Signaling Pathways Important in Developmental Biology

1
Signaling Pathways Important in Developmental
Biology

• Wnt/Frizzled through b-catenin
• Hedgehog
• TGF-b family through Smads
• Growth factors via JAK-STATs
• Notch
• Integrin
• TNF

2
Wnt signaling--another double negative
Wnts wingless and integrated large (350 aa),
very insoluble cysteine-rich glycoproteins. At
least 15 vertebrate members. Wnts bind to
frizzled receptors, 7 TM spanning proteins. Some
might activate heterotrimeric G proteins. Major
signaling occurs through disheveled. Activated
disheveled inhibits GSK-3, which normally
phosphorylates b-catenin, leading to its
degradation. b-catenin can go to nucleus and
increase gene transcription. Check
out http//stke.sciencemag.org/cgi/cm/stkecmCMP_
5533
Gilbert, 6-23
3
Wnt signaling--another double negative
Wnts wingless and integrated large (350 aa),
very insoluble cysteine-rich glycoproteins. At
least 15 vertebrate members. Wnts bind to
frizzled receptors, 7 TM spanning proteins. Some
might activate heterotrimeric G proteins. Major
signaling occurs through disheveled. Activated
disheveled inhibits GSK-3, which normally
phosphorylates b-catenin, leading to its
degradation. b-catenin can go to nucleus and
increase gene transcription. Check out
http//stke.sciencemag.org/cgi/cm/CMP_9763
http//faculty.washington.edu/rtmoon/cell.html
Gilbert, 6-23
4
A new twist for frizzled activation
Varmus group interested in why LRP synergizes
with frizzleds in b-catenin signaling Made
fusion constructs between LRP and TrkN (NGF
receptor) or Fz5 and NT3 (NGF ligand). Transfecte
d constructs into S2 cells (which have endogenous
LRP) and measured signaling by LEF-dependent
transcription of lucerifase gene. These results
suggest the major role of Wnts is to
heterodimerize LRP and Frizzled.
Cong, F. et al. Development 20041315103-5115
5
A new twist for frizzled activation
Cong, F. et al. Development 20041315103-5115
6
Wnt Signaling--Protein Interactions
Very useful website put together by Ron Nusse for
Wnt signalers http//www.stanford.edu/rnusse/wntw
indow.html
7
Wnt8 might signal through G proteins
To explore if Wnts might signal through G
proteins, our own Muslin lab (and Blumer lab)
expressed RGS4 in Xenopus to see if it can
attenuate a Wnt8 overexpression phenotype. Panel
A. 10 pg of Wnt8 rna injected into stage 35
embryo causes axis duplication (two
heads). Panel B. 10 pg of Wnt8 rna AND 1 ng of
rna of mutant RGS4 N128 (lacks GAP
activity). Panel C. 10 pg of Wnt8 rna AND 1 ng
of rna of RGS4.
Wu, et al. Development (2000)
8
Signal Integration--Priming allows for Selective
substrate phosphorylation
Regulating GSK3. (Left) After Wnt binds to its
receptor at the cell surface, proteins such as
FRAT displace axin from GSK3, resulting in an
accumulation of b-catenin in the nucleus and
transcription of Wnt target genes. (Right)
Binding of insulin to its receptor results in
activation of the insulin signaling pathway,
which induces PKB to phosphorylate GSK3 at Ser9
in the amino terminus. The phosphorylated amino
terminus behaves as a pseudosubstrate, competing
with "primed" substrates such as glycogen
synthase (GS) for GSK3's catalytic site,
effectively preventing GSK3 substrates from
becoming phosphorylated. GSK3 bound to axin is
not available for phosphorylation on Ser9 in
response to insulin, thereby restricting the
effects of insulin to a specific subset of GSK3
substrates.
Weston Davis, Science (2001)
9
Hedgehog signaling
Jacob Lum Science 2007
10
Cholesterol modification of hedgehog
Ingestion of Veratrum californicum early in
pregnancy results in cyclopia. The jervine
alkaloid made by this plant inhibits cholesterol
synthesis. What does this have to do with
hedgehog? Cholesterol is covalently linked to
the C-term cleavage product of sonic
hedgehog. Three forms of hedgehog in
vertebrates Indian, desert, and sonic.
11
Hedgehog and Wnt signaling in Cancer
Oncogenes () and tumor suppressor genes ()
have been identified in both pathways. Hedgehog Ba
sal cell ca (50), medullablastoma (25),
fibrosarcoma, rhadbomyosarcoma Wnt Colorectal
(85), Desmoid (74), hepatoblastoma (67)
Taipale Beachy, Nature (2001)
12
TGF-b Signaling
TGF-b (Transforming Growth Factor) family of
ligands mediate multiple developmental (BMPs) and
hormonal signals (Activins). The type of tissue
that develops depends on the concentration of the
morphogen. Synthesized as larger precursors (42
kDa), then processed to form homo- or hetero-
dimers (26 kDa).



C C C C C
C CC CC CC
TGF-b homology
C CC CC CC
Gilbert, 6-13
C CC CC CC
13
TGF-b Signaling
Relative to other signaling pathway, Smads are
fairly straightforward. Check outhttp//stke.scie
ncemag.org/cgi/cm/CMP_9876
Gilbert, 6-20
14
TGF-b Family versus Shh for control of the neural
tube
Two signaling pathways determine the
dorsal/ventral axis of the neural tube. Gradients
of Shh determine the formation of ventral and
motor neurons. An intermediate level (2 nM) of
Shh results in motor neurons, while slightly
higher levels (4 nM) of Shh results in V3
neurons. Members of the TGF-b family determine
dorsal fates.
Gilbert, 12-13
15
Notch Signaling Proteolysis at the membrane
releases transcriptional regulator
Model for the activation of Notch. A ligand
(Delta, Jagged, or Serrate protein) on one cell
binds to the extracellular domain of the Notch
protein on an adjacent cell. This binding causes
a shape change in the intracellular domain of
Notch, which activates a protease. The protease
cleaves Notch and allows the intracellular region
of the Notch protein to enter the nucleus and
activate a transcription factor of the CSL family
(such as Suppressor of hairless or CBF1). The
activated CSL can then transcribe its target
genes.
Gilbert, 6-29
16
Notch and Presenilin
Cell-cell contact allows Notch to interact with
its DSL ligand, Delta, Serrated, or Lag2. Notch
cleavage occurs in three steps S1 Cleavage of
notch by a furin-like protease occurs in the
Golgi. S2 Ligand binding induces proteolytic
clip at site 2 by a metalloproteinase to release
NEXT, Notch extracellular domain truncated. S3
Gamma secretase (presenilin, green) cleaves
within the membrane to release NICD, Notch
intracellular domain. Nicastrin (blue) is
required to get presenilin to the plasma membrane.
17
Proof of NICD
Defined cleavage site Showed cleavage was
required for activity Could get activity by
expressing NICD alone Ligand induced cleavage
Kopan lab Nature 1998
18
JAK-STAT signaling
Ligands a, g- interferons, erythropoietin,
prolactin, growth hormone, GM-CSF, IL-3
Gilbert, 6-21
19
Aberrant STAT signaling leads to severe SOB
Gilbert, 6-22
20
Integrin signaling
Two types of activation by cell adhesion
molecules. (A) Cell-substrate adhesion molecules
such as integrins may transmit a signal from the
cytoplasmic portion of the integrin protein to
the Ras G protein through a cascade involving
caveolin and Fyn proteins. (B) The FGF receptors
may be hijacked by cell adhesion molecules and
dimerized. They may be brought together by the
interaction of opposite cell adhesion molecules,
or the crosslinking of FGF receptors by the
apposing cell membrane may activate their kinase
domains.
Gilbert, 6-37
21
TNF-R1 Signaling A Beautiful Pathway
Ligands include Tumor Necrosis Factor, RANK,
Interleukin 1. Ligands and receptors form
homotrimers. http//stke.sciencemag.org/cgi/cm/
CMP_7107
22
The Integration of Biochemical Networks
23
Ask big questions.
Do great things.