Download S7.Cell Signaling-β-catenin pathway homework

Cell Signaling Pathways – A Case Study Approach
L. Emtage, L. Bradbury, N. Coleman, D. Davenport, A. Dunning and J. Grew
Assignment: The Wnt/ β-catenin pathway
Wnt
Wnt
Fzd
Plasma membrane
LRP
LRP
Fzd
DSH
GSK3
A
x
i
n
Ubiqui n-mediated
degrada on
β-catenin
APC
DSH
GSK3
GBP
A
x
i
n
APC
β-catenin
Ub
Ub
Ub
Ub
β-catenin
β-catenin
TCF
TCF
Note: GSK3, APC and Axin together are called the β-catenin Destruction Complex.
DSH is the abbreviation for Dishevelled. The human genome encodes many Fzd
receptors, many Wnts, several Dsh proteins, one GSK3, and one β-catenin.
Wnts are a family of protein ligands that activate members of the Frizzled (Fzd)
family of receptors. Fzd receptors often associate with co-receptors, such as the
lipoprotein related-receptor protein-5/6, or LRP, shown here. There are several
signaling pathways that may be activated by the binding of a Wnt to a Fzd, but the
canonical pathway, activated by Fzd-LRP, modulates transcription through βcatenin.
β-catenin is constitutively expressed, but in the absence of Wnt signaling, it is
constitutively destroyed in the cytoplasm through the action of the β-catenin
Destruction Complex. The β-catenin Destruction Complex targets β-catenin for
ubiquitination. Once ubiqutinated, β-catenin is broken down by the proteasome.
Upon activation of the Fzd-LRP receptor complex by Wnts, the receptor
complex binds to the β-catenin Destruction Complex via the adaptor Dishevelled
(Dsh). The Destruction Complex is sequestered and prevented from ubiquitinating
β-catenin. The inhibition of the Destruction Complex stabilizes β-catenin, allowing
cytoplasmic and nuclear levels to rise. Although β-catenin does not have a DNA-
Cell Signaling Pathways – A Case Study Approach
L. Emtage, L. Bradbury, N. Coleman, D. Davenport, A. Dunning and J. Grew
binding domain, it is a transcriptional co-activator that associates with the TCF/LEF
family of transcription factors to modulate transcription.
For a recent review of the factors and molecular mechanisms that mediated
canonical Wnt/ β-catenin signaling, see The way Wnt works: Components and
mechanism; Saito-Diaz, et al., Growth Factors, 2013. Freely available at
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697919/
Questions
1. What are the components of the receptor complex? What is the ligand? The
receptor complex is composed of LRP and Fzd. The ligand for Fzd is Wnt.
2. What is the molecular function of β-catenin itself? Apart from its role in
adherens junctions, β-catenin is a transcriptional coactivator. β-catenin has a
transactivation domain, but does not have a DNA binding domain, and so it
activates transcription through binding to transcription factors such as TCF.
3. Where is β-catenin found within the cell? Does its subcellular location ever
change? Low levels of β-catenin are found in the cytoplasm until inhibition of
the β-catenin Destruction Complex allows levels of β-catenin to rise. It is
subsequently found in the cytoplasm and nucleusd.
4. What are the molecular functions of GSK3, APC, and Axin? They work
together to ubiquitinate β-catenin, targeting it for destruction by the
proteasome. GSK3 is a kinase, Axin is a scaffolding protein, the function of
APC is unclear, although it binds to both Axin and β-catenin.
5. Define proteolysis. The break-down of polypeptides to amino acids.
6. Describe the ultimate outcome of Wnt pathway activation. Changes in gene
expression when β-catenin moves into the nucleus.
7. Give two examples of negative regulation found here. Contrast their
molecular mechanisms. β-catenin is ubiquitinated and removed by
proteolysis, ie. it is destroyed. The β-catenin Destruction Complex is
sequestered by the receptor complex upon receptor activation. In contrast to
β-catenin itself, sequestration does not result in the proteolysis of the
destruction complex, its only held and prevented from acting on β-catenin.
8. Describe the effect of loss-of-function mutations in Frizzled, DSH, GSK3, and
β-catenin separately on the transcription of target genes. You can assume
that mutations in any part of the β-catenin Destruction Complex will cause
the loss of function of the entire complex. The loss of Fz, DSH or β-catenin
will prevent the pathway from being activated. The loss of GSK3 will
Cell Signaling Pathways – A Case Study Approach
L. Emtage, L. Bradbury, N. Coleman, D. Davenport, A. Dunning and J. Grew
constitutively activate the pathway. However, because there are many Fz
genes, the loss of any particular Fz is less severe than the loss of β-catenin.
9. What would be the result of a double knock-out of DSH and APC? Of GSK3
and β-catenin? DSH acts upstream of APC, so a DKO of both will result in
constitutive activation of the pathway. Any KO of β-catenin will inactivate the
pathway regardless of the identity of the other gene.