Hippo/YAP-TAZ Signaling: LATS Kinase Cascade, Mechanosensing, TEAD Transcription, and Research Tools

# Hippo/YAP-TAZ Signaling: LATS Kinase Cascade, Mechanosensing, TEAD Transcription, and Research Tools

For Research Use Only. Not for use in humans or animals.

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Introduction

The Hippo signaling pathway is a master regulator of organ size, tissue homeostasis, and regeneration. First identified through mosaic genetic screens in Drosophila that yielded massive overgrowth phenotypes reminiscent of a hippopotamus (giving the pathway its name), the core pathway is deeply conserved across metazoans. In mammals, the pathway culminates in the phosphorylation and cytoplasmic sequestration of YAP (Yes-Associated Protein) and TAZ (Transcriptional co-Activator with PDZ-binding motif, also called WWTR1), two paralogous transcriptional co-activators that lack intrinsic DNA-binding domains but potently activate gene expression through their primary nuclear partners, the TEAD (TEA Domain) transcription factor family.

What distinguishes Hippo/YAP-TAZ from most other signaling pathways is its extraordinary sensitivity to mechanical and physical signals: cell density, extracellular matrix stiffness, cytoskeletal tension, and cell geometry all profoundly influence YAP/TAZ nuclear localization and activity — often independently of the canonical LATS1/2 kinase cascade. This mechanosensing function places YAP/TAZ at the interface of biomechanics and transcriptional regulation, making the pathway relevant not only to cancer biology but also to tissue engineering, regenerative biology, and mechanobiology research.

This review covers the core Hippo kinase cascade (MST1/2-LATS1/2), YAP/TAZ phosphorylation and 14-3-3 sequestration, TEAD-mediated transcription, mechanical regulation of YAP/TAZ, upstream pathway regulators (NF2/Merlin, KIBRA, AMOT), cross-talk with other signaling pathways, and validated research tools.

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Core Hippo Kinase Cascade

MST1 and MST2: Apical Kinases

Mammalian STE20-like protein kinases 1 and 2 (MST1/STK4 and MST2/STK3) are the apical kinases of the canonical Hippo cascade, homologous to Drosophila Hippo (Hpo). MST1/2 are related to the yeast kinase Ste20 and share:

• •N-terminal kinase domain: Activated by autophosphorylation at T183 (MST1) and T180 (MST2) in the activation loop; also phosphorylated by upstream kinases
• •SARAH (Salvador/RASSF/Hippo) domain: C-terminal coiled-coil domain that mediates homodimerization (required for trans-autophosphorylation) and heterodimerization with the scaffold protein Salvador (SAV1/WW45) and RASSF family proteins (RASSF1A, RASSF1C, RASSF5/NORE1A)

SAV1 (Salvador homolog 1) binds MST1/2 via SARAH domain interactions, enhancing MST1/2 activity and recruiting the kinases to LATS1/2 for efficient signal transduction.

RASSF Family: Apoptotic and Growth-Suppressive Inputs

RASSF (Ras Association Domain Family) proteins are scaffolds that link RAS family GTPases to MST1/2 activation:

• •RASSF1A: The most studied; epigenetically silenced in many cancers (promoter hypermethylation); binds MST1/2 via SARAH domains; activates MST1/2 under apoptotic stimuli; links NORE1A-K-RAS signaling to Hippo
• •RASSF5 (NORE1A): Binds active KRAS/HRAS at their effector domain; recruits MST1/2; provides a direct link between oncogenic RAS and Hippo pathway engagement
• •RASSF2/6: Induce apoptosis via MST1/2 activation independently of canonical Hippo-YAP

LATS1 and LATS2: Central Effector Kinases

Large Tumor Suppressor kinases 1 and 2 (LATS1 and LATS2) are NDR (Nuclear Dbf2-Related) family kinases that are the direct upstream kinases for YAP and TAZ. LATS1/2 require two activating phosphorylation events:

1. Hydrophobic motif phosphorylation: MST1/2 phosphorylate LATS1 at T1079 and LATS2 at T1041 in the hydrophobic motif — this is the canonical Hippo cascade phosphorylation event

2. Activation loop phosphorylation: MST1/2 phosphorylate LATS1 at S909 and LATS2 at S872 in the activation loop (with MOB1A/B adapter proteins facilitating this interaction)

MOB1 (MOB Kinase Activator 1): MOB1A and MOB1B are small adapter proteins that bind the N-terminal regulatory domain of LATS1/2, enhancing LATS kinase activity and facilitating MST1/2-LATS interaction. MOB1 is directly phosphorylated by MST1/2 at T35, and this phosphorylation promotes MOB1-LATS interaction. MOB1 is frequently lost in cancer.

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YAP and TAZ: Structure and Phospho-Regulation

YAP Domain Architecture

YAP (65 kDa, isoform YAP1) contains:

• •N-terminal TEAD-binding domain (TBD): The primary interface with TEAD family members; essential for transcriptional co-activation
• •WW domains (1 or 2, depending on isoform): PPxY motif-binding modules; interact with LATS1/2 (PPxY motif in LATS), AMOT family proteins, and PTPN14
• •Transcriptional activation domain (TAD): C-terminal; required for co-activator function with TEAD
• •PDZ-binding motif: Extreme C-terminus; engages PDZ domain scaffolds at tight junctions and adherens junctions
• •Coiled-coil domain: Mediates YAP dimerization; not essential for TEAD interaction

TAZ (WWTR1) Domain Architecture

TAZ (45 kDa) is a structural paralog of YAP:

• •TEAD-binding domain
• •Single WW domain (YAP has 2 WW domains in the 2WW isoform; TAZ has 1)
• •Coiled-coil domain
• •PDZ-binding motif (C-terminal; binds PDZ domains at cell junctions)
• •No SH3-binding domain (unlike YAP)

YAP/TAZ Phosphorylation by LATS1/2

LATS1/2 phosphorylate YAP at five serine residues: S61, S109, S127, S164, S381. The most critical sites are:

LATS1/2 phosphorylate TAZ at S89 (equivalent to YAP S127; 14-3-3 binding and cytoplasmic retention) and S311 (equivalent to YAP S381; CK1/β-TrCP-mediated degradation).

14-3-3 Sequestration

pYAP-S127 and pTAZ-S89 are recognized by 14-3-3 proteins (ζ, σ, η, ε isoforms), which bind the phosphoserine and mask the nuclear localization signal of YAP/TAZ, trapping them in the cytoplasm. This mechanism is rapid and reversible — when LATS1/2 activity decreases, phosphatases (PP2A with B55α subunit, PP1) dephosphorylate YAP/TAZ, freeing them from 14-3-3 and allowing nuclear entry.

β-TrCP-Mediated Degradation

YAP S381 phosphorylation by LATS1/2 creates a priming site for CK1δ/ε, which then phosphorylates S384 and S387. This triply phosphorylated motif (pS381-XX-pS384-pS387) is recognized by β-TrCP (F-box protein in the SCF E3 ubiquitin ligase complex, same as IκBα and GLI3 degradation), leading to YAP ubiquitination at K280 and K497, and subsequent 26S proteasomal degradation. This provides a second tier of YAP suppression beyond cytoplasmic sequestration.

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Upstream Regulators: Switching Hippo On and Off

NF2/Merlin: Tumor Suppressor Link to LATS

NF2 (Neurofibromatosis Type 2, Merlin) is a FERM domain protein and critical upstream activator of the LATS1/2 kinases. NF2 is itself a major tumor suppressor — biallelic NF2 loss causes neurofibromatosis type 2 (schwannomas, meningiomas), and somatic NF2 mutations drive mesothelioma (~50%), clear cell renal carcinoma, and other tumors. NF2 functions by:

1. Directly binding LATS1/2 and facilitating their interaction with MST1/2 at the plasma membrane

2. Stabilizing LATS1/2 by preventing their proteasomal degradation (competing with CRL4-DCAF1)

3. Linking the E-cadherin/α-catenin/β-catenin complex at cell-cell junctions to Hippo pathway activation at high cell density

KIBRA (WWC1): Scaffold for MST-LATS Complex

KIBRA (Kidney and Brain expressed protein, WWC1) is a WW-domain and C2-domain scaffold protein that binds both MST1/2 and LATS1/2, facilitating their interaction and enhancing LATS activation. KIBRA knockout phenocopies partial Hippo pathway loss.

AMOT Family (Angiomotin): YAP Sequestration and Pathway Activation

Angiomotin (AMOT), AMOTL1, and AMOTL2 are scaffolding proteins that interact with YAP/TAZ via WW-PPxY interactions:

• •In the cytoplasm/cell junctions, AMOT proteins sequester YAP/TAZ independent of S127 phosphorylation (a LATS-independent restraint mechanism)
• •AMOT proteins also activate LATS1/2, linking tight junction integrity to Hippo pathway activation
• •Upon F-actin assembly (mechanically active cells), AMOT is titrated away from YAP by actin binding, releasing YAP from AMOT-mediated sequestration

CRL4-DCAF1: Proteasomal Degradation of LATS

The CRL4 E3 ubiquitin ligase complex containing DCAF1 (also called VprBP) polyubiquitinates LATS1/2, targeting them for proteasomal degradation. This is the mechanism by which oncogenic signals (via DCAF1 activation) suppress LATS kinase levels. DCAF1 is also exploited by viral oncoproteins (HIV-1 Vpr, HTLV-1 Tax) to suppress LATS and activate YAP/TAZ.

PTPN14: YAP WW Domain Trapper

PTPN14 (Protein Tyrosine Phosphatase Non-receptor 14) binds directly to the YAP WW domains via its PPXY motifs, sequestering YAP in the cytoplasm independent of LATS-mediated phosphorylation. PTPN14 also facilitates LATS-mediated YAP degradation by promoting CK1/β-TrCP access.

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Mechanical Regulation of YAP/TAZ

A central and distinctive feature of YAP/TAZ biology is their profound sensitivity to mechanical cues, often independent of or parallel to the canonical LATS1/2 kinase cascade.

Cell Density (Contact Inhibition)

At low cell density, YAP/TAZ are nuclear and transcriptionally active, driving proliferation. As cell density increases:

• •Cell-cell contact activates NF2/Merlin → LATS1/2 activation → YAP/TAZ phosphorylation and cytoplasmic retention
• •E-cadherin-mediated adhesion reinforces this via α18-catenin conformational change (tension-sensing) at adherens junctions
• •Tight junction components (AMOT, ZO-1/2) sequester YAP/TAZ at junctions

ECM Stiffness

YAP/TAZ activity scales with substrate stiffness:

• •Soft substrates (0.1–1 kPa, mimicking brain/fat): YAP/TAZ predominantly cytoplasmic; cells adopt round, non-proliferative morphology
• •Stiff substrates (10–40 kPa, mimicking muscle/pre-fibrotic tissue): YAP/TAZ nuclear; cells spread and proliferate
• •Very stiff substrates (>40 kPa, mimicking bone or tumor stroma): YAP/TAZ strongly nuclear; pro-proliferative, pro-invasive gene programs

This stiffness sensing requires integrin-mediated focal adhesion assembly and actomyosin contractility (ROCK/myosin II axis).

Cytoskeletal Tension and F-Actin

Rho GTPases (RhoA, Rac1, Cdc42) and their downstream effectors ROCK1/2 and mDia (formins) regulate F-actin polymerization and actomyosin tension:

• •F-actin assembly → YAP/TAZ nuclear: Active RhoA/ROCK → stress fiber formation → nuclear YAP/TAZ. This occurs via: (1) F-actin sequestering the LATS activator AMOT, reducing LATS activity; (2) increased nuclear pore size/transport in tensed cells; (3) direct YAP/TAZ interaction with filamentous actin
• •F-actin depolymerization → YAP/TAZ cytoplasmic: Latrunculin A or cytochalasin D (G-actin poisoning agents) rapidly relocalize nuclear YAP/TAZ to the cytoplasm
• •Blebbistatin (myosin II inhibitor) → YAP/TAZ cytoplasmic: Confirms that actomyosin contractility (not just F-actin polymerization per se) maintains YAP/TAZ nuclear activity
• •Cell geometry: Cells confined to small micropatterned areas (restricting spreading) show cytoplasmic YAP/TAZ even on stiff substrates; spreading on large patterns promotes nuclear YAP/TAZ

GPCR Signaling to YAP/TAZ

GPCRs couple to YAP/TAZ through Gα12/13-RhoA signaling (promotes nuclear YAP/TAZ) and Gαs-cAMP-PKA signaling (promotes cytoplasmic YAP/TAZ via direct YAP S127 phosphorylation by PKA):

• •Gα12/13-coupled receptors (LPA, S1P, thrombin): → RhoA → ROCK → F-actin → YAP/TAZ nuclear (LATS-independent)
• •Gαs-coupled receptors (glucagon, epinephrine, FSH): → adenylyl cyclase → cAMP → PKA → YAP S127 phosphorylation → cytoplasmic retention. PKA can directly phosphorylate YAP at S127 without requiring LATS1/2.
• •Gαq/11-coupled receptors: Context-dependent; generally promote YAP/TAZ nuclear localization

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TEAD Transcription Factors and Target Genes

TEAD Family

The four TEAD paralogs (TEAD1-4) are the primary nuclear binding partners of YAP and TAZ. TEADs share:

• •TEA/ATTS DNA-binding domain: Recognizes CATTCC or GGATGT (GC-rich TEAD-binding elements in promoters and enhancers)
• •YAP/TAZ-binding domain: C-terminal; binds the TEAD-binding domain of YAP/TAZ through a hydrophobic interface (the target of verteporfin, which disrupts YAP-TEAD interaction)
• •Auto-palmitoylation site: TEAD palmitoylation at a cysteine in the YBD is required for YAP/TAZ interaction; palmitoylation inhibitors (PDEDF inhibitors) can disrupt this
• •Co-activators VGLL1-4 compete with YAP/TAZ for TEAD binding (Vestigial-like proteins act as YAP/TAZ antagonists at TEAD)

Key YAP/TAZ-TEAD Target Genes

CTGF (Connective Tissue Growth Factor/CCN2) and CYR61 (CCN1) are the canonical YAP/TAZ target genes used as positive pathway activity readouts in most experimental settings.

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Research Tools for Hippo/YAP-TAZ Signaling

Small Molecule Research Probes

Key Research Antibodies

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Experimental Protocols

Protocol 1: YAP/TAZ Nuclear-Cytoplasmic Localization by Immunofluorescence

Objective: Quantify YAP/TAZ nuclear vs. cytoplasmic localization as a function of cell density, substrate stiffness, or pharmacological manipulation.

Materials: Anti-YAP (D8H1X), secondary antibody (Alexa Fluor 488), DAPI, polyacrylamide hydrogels of defined stiffness (0.5, 5, 40 kPa; BioCoat Matrigel or custom-fabricated; functionalized with fibronectin or collagen I), confocal microscope.

Procedure:

1. Cell density experiment: Plate MCF10A, Caco-2, or primary MEFs at: (a) sparse (500 cells/cm², ~30% confluent), (b) medium (~70% confluent), (c) high density (>90% confluent, post-confluence). Allow 24h attachment before fixation.

2. Substrate stiffness experiment: Prepare polyacrylamide gels (0.5 kPa: 3% acrylamide/0.06% bis; 5 kPa: 8%/0.048%; 40 kPa: 15%/0.45%) cast on silanized glass, functionalized with 0.1 mg/mL fibronectin via sulfo-SANPAH photoactivation. Plate 5,000 cells/gel; incubate 24–48h before fixation.

3. Pharmacological perturbations: Treat cells with: Latrunculin A (1 μM, 1h), blebbistatin (50 μM, 2h), Y-27632 (10 μM, 2h), LPA (10 μM, 1h), verteporfin (1 μM, 4h). Fix at time of peak effect.

4. Immunofluorescence: Fix in 4% PFA (15 min), permeabilize (0.3% Triton X-100, 5 min), block (5% BSA, 1h). Anti-YAP primary overnight (4°C), Alexa Fluor 488 secondary (1h). Mount with DAPI.

5. Quantification: Acquire ≥20 fields per condition (20× or 40× objective). Using ImageJ or CellProfiler: (a) segment nuclei by DAPI; (b) define cytoplasmic ring around each nucleus; (c) measure mean YAP fluorescence intensity in nuclear and cytoplasmic compartments; (d) calculate N:C ratio per cell. Score ≥100 cells per condition. Classify: N:C >1.5 = predominantly nuclear; N:C <0.7 = predominantly cytoplasmic; 0.7–1.5 = mixed.

Expected outcomes: Sparse cells on stiff substrate → >80% cells with nuclear YAP; high density or Latrunculin A treatment → >80% cytoplasmic YAP. Stiffness gradient (0.5→40 kPa): progressive increase in nuclear YAP fraction. LPA treatment → rapid nuclear translocation. Blebbistatin/Y-27632 → cytoplasmic shift within 1–2h.

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Protocol 2: Phospho-YAP Western Blot — Hippo Kinase Cascade Activity

Objective: Monitor MST1/2→LATS1/2→YAP phosphorylation cascade under pathway-activating (high density, cell-cell contact) and pathway-inactivating (LPA, low density) conditions.

Materials: Anti-phospho-YAP S127, anti-total YAP, anti-phospho-LATS1 T1079, anti-total LATS1, anti-phospho-MST1/2 T183/T180, anti-total MST1, anti-MOB1 phospho-T35, anti-β-actin; lysis buffer (RIPA + phosphatase inhibitors: PhosSTOP, NaF, Na₃VO₄).

Procedure:

1. Plate cells at sparse (10% confluent) vs. high density (90%+ confluent, allowed to contact-inhibit for 24h) in matched conditions.

2. Kinase cascade activation: To activate Hippo, treat cells with: MST1/2 agonist (Okadaic acid, phosphatase inhibitor that amplifies kinase cascade, 100 nM, 30 min), or high density (endogenous activation).

3. Lyse cells immediately in cold RIPA + phosphatase inhibitors. BCA quantification.

4. Resolve on 10% SDS-PAGE; transfer. Probe sequentially (strip and reprobe, or use multiplexed fluorescent detection): phospho-MST1 T183 / total MST1, phospho-LATS1 T1079 / total LATS1, phospho-YAP S127 / total YAP.

5. LATS inhibitor (TRULI): Pretreat high-density cells with TRULI (LATS1/2 inhibitor, 0.1–1 μM, a recently described tool compound) to confirm LATS dependency of YAP S127 phosphorylation. Note: LATS inhibitors can paradoxically increase total YAP protein by reducing S381-dependent degradation.

6. Kinase independence control: Treat LPA (10 μM, 1h) at high density — should reduce phospho-YAP S127 without reducing phospho-LATS T1079 in all contexts (demonstrating that some YAP regulation bypasses LATS).

Expected outcomes: High density → elevated phospho-MST1 T183, phospho-LATS1 T1079, phospho-YAP S127, cytoplasmic YAP by IF. Low density → reduced phosphorylation cascade, nuclear YAP. Okadaic acid → amplified phospho-MST/LATS/YAP signal. LPA → reduces phospho-YAP S127 at high density despite maintained phospho-LATS (LATS-independent YAP nuclear activation).

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Protocol 3: YAP/TAZ Target Gene Induction — RT-qPCR and Reporter Assay

Objective: Quantify YAP/TAZ transcriptional activity through target gene mRNA measurement and TEAD-responsive luciferase reporter.

Materials: RT-qPCR reagents, primers for CTGF, CYR61, ANKRD1, AMOTL2, BIRC5; 8×GTIIC-Luciferase (TEAD reporter containing 8× CATTCC TEAD binding sites), verteporfin, IBS-008CSO.

Procedure:

1. Density-dependent target gene induction: Harvest RNA from sparse (24h post-seeding) vs. high-density (72h post-seeding) cells. Synthesize cDNA (SuperScript IV), qPCR with primers for CTGF (exon junction-spanning), CYR61, ANKRD1, AMOTL2. Normalize to GAPDH and HPRT1. Sparse cells should show 3–15× higher CTGF/CYR61/ANKRD1 vs. high-density cells.

2. Verteporfin inhibition: Treat sparse cells with verteporfin (0.1, 0.3, 1, 3 μM) for 6h. Assess YAP target gene suppression. Note: verteporfin has photosensitizing activity; perform all steps in dim light.

3. TEAD reporter: Transfect 8×GTIIC-Luc + Renilla control (10:1 ratio) into target cells. After 24h, impose sparse vs. dense conditions for 48h. Measure reporter activity (firefly:Renilla ratio). Sparse: high reporter; dense: low reporter. Verteporfin (1 μM) should suppress reporter in sparse cells.

4. YAP overexpression: Transfect YAP-5SA (constitutively active, all 5 LATS phosphorylation sites mutated to alanine) alongside TEAD reporter. YAP-5SA should maximally activate reporter independent of cell density. Confirm verteporfin sensitivity of YAP-5SA-driven reporter.

5. TEAD inhibitor comparison: Compare verteporfin, IBS-008CSO (50–500 nM), and TED-347 (30–300 nM) side-by-side on GTIIC reporter in YAP-5SA-expressing cells. Calculate IC50 for each compound.

Expected outcomes: Sparse cells: 5–20× GTIIC reporter over dense cells. Verteporfin IC50 ~0.5–2 μM on reporter; IBS-008CSO IC50 ~100–300 nM; TED-347 IC50 ~50–200 nM. YAP-5SA overexpression → 20–100× reporter induction; fully blocked by all TEAD inhibitors.

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Protocol 4: Hydrogel Stiffness Model — Mechanosensing Quantification

Objective: Establish quantitative relationship between substrate stiffness and YAP/TAZ nuclear localization with mechanistic dissection.

Materials: Custom polyacrylamide hydrogels (0.2, 1, 4, 12, 40 kPa), sulfo-SANPAH (photoactivatable crosslinker), fibronectin (coating protein), atomic force microscopy (AFM) or parallel plate rheometry for gel stiffness verification, blebbistatin, Y-27632, TRULI.

Procedure:

1. Gel fabrication: Prepare acrylamide/bis-acrylamide solutions for each stiffness, cast between silanized glass slide and non-silanized coverslip spacer system. Polymerize with TEMED/APS. Verify Young's modulus by AFM nanoindentation (target values ±20% of intended stiffness). Transfer gels to tissue culture plates.

2. Fibronectin functionalization: Activate gels with UV-activated sulfo-SANPAH (0.5 mg/mL, UV 365 nm, 5 min), wash 3× PBS, incubate with fibronectin (0.1 mg/mL, 1h, 37°C), wash 3× PBS. Block with 1% BSA (30 min).

3. Cell seeding and analysis: Plate cells at identical density (5,000 cells/cm²) on each stiffness gel. Fix at 24h with 4% PFA. Co-stain with anti-YAP + DAPI + anti-paxillin (focal adhesion marker). Quantify: (a) YAP N:C ratio (primary readout); (b) focal adhesion area (paxillin+ structures); (c) cell spread area (by WGA or CellMask staining); (d) nuclear area (by DAPI).

4. Inhibitor interventions on gels: On stiff (40 kPa) gels, treat with blebbistatin (50 μM), Y-27632 (10 μM), or Latrunculin A (0.5 μM) for 2h before fixation. Confirm that F-actin disruption relocates YAP to cytoplasm even on stiff substrates.

5. Correlative analysis: Plot YAP N:C ratio vs. log(stiffness): expect sigmoidal relationship with inflection ~5–10 kPa. Plot focal adhesion area vs. YAP N:C ratio per cell for correlative mechanosensing analysis.

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Protocol 5: YAP-TEAD Co-Immunoprecipitation

Objective: Confirm YAP-TEAD complex formation and test small molecule disruption of the interaction.

Materials: Anti-TEAD1 (rabbit, for IP), anti-YAP (mouse, for detection), anti-TEAD1 (mouse, for detection), protein A/G magnetic beads, co-IP lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, protease inhibitors — no SDS), verteporfin, IBS-008CSO.

Procedure:

1. Plate sparse cells (nuclear YAP, active YAP-TEAD complex) in 10 cm dishes; collect ~3×10⁶ cells per condition. Treat with verteporfin (1 μM), IBS-008CSO (0.5 μM), or DMSO vehicle for 4h.

2. Lyse cells in non-denaturing co-IP buffer (0.5% NP-40, NO SDS). Centrifuge 13,000g × 10 min. Pre-clear with 20 μL protein A/G beads (1h, 4°C rotating).

3. Input: save 5% of lysate. IP: incubate pre-cleared lysate with 3–5 μg anti-TEAD1 antibody overnight (4°C). Add 40 μL protein A/G beads (2h, 4°C), wash 5× co-IP buffer. Elute with Laemmli sample buffer (95°C, 5 min).

4. Resolve IP and Input on 10% SDS-PAGE. Probe for YAP (co-IP detection): strong YAP band in anti-TEAD1 IP from vehicle-treated cells; reduced or absent in verteporfin- or IBS-008CSO-treated cells.

5. Reverse co-IP: Anti-YAP IP, detect TEAD1 by Western blot. Confirm bidirectional disruption.

6. VGLL4 competitive IP: Overexpress VGLL4 (TEAD competitor of YAP) by transient transfection; confirm reduced YAP-TEAD co-IP in VGLL4-expressing cells (VGLL4 competes with YAP for the same TEAD interface).

Expected outcomes: Strong YAP co-IP with TEAD1 in sparse cells. Verteporfin (1 μM) reduces YAP-TEAD co-IP by 60–90%. IBS-008CSO (500 nM) reduces co-IP by >80%. Dense cells show reduced YAP-TEAD co-IP compared to sparse (less nuclear YAP available for TEAD interaction). VGLL4 overexpression → competitive reduction of YAP-TEAD co-IP.

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Pathway Cross-Talk

Hippo-WNT

YAP/TAZ interact with the WNT destruction complex: when WNT is absent, β-catenin is phosphorylated by the destruction complex (APC/AXIN/CK1/GSK3β) and degraded. TAZ is a component of this complex and promotes β-catenin degradation (TAZ as a negative regulator of WNT). Paradoxically, nuclear YAP can also potentiate β-catenin-TCF transcription at shared target genes (e.g., CTGF, CYR61, CCN2) in WNT-active cells.

Hippo-TGF-β

As discussed in the TGF-β/SMAD article, YAP/TAZ are potent co-activators of SMAD2/3 in the nucleus, driving EMT and pro-fibrotic programs. Nuclear YAP/TAZ physically interact with SMAD2/3 and SMAD7 to modulate TGF-β transcriptional responses.

Hippo-HH

LATS2 phosphorylates GLI2 and promotes its cytoplasmic retention, linking Hippo pathway activation to HH pathway suppression. Conversely, active YAP/TAZ can promote GLI1 expression.

Hippo-mTOR

YAP/TAZ promote mTORC1 activity by inducing the expression of Leucine transporters (LAT1/SLC7A5), increasing amino acid uptake and mTORC1 activation. This creates a metabolic amplification axis in proliferating cells.

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References

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This article is intended for research use only (RUO). All compounds, reagents, and biological tools discussed herein are for in vitro laboratory investigation. No information in this article constitutes medical advice, dosing guidance for human or animal use, or clinical protocol recommendations.