1) How the Hippo pathway was discovered?
genetic mosaic screens in Drosophila for overgrowth mutants, identified four tumor suppressors: hippo ( serine-threonin Ste-20 like protein kinase), Warts (Lats) NDR family kinase Salvador (WW domain adaptor protein) Mob (adaptor protein)
—> overgrowth = big organs = hippo
2) Describe the core conserved components of Hippo/YAP-TAZ Pathway.
Hippo -> P on Warts (with adaptors Salvador and Mob)
Warts -> P on Yorkie -> inhibition -> Scalloped no more nuclear translocation no transcription of genes for proliferation/apoptosis/cell fate (high phosphorylation = signal for degradation)
3) How Hippo kinase cascade is activated under the membrane?
-> Salvador is membrane bound —> depending of cell cell contact and polarity mediated by cadherins / catenin
-> Heterotetramer forming-> binding hippo/MST -> activation via auto-transphosphorylation
-> Binding of phosphorylation sites by Mats/MOB helps recruit Wts/LATS together with KIBRA/Merlin [Apical polarity proteins] complex formation
-> Wts/LATS phosphorylated by Hpo/MST
Then Kinase cascade Lats -> YAP/TAZ there its Phosphorylation leads to Cytoplasmic Retention by binding to 14-3-3 protein
STRIPAX Complex inhibits Hippo Phosphorylation (includes PP2A)
4) How nuclear YAP/TAZ affects transcription?
HIPPO off: YAP/TAZ/Yki unphosphorylated = goes to the nucleus, binds TFs TEAD/Sd and regulates transcription of target genes by binding to Hippo response elements (HREs)
Recruitment of Co-activators and Chromatin remodeling complexes (Methyltransferases, mediator Coaktivator …)
Transcription of genes like CYR61, CTGF, and AREG, which are involved in cell proliferation and growth, Anti-Apoptosis
HIPPO on: YAP/TAZ/Yki phosphorylated -> cytoplasmic retention by 14-3-3 /degradation ß-TRCP Ubiquitin-ligase
TEAD/Sd bound to Corepressor -> Default repression
Similar mechanism to WNT signaling —> in absense of the ligand ß-catenin destruction but upon activation ß-catenin accumulates and enters nucleus
5) Which proteins directly interact with YAP and TAZ?
Upstream LATS (with Mob) phosphorylation
Cytoplasmic retention -> 14-3-3
Degradation -> CK , SCF
Nuclear translocation -> TEAD (Scalloped)
Other pathways: WNT (Axin, TrCPB) , SMADs , Abl,SRC, ß-catenin …
6) What is the role of cell polarity proteins in regulation of YAP/TAZ, in the epithelia?
Intact cell polarity (=Scibble on membrane, catenins bound) -> Hippo signal ON -> YAP/TAZ cytoplasmic retention / degradation
EMT or cells uncontrolled (=Scribble delocalization) -> Hippo signal OFF -> high levels YAP/TAZ -> cancer stem cell related traits
Factors important for establishing epithelial cell polarity activate Hippo -> YAP/TAZ is excluded from the nucleus.
Apical: Kibra, Merlin (NF2), Lateral: Tight junctions, Adherens junctions ( E-cad) Baso-lateral: Scribble
7) How does EMT (Epithelial-to mesenchymal transition) regulate YAP/TAZ localization and activity?
8) Which mechanical stimuli promote YAP/TAZ nuclear localization?
High cell density / soft substrate / small adhesive area -> round cell ‘= low mechanical stimuli -> YAP/TAZ in cytoplasm
Large adhesive area, stiff substrate, low cell density, stretching of monolayer
Flat cell, high mechanical stimuli -> nucleus (e.g. for wound healing proliferation needed)
9) How focal adhesion kinases regulate YAP/TAZ?
cells respond to ECM stiffness through integrin-mediated Focal adhesions which connect the ECM with the intracellular F-actin cytoskeleton
Stiff matrix → Integrins cluster → Activation of FAK and Src -> induces force-dependent conformational changes in ECM adhesion complexes -> increasing actin polymerization and stress fiber contractility (actomyosin tension)-> active YAP/TAZ
Focal adhesions activate Rho-GTPases → Activate ROCK → F-actin polymerization → YAP/TAZ activation.
Triggering Actomyosin force -> Cytoskeleton remodeling -> YAP/TAZ dephosphorylated -> Translocates to nucleus & promotes target gene expression
F-actin polymerization inhibits Hippo pathway → Unable to inhibit YAP/TAZ → YAP/TAZ nuclear translocation
10) Give two examples of cell differentiation induced by mechanical stimuli.
High stiffness/ high mechanical stimuli -> MSC differentiate into osteocytes
softness / low mechanical stimuli
-> MSC differentiate into adipocytes
hESC into neurons
11) How do YAP and TAZ interact with canonical WNT pathway?
YAP/TAZ associates with ß-Catenin destruction complex
If Components of destruction complex k/o -> YAP/TAZ released 6 translocates to nucleus
same with Wnt signaling active
Wnt off: YAP/TAZ recruits E3 ubiquitin ligase ß-TRCP to complex -> ß-Catenin inhibition 6 degradation
-> if you use YAP/TAZ RNAi -> ß-catenin will translocate into nucleus in abscence of Wnt
Synergistic effect between ß-catenin and YAP/TAZ
12) Give two examples of Hippo pathway biological effects.
Regeneration upon injury in intestinal crypts
-> upon injury TAZ levels rise to revive SC and make them proliferate
Trophectoderm specification in mouse blastocyst
-> Cell contact and cell polarity lead to differential activation of Lats kinase in the inside versus the outside cells
-> in outer cell layer hippo off -> YAP nuclear -> activates trophoectoderm gene expression
-> inside Lats phosphorylates YAP -> Hippo on -> YAP cytoplasm
-> Reversed to epithelial cells (apical-basal restricts nuclear YAP/TAZ)
13) Which therapeutic strategies can be developed to control YAP/TAZ activity in cancer?
using small molecules
1) Disruption YAP/TEAD complex (e.g targeting downstream transcriptional machinery)
2) Targeting Rho GTPases or their effector ROCK
3) Using Wnt signaling inhibitors
4) Using GPCR inhibitors
Active Hippo / Salvador complex (and Mob Warts) act as Tumor suppressor —> drugs to stabilize
YAP/TAZ , Yorkie, TEAD (Scalloped) act as oncogens -> inhibit
F-actin and Integrins inhibit Hippo -> drugs to inhibit those or that effect?
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