Transcript Wnt Signaling Pathway

Signaling Pathways that Depend
on Regulated Proteolysis
7 May 2007
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Identified through genetic studies in
drosophila.
Highly conserved in evolution and are very
important in animal development
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Wnt
Notch
Hedgehog
NFB
Wnts
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Wnt proteins released from or presented on the
surface of signaling cells act on target cells by
binding to Frizzled (Fz)/LDL-related protein
(LDR) complex at the cell surface.
Receptors
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Frizzled receptors, like GPCRs, are transmembrane
proteins that span 7 times the plasma membrane.
Their ligand-binding site is exposed outside the
surface of the cell.
Their effector site extends into the cytosol.
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Ligands
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Their ligands are Wnt
proteins. These get
their name from two
of the first to be
discovered, proteins
encoded by
wingless (wg) in
Drosophila and its
homolog
Int-1 in mice.
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Fz receptors transduce
signals to intra-cellular
proteins including Dsh,
GSK-3ß, Axin, APC and ßcatenin
Nuclear ß-catenin
interacts with lymphoid
enhancer-binding factor
1/T cell-specific
transcription factor
(LEF/TCF) to affect
transcription.
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In vertebrates, Wnt
proteins are inhibited by
direct binding to either
secreted frizzled-related
protein (SFRP) or Wnt
inhibitory factor (WIF).
SFRP is similar in
sequence to the cysteinerich domain (CRD) of
Frizzled, one of the Wnt
receptors.
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GSK-3b dependent
phosphorylation,
ubiquitination and
complex formation with
the proteins axin and APC
are important to regulate
the cytoplasmic stability
of beta-catenin protein in
the wnt-signal
transduction pathway
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Interaction with TCF
transcription factors
and the
transactivation
domains of betacatenin are
instrumental to
activate/derepress
wnt-target genes in
the nucleus.
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Complex formation with cadherins and alpha-catenin at
the plasma membrane is essential for the role of betacatenin in cell adhesion.
In vertebrate development, loss of a single Wnt gene
can produce dramatic phenotypes that range from
embryonic lethality and CNS abnormalities to kidney and
limb defects
These diverse phenotypes indicate that the Wnt pathway
has distinct transcriptional outputs.
In many cases, the cell, rather than the signal,
determines the nature of the response, and up- or
down-regulation of Wnt target genes is cell-type specific.
WNT TARGET GENES AND
FEEDBACK LOOPS
WNT SIGNALING IN CANCER
AND HUMAN DISEASE
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In adults, mis-regulation of the Wnt
pathway also leads to a variety of
abnormalities and degenerative diseases
From crypt physiology to colon
cancer
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Current evidence indicates that
the Wnt cascade is the single
most dominant force in
controlling cell fate along the
crypt-villus axis.
In Tcf4 -/- neonatal mice, the
villus epithelial compartment
appears unaffected but the
crypt progenitor compartment
is entirely absent, implying
that physiological Wnt
signalling is required for
maintenance of the crypt
progenitor phenotype.
The Wnt pathway in colon cancer
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The APC gene was originally discovered to be
the culprit in a hereditary cancer syndrome
termed familial adenomatous polyposis (FAP).
FAP patients, inheriting one defective APC allele,
develop large numbers of colon polyps, or
adenomas, early in life.
Individual polyps are clonal outgrowths of
epithelial cells in which the second APC allele is
inactivated.
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Mutational inactivation of APC leads to the
inappropriate stabilization of b-catenin, implying
that the absence of functional APC transforms
epithelial cells through activation of the Wnt
cascade.
In some cases of colorectal cancer in which APC is
not mutated, the scaffolding protein axin 2 is
mutant, or activating (oncogenic) point mutations
in b-catenin remove its N-terminal Ser/Thr
destruction motif.
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Multiple studies have used a candidate
gene approach to address the nature of
the Tcf4 target gene programme in
colorectal cancer.
“Non-canonical” wnt signaling
Non-canonical wnt signaling
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Involved in different development processes, but
its intracellular mediators are ill-known.
In vertabrates, Wnt-11 & Wnt-5a can activate
Wnt/JNK pathway (planar cell polarity?)
Wnt/Ca2+ pathway (described only in Xenopus &
Zebrafish)
Additional wnt pathways (described only in
drosophila)
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In drosophila, non-canonical wnt signaling regulates
“cell motility” through focal adhesion kinase
(FAK)Planar cell polarity involved in tissue engineering
In vertabrates, non-canonical wnt signaling regulates
“gastrulation” through protein kinase C (PKC)Planar
cell polarity involved in tissue engineering
Wnt/Ca2+ signaling regulates dorso-ventral axis
patterning
(Wnt5aCa2+CalcineurinNFATActivation of target
genesNegative regulation of canonical wnt
signalingVentralization)
Notch Signaling Pathway
A brief history
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In 1917, Thomas Hunt Morgan described a
strain of Drosophila with notches at the
end of their wing blades, which result
from haploinsufficiency
Notch gene was cloned in the mid-1980s
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Notch is an essential gene encoding a signalling
receptor that is required throughout development
to regulate the spatial patterning, timing and
outcomes of many different cell fate decisions in
both vertebrate and invertebrate species.
Notch is a single spanning transmembrane
protein,which has a modular architecture.
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Ligands: Delta1, 3 and 4; Jag1, 2
Receptors: Notch1-4
Notch signaling has effects in many
different organs
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•Notch signalling can maintain stem cells or
precursor populations in an undifferentiated
state
•Notch signalling influences binary cell-fate
decisions via lateral or inductive signalling
•A third property of Notch is its ability to
influence differentiation and cell-cycle
progression
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The best known role of notch signaling is in
nerve cell development
Nerve cells arise within an epithelial sheet of
precursor cells.
Each future nerve cells signals to its immediate
neighbours not to develop in the same way at
the same time
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Lateral inhibition
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Lateral inhibition is a
contact-dependent
signaling that is mediated
by the ligand delta.
Delta displayed on the
surface of future neural
cell binds to notch on
neighbours
If the signal is defective,
excess production of
neurons at the expense
of epidermal cell causes
lethality.
Processing and Activation of Notch
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S1 cleavage occurs
within the secretory
pathway so that a
processed
heterodimeric form is
transported to the cell
surface (furin
protease in the golgi
apparatus.
Processing and Activation of Notch
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S2 cleavage occurs
following ligand binding
by Delta or
Serrate(Jagged in
mammals) through their
DSL domains
(Delta/Serrate/Lag2), and
releases a membrane
tethered form of the
Notch intracellular
domain.
The latter is a constitutive
substrate for the S3
cleavage, which releases
the soluble intracellular
domain of Notch (NIc).
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NIc is translocatedto the
nucleus where it binds via
the RAM domainand ankyrin
repeats to a transcription
factor, Suppressor of
Hairless (Su(H)), or CBF1 in
vertebrates.
In the absence of a Notch
signal, Su(H)/CBF1 can
repress transcription through
the recruitment of a histone
deacetylase (HDAC)
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Binding of NIc
displaces HDAC
and allows
recruitment of
histone acetylases
and the nuclear
protein
Mastermind,which
together activate
transcription
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The signal is
terminated through
ubiquitination of NIc by
a complex including
Sel-10, followed by
proteosome-dependent
degradation.
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The NICD-RBP-Jk complex up-regulates expression of
primary target genes of Notch signaling such as HES in
mammals, and E(spl) (for Enhancer of Split) in Drosophila.
The HES/E(spl) family is a basic helix-loop-helix (bHLH) type
trancriptional repressor and acts as Notch effectors by
negatively regulating expression of downstream target
genes such as tissue-specific transcription factors.
Consistent with this view, HES1 and HES5, for instance,
were shown to be upregulated by NICD and necessary to
prevent neuronal differentiation of neural precursor cells
from mouse embryos
Modulators of Notch signalling
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Fringe: regulate notch
Numb: notch inhibitor
Neuralized (E3 ubiquitin ligases): regulate
ligand Delta.
Mib (mindbomb): (E3 ubiquitin ligase):
function on Delta
Fringe
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Fringe encodes a glycosyltransferase that adds
O-fucoseglycans to the Notch EGF repeats.
Fringe proteins might differentially modulate the
response of Notch receptors to different DSL
(Delta,Serrate and LAG2) ligands. Fringe can
enhance Delta binding to the notch receptor.
Whether fringe modification of Notch inhibits
Serrate/Jagged binding is much less certain.
Function autonomously
Numb
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Numb domains:
 1. PTB domain; N-terminal phosphotyrosine binding
domain
 2. proline-rich C-terminal region.
In vitro studies have shown that Numb binds directly to
NICD. The Cterminal half of the PTB domain and the Nterminus of Numb are required to inhibit Notch. Numb
also has two motifs associated with endocytic proteins.
mammalian Numb (mNumb) localizes to clathrin coated
pits and early endosomes, might target endocytosed
NICD for proteosomal destruction.
Numb acts either upstream of S3 cleavage site of Notch
or inhibit the endocytosis of membrane-bound activated
Notch.
Hedgehog Signaling
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Sonic
Desert
Indian Hedgehog
Processing of Hedgehog (Hh) precursor
protein
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Adding cholesterol to a
glycine residue,
Splitting the molecule into
two fragments,
Leaving the N-terminal
signaling fragment with
an attached hydrophobic
cholesterol moiety.
Addition of a palmitoyl
group to the N-terminus
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Findings from genetic studies in Drosophila
indicate that two membrane proteins,
Smoothened (Smo) and Patched (Ptc),
are required to receive and transduce a
Hedgehog signal to the cell interior.
They are required to activate transcription of the
same target genes (e.g., wingless) during
embryonic development.
Model of the
Hedgehog (Hh) signaling pathway
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In the absence of Hh, Patched
(Ptc) protein inhibits
Smoothened (Smo) protein. In
the absence of Smo signaling,
a complex containing the
Fused (Fu), Costal-2 (Cos2),
and Cubitis interuptus (Ci)
proteins binds to
microtubules.
Ci is cleaved in a process
requiring the
ubiquitin/proteasome-related
F-box protein Slimb,
generating the fragment Ci75,
which functions as a
transcriptional repressor.
Model of the
Hedgehog (Hh) signaling pathway
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In the presence of Hh,
inhibition of Smo by Ptc is
relieved. Signaling from
Smo causes
hyperphosphorylation of Fu
and Cos2, and
disassociation of the
Fu/Cos2/Ci complex from
microtubules.
This leads to the
stabilization of a full-length,
alternately modified Ci,
which functions as a
transcriptional activator
inconjunction with CREB
binding protein (CBP).
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Hedgehog signaling is conserved throughout the
animal kingdom, and functions in the formation
of many tissues and organs.
Mutations in components of the Hedgehog
signaling pathway have been implicated in birth
defects such as cyclopia, a single eye resulting
from union of the right and left brain primordia,
and in multiple forms of human cancer (basal
cell carcinoma).