Notch receptor activation and function

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Notch receptor activation and function
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Notch signaling completely changes the way that
cells respond to other signaling pathways
Flores et al., Cell 10375-85 (2000)
Lz
R7 Photoreceptor
D-Pax2
Lz
Cone cell
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Question What are Notch receptors normally used
for? Answer Lateral Specification Required to
generate two distinct cell types in the correct
ratio and configuration e.g. spacing of neurons
vs epidermal cells. Inductive Signaling
Induces differentiation of a new cell fate in
response to Notch activation e.g. induction of
wing margin tissue in a fruit fly. Cellular
Migration e.g. Axonal pathfinding in
flies Cell Division e.g. Mitosis in the C.
elegans male gonad. Cell Adhesion e.g.
Adhesion between the Oocyte and Follicle cells in
Drosophila. Mesenchymal/Epithelial Transitions
e.g. maintenance of epithelial tissues in
Drosophila Cell Survival/Death e.g. Regulates
cell death in the developing fly eye
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Question Maybe Notch receptors control
everything? Answer Probably involved in most
biological processes in complex
animals. Development We know from temperature
sensitive mutants of Notch in both flies and
worms that Notch receptors are required for
development of every tissue (blood vessels,
nervous system, white blood cells, epithelial
tissues, etc.). Diseases Cancer (Notch
oncogenes, EBNA2 oncogene from EBV mimics
NotchIC, Deltex, AF6 Stroke caused by mutations
in Notch3 Dementia caused by mutations in
Presenilin (PS) genes and in Notch3). Both
oncogene and anti-oncogene.
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Notch A large Transmembrane Receptor/Transcriptio
n factor that regulates development of every
tissue in complex animals. First analyzed in
Drosophila (1930s). Extracellular domains
Mammalian Notch 1, 2, 3 and 4 contain 36, 36, 34
and 29 highly conserved EGF-like repeats
respectively. EGF-like repeats are 40 amino
acids in length and contain 6 cysteine residues
(forming 3 C-C bonds). Three LNR repeats
(Lin-12, Notch Repeats) are also present in the
Notch extracellular domain. LNR repeats are
cysteine-rich domains found only in Notch family
receptors (Note that Lin-12 is a C. elegans Notch
receptor).
EGF Repeats
LNR Repeats
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Notch A large Transmembrane Receptor/Transcriptio
n factor. The Ram23 domain is 50 amino acids
in length. It binds to a CSL DNA-binding protein
(CSL CBF-1, Suppresser of Hairless, and Lag-1
which are the mammalian, fly and worm versions of
this protein) see below. Cdc10 repeats are 33
amino acids in length and bind to a number of
proteins involved in chromatin remodeling such as
Mastermind, Lag-3/Sel-8, or EMB-5, and also to
Deltex.
NLS
PEST Sequences
Ram23 domain
Cdc10/ankyrin repeats
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Notch A large Transmembrane Receptor/Transcriptio
n factor. Each domain of Notch is important for
at least one function -Receptor
Trafficking/Processing or Activation -Receptor
Signaling -Receptor Regulation ( or -)
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Notch is made as a very large single-chain
precursor that must be glycosylated and cleaved
before it appears on the cell surface.
Glycosylation of Notch starts in the ER and is
finished in the Golgi. Cleavage to generate the
mature heterodimeric form on the plasma membrane
occurs in the Golgi. This cleavage reaction at
site 1 (S1) is carried out by a Furin protease.
S1 cleavage
Furin Protease
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Notch
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Question What is Notch? Answer A Receptor, an
Adhesion Molecule, and a Transcription
factor. Question How is Notch
regulated? Answer Just about every way that you
can imagine Synthesis Folding Glycosylation Proc
essing Subcellular localization Ligand
Activation Proteolytic cleavage at
activation Endocytosis Translocation to the
Nucleus Binding to partners DNA
binding/Transcription Degradation
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Serrate
Notch
Delta
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Serrate OFUT1
Notch OFUT1 (O-fucosyltransferase)
Delta OFUT1
O-linked Fucose
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Serrate Fringe
Notch Fringe
Delta Fringe
GlcNac - Galactose - Sialic Acid
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Serrate
Jagged 1,2
Notch
Notch 1,2,3,4
Fringe
Delta
Lunatic Fringe Manic Fringe Radical Fringe
Dll 1, 4 Dll3, Dlk/Pref1 antagonists
The mammalian Notch receptors, ligands, and
Fringes
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TACE or Kuzbanian Disintegrin protease
S2 cleavage
Neuralized?
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S3 cleavage by a membrane protease complex that
includes PS proteins and Nicastrin (in
endocytic compartment?)
Endocytosis
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Endocytosis
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Endocytosis
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CSL/RBPJk
Hes genes
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CSL/RBPJk
Hes genes
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CSL/RBPJk
Hes genes
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The NotchIC (intracellular domain) switches on
genes that were repressed by CSL/RBPJk/Su(H)
through conversion of a repressor complex into a
transcriptional activation complex.
CSL/RBPJk
Hes genes
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Prior to Notch activation, the CSL protein was
sitting on DNA in a complex with Ski-interacting
protein (SKIP) and a transcriptional co-repressor
termed SMRT. The CSL-Skip-SMRT complex represses
gene expression and therefore CSL was repressing
expression of a set of genes prior to Notch
activation. Once NotchIC arrives in the nucleus,
it binds to the CSL-Skip complex, displacing SMRT
and recruiting transcription activating proteins
such as Mastermind, Lag-3/Sel-8, and/or EMB-5.
Consequently, Notch activation turns on the genes
that were repressed by CSL. Many of these Notch
target genes are themselves transcription factors
that regulate differentiation (e.g. HES).
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Notch signaling
Jagged 1Ligand
CSL/RBP-J
Notch 1Receptor
MAML
CBP
HES proteins mRNA
Nicastrin
CSL/RBP-J
PEN2
APH1
?-secretase
RAM
CBP
Anky
TAD
Pest
MAML
Numb
Nuclear localization signal
EGF-like repeats
PEST domain
Pest
Delta/Serrate domain
DSL
Lin/Notch repeats
TAD
Transactivation domain
LNR
Von Willebrand Factor cysteine-rich domain
RAM domain
vWF
Ankyrin repeats
RAM
Anky
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Bray Nature Reviews Molecular Cell Biology 7,
678689 (September 2006) doi10.1038/nrm2009
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Question How can we find other proteins that are
involved in Notch signaling? Answer Genetics,
Developmental Biology, Biochemistry, and Cell
Biology.
Positive components of the pathway Ca2-ATPase
Required to maintain high levels of Ca2 in the
ER (for Notch folding?) Rab6 Required for
correct sorting and transport of Notch through
the Golgi/TGN for S1 cleavage and cell surface
expression Furin Protease that cleaves at
S1 OFUT1 O-fucosyltransferase that adds
O-linked fucose to EGF repeats of Notch
ligands, also functions as a chaperone. Fringe
?1,3-N-acetylglucosaminyltransferase that extends
O-Fucose residues on Notch to control whether
Delta or Serrate ligands activate Notch in
vivo. Delta Ligand Serrate Ligand
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Question How can we find other proteins that are
involved in Notch signaling? Answer Genetics,
Developmental Biology, Biochemistry, and Cell
Biology.
Positive components of the pathway F3/contactin
Ligand for Notch during oligodendrocyte
maturation (signals through Deltex) DNER
Delta/Notch-like EGF-related receptor (DNER), a
neuron-specific transmembrane protein, as a
previously unknown ligand of Notch during
cellular morphogenesis of Bergmann glia in the
mouse cerebellum (signals through
Deltex) Mindbomb An E3 Ubiquitin ligase
(Required for endocytosis of all Notch ligands)
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Positive components of the pathway
(cont.) Deltex Binds to the cdc10 repeats and
facilitates activation. Deltex also binds to the
p300 transcription co-activator protein blocking
the interaction between p300 and E-box activators
like the achaete/scute bHLH class of
transcription factors. Deltex signaling is
blocked by Dishevelled which binds to the
C-terminus of Notch (Wingless/Wnt
signaling). TACE and Kuzbanian Proteases that
cleaves at S2 (Kuz also cleaves Delta
ligands) Presenilin proteins/Sel-12 A protease
or part of the protease that cleaves at
S3 Nicastrin/aph-2 A Presenilin-binding protein
that is also involved in S3 cleavage (and
AD) Aph-1 a novel seven transmembrane protein
involved in Notch activation (S3 cleavage?) GSK3
kinase Phosphorylates the C-terminal tail of
Notch and stabilizes NotchIC
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Positive components of the pathway
(cont.) EMB-5 A chromatin regulator that binds
to the cdc10 repeats Mastermind/Lag-3/Sel-8
Transcriptional activators that bind to NotchIC
cdc10 repeats and to the CSL proteins HES/Hey A
family of basic helix-loop-helix transcriptional
repressor proteins that are activated by Notch to
block differentiation Groucho A transcriptional
co-repressor protein that binds to the Notch
induced HES protein Sandpodo A Tropomyosin
homologue that binds to actin and positively
regulates Notch signaling downstream of
Numb Wasp an actin regulating molecule that is
required for Notch signaling in flies Gp150 a
transmembrane protein that may regulate
trafficking of Notch/Delta in the endocytic
compartment Neuralized A membrane localized E3
Ubiquitin ligase (Ubiquitinylates Delta and is
required for Notch activation)
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Negative components of the pathway p24/Sel-9
Reduces Notch transport to the cell surface in
order to ensure proper Notch folding in the
ER/Golgi quality control system Sel-1 (A
Protein involved in destroying unfolded proteins
in the ER presumably destroys poorly folded
Notch) Sel-10 and Notchless WD40 repeat
protein that binds to the phosphorylated
Notch intracellular domain. Sel-10 localizes to
the nucleus, antagonizes NotchIC signaling, and
induces ubiquitinylation/destruction of Notch (it
is an E3 ubiquitin ligase). This effect requires
the C-terminus of Notch (including the PEST
sequences). Also binds to Sel-12. Suppresser of
Deltex/Itch An E3 ubiquitin ligase proteins
containing a membrane- targeting C2 domain, WW
domains and a HECT domain (WW domains may bind
to the proline-rich region of Deltex or to the
Notch cytoplasmic domain directly it may then
target Notch and/or Deltex for Ubiquitination) Sc
abrous A secreted protein that inhibits Notch
activation by Delta (and slightly by Serrate)
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Negative components of the pathway (cont.) Numb
An endocytic protein with a PTB domain,
polyproline sequences (including the PxxP SH3
binding motifs) and NPF motifs (which bind to EH
domains in the endocytic protein Eps15). Numb
suppresses Notch signaling (it also binds to Lnx
and MDM2 E3-ligases) Kurtz (Krz) non-visual
?-Arrestin that binds to Deltex and facilitates
internalization/degradation of Notch Dlk/Pref1
Notch antagonist or inhibitory ligand
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Ubiquitin and Notch receptor signaling/regulation
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Other Regulators Bearded and M4 family proteins
a family of small proteins which are directly
activated by the NotchIC-CSL complex. These
proteins can both positively and negatively
regulate Notch signaling Wingless/Wnt proteins
Morphogens that bind to the Notch extracellular
domain to either induce a distinct signaling
pathway downstream from Notch, or to negatively
regulate Notch in some way. Disheveled A
component of the Wingless/Wnt and Planar polarity
signaling pathways that binds to the Notch
C-terminus Pecanex - A very large
membrane-spanning protein involved in some Notch
dependent developmental processes Brainiac A
homologue of mammalian ?1,3-Galactosyltransferases
that regulates Notch dependent adhesion
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Other Regulators (cont.) Kuzbainian A
TACE-like protease that may cut Notch at S2 in
some tissues and cleaves Delta AF6/Canoe - A
Ras-GTP binding protein involved in Notch
dependent development Strawberry Notch A
nuclear protein involved in some Notch signaling
events Disabled An adapter protein that binds
to the cytoplasmic domains of Notch for axonal
guidance Bigbrain a H2O channel involved in
Notch signaling Hairless Blocks CSL proteins
from binding DNA (which can block the repression
and activation of Notch-responsive promoters)
Skip Involved in recruiting co-repressors to
CSL proteins in the absence of Notch and also
found in NotchICCSL activation complexes (i.e.
may have positive and negative functions)
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Other Regulators (cont.) the K box (UGUGAU) a
sequence motif present in many Notch pathway mRNA
3 UTRs which destabilize the corresponding
transcript
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Signal Integration
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Notch signalling completely changes the way that
cells respond to other signaling pathways
Flores et al., Cell 10375-85 (2000)
Lz
R7 Photoreceptor
D-Pax2
Lz
Cone cell
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Wingless
Delta
Arrow/Frizzled
Notch
Notch
Dishevelled
Su(H)
Deltex
E(spl)
Armadillo/b-catenin
Groucho
ac-sc
ac-sc
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LEF1-mediated regulation of Delta-like1 links Wnt
and Notch signaling in somitogenesis. Wnt
signaling, which is mediated by LEF1/TCF
transcription factors, has been placed upstream
of the Notch pathway in vertebrate somitogenesis.
Here, we examine the molecular basis for this
presumed hierarchy and show that a targeted
mutation of Lef1, which abrogates LEF1 function
and impairs the activity of coexpressed TCF
factors, affects the patterning of somites and
the expression of components of the Notch
pathway. LEF1 was found to bind multiple sites in
the Dll1 promoter in vitro and in vivo. Moreover,
mutations of LEF1-binding sites in the Dll1
promoter impair expression of a Dll1-LacZ
transgene in the presomitic mesoderm. Finally,
the induced expression of LEF1-beta-catenin
activates the expression of endogenous Dll1 in
fibroblastic cells. Thus, Wnt signaling can
affect the Notch pathway by a LEF1-mediated
regulation of Dll1. Galceran J, Sustmann C, Hsu
SC, Folberth S, Grosschedl R. Genes Dev. 2004 Nov
1518(22)2718-23.
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Mouse Nkd1, a Wnt antagonist, exhibits
oscillatory gene expression in the PSM under the
control of Notch signaling. During vertebrate
embryogenesis, the formation of reiterated
structures along the body axis is dependent upon
the generation of the somite by segmentation of
the presomitic mesoderm (PSM). Notch signaling
plays a crucial role in both the generation and
regulation of the molecular clock that provides
the spatial information for PSM cells to form
somites. In a screen for novel genes involved in
somitogenesis, we identified a gene encoding a
Wnt antagonist, Nkd1, which is transcribed in an
oscillatory manner, and may represent a new
member of the molecular clock constituents. The
transcription of nkd1 is extremely downregulated
in the PSM of vestigial tail (vt/vt), a
hypomorphic mutant of Wnt3a, whereas nkd1
oscillations have a similar phase to lunatic
fringe (L-fng) transcription and they are
arrested in Hes7 (a negative regulator of Notch
signaling) deficient embryos. These results
suggest that the transcription of nkd1 requires
Wnt3a, and that its oscillation patterns depend
upon the function of Hes7. Wnt signaling has been
postulated to be upstream of Notch signaling but
we demonstrate in this study that a
Wnt-signal-related gene may also be regulated by
Notch signaling. Collectively, our data suggest
that the reciprocal interaction of Notch and Wnt
signals, and of their respective negative
feedback loops, function to organize the
segmentation clock required for
somitogenesis. Ishikawa A, Kitajima S, Takahashi
Y, Kokubo H, Kanno J, Inoue T, Saga Y. Mech Dev.
2004 Dec121(12)1443-53.
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Wnt3a plays a major role in the segmentation
clock controlling somitogenesis. The vertebral
column derives from somites generated by
segmentation of presomitic mesoderm (PSM).
Somitogenesis involves a molecular oscillator,
the segmentation clock, controlling periodic
Notch signaling in the PSM. Here, we establish a
novel link between Wnt/beta-catenin signaling and
the segmentation clock. Axin2, a negative
regulator of the Wnt pathway, is directly
controlled by Wnt/beta-catenin and shows
oscillating expression in the PSM, even when
Notch signaling is impaired, alternating with
Lfng expression. Moreover, Wnt3a is required for
oscillating Notch signaling activity in the PSM.
We propose that the segmentation clock is
established by Wnt/beta-catenin signaling via a
negative-feedback mechanism and that Wnt3a
controls the segmentation process in
vertebrates. Aulehla A, Wehrle C, Brand-Saberi B,
Kemler R, Gossler A, Kanzler B, Herrmann BG. Dev
Cell. 2003 Mar4(3)395-406.
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Notch controls developmental decisions together
with -TGF? -BMPs -HH -FGFs -EGFs -VEGFs -
Integrins -Wnts -Hypoxia signaling -etc
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Notch receptors and ligands(Human)
Jagged 1 1033 aa
Jagged 2 1053 aa
Delta-like 1 706 aa
Delta-like 3 591 aa
Delta-like 4 658 aa
Notch 1 2537 aa
Notch 2 2445 aa
Notch 3 2281 aa
Notch 4 1979 aa
Nuclear localization signal
EGF-like repeats
PEST domain
Pest
Delta/Serrate domain
Lin/Notch repeats
DSL
TAD
Transactivation domain
LNR
RAM domain
Von Willebrand Factor cysteine-rich domain
RAM
Ankyrin repeats
vWF
Anky
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