hh, the cytoplasmic domain of the receptor is sequentially phosphorylated by PKA and CK1 (green arrows) preventing phosphorylation of Ci-155. Similar events occur in Wnt signalling (Figure 14.11a). Thus, phosphorylation of both Wnt and Hh receptors prevents transcriptional repression. This is in striking contrast to the tyrosine kinase containing cytokine receptors, in which phosphorylation invariably initiates a cascade of activation events.75,76
Wnt and the epithelial to mesenchymal transition
Wnt has a permissive role in -catenin signalling. The cellular localization of -catenin determines the fate of mammalian epithelial cells. At the membrane it promotes a differentiated epithelial cell having numerous cell to cell contacts, while in the nucleus it signals a loosely attached cell having a propensity to proliferate and degrade its extracellular matrix. Many of the Wnt-mediated effects on transcription resemble those that occur in the
epithelial mesenchymal transformation. The cadherin/ -catenin complex may be regarded as the guardian of the differentiated epithelial phenotype.77,78
The factors that determine whether cells differentiate or de-differentiate are listed in Table 14.2 (page 420). Key to this is the formation of stable cell–cell adhesions which depends on the association of the cytoplasmic domains of type 1 cadherin with -catenin, and of -catenin with -catenin (the core complex). The binding of -catenin to these partners depends on its phosphorylation status. This is regulated by receptor tyrosine protein
phosphatases (PTP , VEPTP, or LAR) and soluble tyrosine phosphatases (PTP1B, LMW-PTP, SHP2, and DEP1). Opposing effects have been reported for a number of cytokines. The receptors for EGF, FGF, HPG, and TGF , by activation of cytoplasmic tyrosine kinases such as Src, Fer, Fyn, or Abl directly or indirectly phosphorylate components of the cadherin core complex resulting in its disruption.79 Moreover, phosphorylation of -catenin by the EGFR increases binding to the TATA-box-binding protein (TBP), whereas phosphorylation by Fer, Fyn, or Met promotes binding to Bcl-9. Either way, these phosphorylations favour nuclear localization of -catenin (Figure 14.12). Not only this, FGF, EGF, and TGF also induce expression of numerous transcription factors (E12, Twist, SIP1,EF1, Snail1, and Slug) that repress transcription of E-cadherin. The cytokines also induce the pro-migratory adhesion molecule N-cadherin (figure 14.13).
Transfer of -catenin to the nucleus is normally limited by its attachment to its second cytosolic partner, axin/APC (see Figure 14.5). This ensures its destruction. To be effective, -catenin therefore requires a second signal to prevent this.
Herein lies the crucial role of Wnt (see Figure 14.10). By disabling the destruction complex, entry of -catenin into the nucleus is favoured, there to reinforce (or enable) the FGF, EGF, or TGF signal by removing the repressor machinery
associated with Groucho (and leaving the chromatin in an open configuration).
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Adhesion molecules in the regulation of cell differentiation: Mainly about Wnt
FIG 14.12 Determination of cell fate: phosphorylation and dephosphorylation of the cadherin core complex.
Phosphorylation of -catenin plays an important role in the determination of cell fate. Serine/threonine phosphorylations (indicated in red) prepare the protein to bind to the SCFTrcp1 E3-ubiquitin ligase leading to its destruction and favouring differentiation. Serine phosphorylation of E-cadherin stabilizes the core complex and promotes formation of adherent junctions. Numerous tyrosine phosphatases are also involved. These stabilize the cadherin core complex by maintaining -catenin in a tyrosine-dephosphorylated state.
De-differentiation is favoured by tyrosine phosphorylations (dark blue) of -catenin and E-cadherin. These destabilize the cadherin core complex by dissociating -catenin from cadherin. It is of note that Src, a known oncogene, can phosphorylate -catenin at Y654.
FIG 14.13 Suppression of E-cadherin expression by growth factors and Wnt.
(a) Activation of the receptors for growth factors such as EGF, FGF (rPTK), and TGF 1 (a receptor serine/threonine kinase) induces suppressors of E-cadherin expression. The growth factor signals are strongly enhanced by the Wnt signal. (b) and (c) Expression of the transcriptional suppressor Snail is inversely related to that of E-cadherin in mouse embryos at 8.5 days of development (mRNA revealed by an in situ hybridization procedure using anti-sense DNA). (d) and (e) Ectopic expression of Snail in epidermal keratinocytes causes an epithelial mesenchymal transformation, indicated by the loss of adherent junctions and epithelial structure, and the appearance of detached, non-polarized cells. Expression of protein was detected by antibodies coupled to a fluorescent dye. Figures b–e from Cano et al.80
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