Signaling Pathways that Signaling Pathways that Depend on Regulated Depend on Regulated

ProteolysisProteolysis

7 May 20077 May 2007

Identified through genetic studies in Identified through genetic studies in drosophila.drosophila.

Highly conserved in evolution and Highly conserved in evolution and are very important in animal are very important in animal developmentdevelopment

WntWnt NotchNotch HedgehogHedgehog NFNFBB

WntsWnts

Wnt proteins released from or presented on Wnt proteins released from or presented on the surface of signaling cells act on target the surface of signaling cells act on target cells by binding to Frizzled (Fz)/LDL-related cells by binding to Frizzled (Fz)/LDL-related protein (LDR) complex at the cell surface.protein (LDR) complex at the cell surface.

ReceptorsReceptors Frizzled receptors, like GPCRs, are Frizzled receptors, like GPCRs, are

transmembrane proteins that span transmembrane proteins that span 7 times7 times the the plasma membrane. plasma membrane.

Their ligand-binding site is exposed outside the Their ligand-binding site is exposed outside the surface of the cell. surface of the cell.

Their effector site extends into the cytosol.Their effector site extends into the cytosol.

LigandsLigands Their ligands are Their ligands are

Wnt proteins. These Wnt proteins. These get their name from get their name from two of the first to be two of the first to be discovered, proteins discovered, proteins encoded by encoded by

winglesswingless ( (wgwg) in ) in Drosophila and its Drosophila and its homolog homolog

Int-1Int-1 in mice. in mice.

Fz receptors transduce Fz receptors transduce signals to intra-cellular signals to intra-cellular proteins including proteins including Dsh, GSK-3ß, Axin, Dsh, GSK-3ß, Axin, APC and ß-cateninAPC and ß-catenin

Nuclear ß-catenin Nuclear ß-catenin interacts with interacts with lymphoid enhancer-lymphoid enhancer-binding factor 1/T cell-binding factor 1/T cell-specific transcription specific transcription factor (LEF/TCF) to factor (LEF/TCF) to affect transcription.affect transcription.

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 cysteine-rich domain (CRD) of Frizzled, one of the Wnt receptors.

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

Interaction with TCF transcription factors and the transactivation domains of beta-catenin are instrumental to activate/derepress wnt-target genes in the nucleus.

Complex formation with cadherins and alpha-catenin at the plasma membrane is essential for the role of beta-catenin 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

In adults, mis-regulation of the Wnt pathway also leads to a variety of abnormalities and degenerative diseases

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.

From crypt physiology to colon cancer

The Wnt pathway in colon cancer

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.

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.

Multiple studies have used a candidate gene approach to address the nature of the Tcf4 target gene programme in colorectal cancer.

““Non-canonical” wnt Non-canonical” wnt signalingsignaling

Non-canonical wnt signalingNon-canonical wnt signaling

Involved in different development processes, Involved in different development processes, but its intracellular mediators are ill-known.but its intracellular mediators are ill-known.

In vertabrates, Wnt-11 & Wnt-5a can activate In vertabrates, Wnt-11 & Wnt-5a can activate Wnt/JNK pathway (planar cell polarity?)Wnt/JNK pathway (planar cell polarity?)

Wnt/CaWnt/Ca2+2+ pathway (described only in pathway (described only in Xenopus & Zebrafish)Xenopus & Zebrafish)

Additional wnt pathways (described only in Additional wnt pathways (described only in drosophila)drosophila)

In drosophila, non-canonical wnt signaling regulates In drosophila, non-canonical wnt signaling regulates ““cell motilitycell motility” through focal adhesion kinase ” through focal adhesion kinase ((FAKFAK))Planar cell polarity involved in tissue Planar cell polarity involved in tissue engineeringengineering

In vertabrates, non-canonical wnt signaling regulates In vertabrates, non-canonical wnt signaling regulates ““gastrulationgastrulation” through protein kinase C ” through protein kinase C ((PKCPKC))Planar cell polarity involved in tissue Planar cell polarity involved in tissue engineeringengineering

Wnt/CaWnt/Ca2+2+ signaling regulates dorso-ventral axis signaling regulates dorso-ventral axis patterning patterning (Wnt5a(Wnt5aCaCa2+2+CalcineurinCalcineurinNFATNFATActivation of Activation of target genestarget genesNegative regulation of canonical wnt Negative regulation of canonical wnt signalingsignalingVentralization)Ventralization)

Notch Signaling PathwayNotch Signaling Pathway

A brief history

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

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.

Ligands: Delta1, 3 and 4; Jag1, 2 Receptors: Notch1-4

Notch signaling has effects in many different organs

•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

The best known role of notch signaling is The best known role of notch signaling is in nerve cell developmentin nerve cell development

Nerve cells arise within an epithelial sheet Nerve cells arise within an epithelial sheet of precursor cells.of precursor cells.

Each future nerve cells signals to its Each future nerve cells signals to its immediate neighbours not to develop in immediate neighbours not to develop in the same way at the same timethe same way at the same time Lateral inhibitionLateral inhibition

Lateral inhibition is a Lateral inhibition is a contact-dependent contact-dependent signaling that is signaling that is mediated by the ligand mediated by the ligand delta.delta.

Delta displayed on the Delta displayed on the surface of future neural surface of future neural cell binds to notch on cell binds to notch on neighboursneighbours

If the signal is defective, If the signal is defective, excess production of excess production of neurons at the expense neurons at the expense of epidermal cell causes of epidermal cell causes lethality.lethality.

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 Processing and Activation of NotchNotch

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).

Processing and Activation of Processing and Activation of NotchNotch

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)

Binding of NIc displaces HDAC and allows recruitment of histone acetylases and the nuclear protein Mastermind,which together activate transcription

The signal is terminated through ubiquitination of NIc by a complex including Sel-10, followed by proteosome-dependent degradation.

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

Fringe: regulate notch Numb: notch inhibitor Neuralized (E3 ubiquitin ligases):

regulate ligand Delta. Mib (mindbomb): (E3 ubiquitin

ligase): function on Delta

Fringe

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 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 N-terminus 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

SonicSonic DesertDesert Indian HedgehogIndian Hedgehog

Processing of Hedgehog (Hh) precursorprotein

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

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 theHedgehog (Hh) signaling

pathway 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.

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).

Model of theHedgehog (Hh) signaling

pathway

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).