SRC/FAK/Integrin Signaling: Focal Adhesion Kinase Activation, Mechanosensing, and Anoikis Resistance in Cancer Research

# SRC/FAK/Integrin Signaling: Focal Adhesion Assembly, Kinase Cascades, and Research Tools

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

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Introduction

Integrin-mediated adhesion to the extracellular matrix (ECM) is a fundamental requirement for the survival, proliferation, migration, and differentiation of most non-hematopoietic cells. Integrins are heterodimeric transmembrane receptors that physically bridge the ECM to the intracellular actin cytoskeleton through large multiprotein assemblies called focal adhesions (FAs). At the core of focal adhesion signaling are two non-receptor tyrosine kinases — Focal Adhesion Kinase (FAK/PTK2) and SRC family kinases (particularly c-SRC) — whose cooperative activation generates a phosphotyrosine scaffold that recruits dozens of signaling and structural proteins, ultimately activating downstream PI3K/AKT, RAS/MAPK, and Rho GTPase pathways.

Understanding SRC-FAK-integrin signaling is essential to mechanobiology, cancer invasion and metastasis research, angiogenesis studies, and the investigation of anoikis (apoptosis triggered by loss of matrix attachment). FAK is amplified or overexpressed in numerous cancers, and elevated FAK activity correlates with invasive, metastatic phenotypes. SRC itself is overactivated in ~50% of colon, breast, and liver cancers. This review covers integrin structure and ECM engagement, focal adhesion assembly, FAK Y397 autophosphorylation and the FAK-SRC complex, downstream effectors (PI3K, GRB2-SOS-RAS, paxillin, vinculin, DOCK180-RAC1), anoikis resistance mechanisms, and validated research tools.

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Integrin Structure and ECM Engagement

Integrin Heterodimer Architecture

Integrins are non-covalent αβ heterodimers; humans express 18 α subunits and 8 β subunits that combinatorially generate 24 distinct integrins with different ECM ligand specificities:

Each integrin subunit consists of an N-terminal extracellular domain (~700–1100 residues for α; ~600–800 for β), a single-pass transmembrane domain, and a short cytoplasmic tail (~30–50 residues). The integrin β subunit cytoplasmic tail is the critical signaling interface, containing binding sites for talin, kindlin, filamin, and FAK.

Inside-Out and Outside-In Signaling

Integrins are regulated by bidirectional signaling:

• •Inside-out activation: Intracellular signals (e.g., talin-1 head domain binding to the β-tail NPxY motif, or kindlin-2 binding the distal NPxY motif) induce a conformational change from the bent/inactive to the extended/active ectodomain conformation, increasing ligand-binding affinity by 1000-fold or more. In platelets, GPCRs activate talin via PIP2/PI(4,5)P₂ hydrolysis → PKC → talin activation.
• •Outside-in signaling: ECM ligand binding to the extended integrin ectodomain induces integrin clustering, further conformational changes in the cytoplasmic tails, and recruitment of signaling complexes to form nascent adhesions → focal complexes → mature focal adhesions.

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Focal Adhesion Assembly: From Nascent Contacts to Mature Focal Adhesions

Focal adhesions evolve through distinct morphological and molecular stages:

Stage 1: Nascent Adhesions

Small (~100–200 nm) integrin clusters at the lamellipodium edge, formed within seconds of ECM contact. Contain: integrins, talin, paxillin, FAK, and early SRC activity. Nascent adhesions are highly dynamic, turning over in ~1 min; most disassemble while some mature into focal complexes.

Stage 2: Focal Complexes

Larger (~0.5–1 μm) and slightly more stable structures at the lamellipodium/lamellum boundary. Require Rac1/Cdc42 activity (via PAK1 → MLCK), vinculin recruitment, and initial FAK-SRC complex formation. Transition to focal adhesions requires RhoA/ROCK-mediated actomyosin contractility.

Stage 3: Mature Focal Adhesions

Large (1–5 μm), highly stable structures anchoring stress fibers under high myosin II contractility. Contain a stratified molecular architecture (integrin→talin→vinculin→paxillin→FAK/SRC layer→actin) with ~150+ proteins. Require RhoA/ROCK activity, high actomyosin tension, and sustained FAK-SRC signaling.

Stage 4: Fibrillar Adhesions

Large, elongated structures along the cell interior formed from mature FAs by α5β1 integrin translocation along fibronectin fibrils, driven by myosin IIA contractility. Enriched in tensin; depleted of FAK and SRC; mark fibronectin matrix assembly.

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FAK: Structure and Y397 Autophosphorylation

FAK Domain Organization

Focal Adhesion Kinase (FAK/PTK2) is a 125 kDa non-receptor tyrosine kinase with a modular domain structure:

• •N-terminal FERM domain (residues 1–352): Band 4.1, Ezrin, Radixin, Moesin homology domain; directly binds the β1-integrin cytoplasmic tail (interacts with the membrane-proximal NPIY motif); also binds growth factor receptors (EGFR, PDGFR), p53, and PIP2 at the inner leaflet. In the autoinhibited resting state, the FERM domain binds and inhibits the kinase domain.
• •Central kinase domain (residues 416–676): Bilobal kinase fold; contains the activation loop with the critical Y576/Y577 residues (phosphorylated by SRC for full kinase activation); autophosphorylation at Y397 (between FERM and kinase domains in the FERM-kinase linker).
• •KAKTLRK motif: Basic residue cluster between FERM and kinase; contributes to electrostatic interactions with the plasma membrane.
• •Proline-rich regions (PRRs 1–3, residues 712–898): Bind SH3 domains of GRB7, GRAF, and other effectors.
• •C-terminal FAT domain (Focal Adhesion Targeting, residues 920–1052): Four-helix bundle that directly binds paxillin LD motifs (LD2 and LD4) and talin; required for focal adhesion localization.

Y397 Autophosphorylation: The Initiating Event

When ECM-bound integrins cluster and the FERM-kinase autoinhibitory interaction is released (by talin-integrin recruitment competing with FERM-kinase interaction, and by membrane PIP2 binding to the FERM basic patch), FAK undergoes trans-autophosphorylation at Y397 in the linker between FERM and kinase domains. This creates a high-affinity SH2 domain binding site (pY397-VAE motif) with selectivity for the SRC-family kinase SH2 domain (Kd ~50 nM). Phospho-Y397 is the canonical marker of FAK activation in every published Western blot and IF experiment using this pathway.

Phospho-FAK Y397 is detected by widely used antibodies (e.g., Cell Signaling Technology #3283 and #8556) and is used to assess:

• •Integrin-ECM engagement
• •Cell adhesion and spreading
• •Mechanical force transduction
• •Growth factor receptor-FAK crosstalk

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The FAK-SRC Complex

SRC Recruitment and Activation

c-SRC (Proto-oncogene tyrosine-protein kinase Src) is recruited to focal adhesions via:

1. Its SH2 domain binding phospho-FAK Y397 (primary recruitment mechanism)

2. Its myristoyl and palmitate lipid modifications anchoring it to the inner plasma membrane leaflet

3. Its SH3 domain binding PRR sequences in FAK and paxillin

SRC itself is regulated by intramolecular autoinhibition:

• •Inactive state: The SRC SH2 domain binds intramolecularly to pY527 in the SRC C-terminal tail (phosphorylated by CSK, C-terminal Src Kinase); the SH3 domain simultaneously binds the SH2-kinase linker intramolecularly. This doubly-locked conformation maintains SRC in an inactive state (low basal activity).
• •Activation: Dephosphorylation of pY527 by receptor-linked phosphatases (PTP1B, SHP1/2, CD45 in immune cells) releases SH2 autoinhibition. SRC then trans-autophosphorylates at Y416 in the activation loop (increases catalytic activity 8–10-fold). When recruited to FAK via SH2-pY397 interaction, the intramolecular SH2-pY527 is displaced, enabling full SRC activation.

SRC-FAK Cooperative Activation

The FAK-SRC complex is mutually activating:

1. FAK autophosphorylates Y397 → recruits SRC SH2 domain

2. SRC phosphorylates FAK activation loop at Y576/Y577 (FAK activation loop; increases FAK kinase activity ~2-fold above pY397 alone)

3. SRC phosphorylates FAK at Y861 (paxillin recruitment scaffold expansion) and Y925 (GRB2-SOS recruitment → RAS→ERK activation)

4. SRC phosphorylates paxillin at Y31/Y118 (creates additional SH2 binding sites for CRK, GIT proteins, and further signaling)

5. SRC phosphorylates vinculin, p130Cas, and DOCK180, expanding the signaling scaffold

The resulting FAK-SRC signaling complex is a multi-protein assembly that functions as a "signalosome" organizing downstream pathway activation.

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Focal Adhesion Scaffold Proteins

Paxillin

Paxillin is a ~68 kDa scaffolding protein with five leucine-aspartate (LD) motifs in the N-terminus (binding FAK, vinculin, actopaxin/parvin, and GIT proteins) and four LIM domains in the C-terminus (binding tubulin and the PTPN12 phosphatase). SRC-mediated paxillin phosphorylation at Y31 and Y118 recruits:

• •CRK (SH2 binding pY31): → DOCK180 (GEF) → RAC1 activation → lamellipodia
• •GIT1/2 (bind paxillin LD motifs): → PAK-PIX complex → CDC42 and RAC1 activation → nascent adhesion regulation

Talin

Talin is a large (270 kDa) scaffolding protein with an N-terminal head domain (FERM-like; binds β-integrin NPxY motifs and activates integrins inside-out) and a C-terminal rod domain (~60 helical bundle subdomains). The talin rod contains:

• •Multiple vinculin-binding sites (VBS, ~11 sites) that become exposed under actomyosin tension → vinculin recruitment → FA strengthening
• •Actin-binding sites (ABS2, ABS3) for direct F-actin engagement
• •RIAM-binding sites for PI(3,4,5)P₃-dependent talin activation
• •Dimerization domain (C-terminal)

Talin is the molecular clutch between integrin and F-actin; force applied to FAs by actomyosin unfolding of talin rod domains progressively exposes more vinculin-binding sites, enabling force-dependent FA strengthening (catch-bond behavior at FAs).

Vinculin

Vinculin (124 kDa) adopts an autoinhibited closed conformation in which the head (Vh) domain binds and suppresses the tail (Vt) domain. Both talin (binding Vh) and PIP2 (binding Vt) cooperate to open vinculin and expose its F-actin binding site in Vt. Open vinculin acts as a reinforcing clamp linking talin (via Vh) to F-actin (via Vt), mechanically strengthening FAs under tension.

p130Cas (BCAR1)

p130Cas (Breast Cancer Anti-estrogen Resistance 1) is a large scaffold protein containing 15 SH3-binding YXXP motifs in its substrate domain that, when phosphorylated by SRC, create binding sites for the CRK SH2 domain. The p130Cas-CRK interaction activates DOCK180 (a Rac1 GEF), stimulating Rac1 and lamellipodia formation. Force-dependent p130Cas substrate domain stretching enhances its phosphorylation by SRC, providing a mechanosensitive switch amplifying SRC signaling under tension.

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Downstream Signaling Pathways

PI3K/AKT Activation

FAK-pY397 recruits the p85 regulatory subunit of PI3K via SH2 domain interaction, activating PI3K at focal adhesions and generating PIP3 at the inner leaflet. Focal adhesion-localized PIP3 recruits AKT (via PH domain) and PDK1, enabling AKT T308 phosphorylation and activation. Integrin-FAK-PI3K-AKT signaling promotes:

• •Cell survival (BAD S136 phosphorylation, MDM2 activation, NF-κB activation)
• •mTORC1 activation → protein synthesis
• •Anoikis resistance (critical for cancer metastasis)

GRB2-SOS-RAS-ERK Pathway

FAK pY925 (SRC-phosphorylated site in the FAT domain) creates a binding site for the GRB2 SH2 domain. GRB2 recruits SOS (Son Of Sevenless), a Ras GEF that activates KRAS/NRAS → RAF → MEK → ERK, driving proliferation and survival downstream of ECM adhesion.

RHO GTPase Pathways

FAK-SRC signaling activates all three major Rho GTPases through distinct mechanisms:

• •RhoA: FAK activates GEF-H1 (microtubule-bound RhoA GEF) and suppresses the RhoA GAP p190RhoGAP (via SRC-mediated p190RhoGAP tyrosine phosphorylation); net effect is RhoA activation → ROCK → myosin II → stress fibers and FA maturation
• •Rac1: Paxillin pY31→CRK→DOCK180→Rac1; p130Cas pY→CRK→DOCK180→Rac1; FAK FERM→p85→PI3K→PIP3→Tiam1/DOCK180→Rac1 → lamellipodia
• •CDC42: FAK→paxillin→GIT1/2-PIX→CDC42 → filopodia and polarity

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Anoikis Resistance

Anoikis (from the Greek "homelessness") is the apoptotic death of anchorage-dependent cells deprived of ECM contact. Under normal conditions:

• •Loss of integrin engagement → FAK dephosphorylation (Y397) → loss of PI3K/AKT survival signaling → BAD activation → cytochrome c release → caspase-3/7 activation → apoptosis
• •Loss of integrin clustering → activation of pro-apoptotic BH3-only proteins (BIM, BMF) that are normally sequestered by cytoskeletal F-actin/myosin

Cancer cells acquire anoikis resistance through:

• •FAK overexpression/amplification: Constitutive Y397 autophosphorylation even at low cell density or in suspension
• •Integrin upregulation/switching: Expression of integrins (e.g., αvβ3, αvβ5) that engage fibronectin secreted by the tumor cells themselves (autocrine matrix loop)
• •ErbB2/HER2 overexpression: HER2 trans-activates FAK via SRC; HER2-SRC-FAK axis bypasses ECM requirement for survival signaling
• •SRC activation: Activated SRC phosphorylates FAK Y576/Y577 and paxillin independently of integrin ligation
• •EGFR/PDGFR mutation/amplification: Growth factor receptors directly activate PI3K/AKT survival signals that partially overlap with integrin-FAK signaling

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Research Tools for SRC/FAK/Integrin Signaling

Small Molecule Research Probes

Key Research Antibodies

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Experimental Protocols

Protocol 1: Adhesion-Dependent FAK Y397 Phosphorylation Assay

Objective: Characterize integrin-engagement-dependent FAK activation during cell adhesion to defined ECM substrates.

Materials: Fibronectin, vitronectin, collagen I, poly-L-lysine (PLL, non-integrin control), defactinib, PF-573228, anti-phospho-FAK Y397, anti-FAK total.

Procedure:

1. Coat 6-well plates with fibronectin (10 μg/mL, 1h, 37°C), vitronectin (5 μg/mL), collagen I (20 μg/mL), PLL (non-ECM control, 0.1 mg/mL), or BSA (1 mg/mL, negative control). Block uncoated areas with 1% BSA.

2. Trypsinize cells and hold in suspension (1.5% methylcellulose medium, 37°C, 30 min) to eliminate basal adhesion-dependent FAK signaling. Verify loss of phospho-FAK Y397 in suspension by Western blot.

3. Re-plate cells on coated surfaces at equal density (5×10⁴/well). Collect at 0 (suspension), 15, 30, 60, 120 min post-plating.

4. Lyse in RIPA + phosphatase inhibitors. Western blot: phospho-FAK Y397 (expect rapid increase upon ECM contact, >10-fold over PLL or BSA at 30–60 min), phospho-SRC Y416 (SRC activation co-incident with FAK), phospho-paxillin Y118, total FAK (loading control).

5. FAK inhibitor control: Pretreat with defactinib (1 μM, 30 min in suspension) before plating on fibronectin. Confirm complete phospho-FAK Y397 abolition and downstream signaling loss.

6. Integrin specificity: Pretreat cells with function-blocking anti-β1 integrin (P5D2 clone, 25 μg/mL) or GRGDSP peptide (500 μM) before replating on fibronectin. Confirm reduced phospho-FAK Y397 vs. isotype/scrambled peptide controls.

Expected outcomes: FAK Y397 phosphorylation: robust induction on fibronectin (5–20× vs. BSA control) within 15–30 min; parallel SRC Y416 activation; paxillin Y118 phosphorylation at 30–60 min. PLL or BSA coatings: minimal FAK activation despite cell attachment (non-integrin adhesion). Defactinib completely blocks Y397 without affecting cell attachment (cells still spread but cannot form mature FAs). Anti-β1 or GRGDSP reduces fibronectin-induced FAK activation by 50–80%.

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Protocol 2: Focal Adhesion Immunofluorescence — Structure-Function Analysis

Objective: Visualize focal adhesion morphology, molecular composition, and the effect of kinase inhibitors on FA maturation.

Materials: Fibronectin-coated coverslips, anti-paxillin, anti-vinculin, anti-phospho-FAK Y397, anti-phospho-paxillin Y118, phalloidin (F-actin), DAPI; defactinib, PP2 (SRC inhibitor), blebbistatin, Y-27632.

Procedure:

1. Plate cells on fibronectin-coated (10 μg/mL) glass coverslips. Allow 2h for FA formation and maturation.

2. Pharmacological perturbations: Add inhibitors 30 min before fixation: defactinib (1 μM), PP2 (10 μM), blebbistatin (50 μM), Y-27632 (10 μM), or vehicle (DMSO).

3. Fix in 4% PFA (10 min, RT). Permeabilize (0.3% Triton X-100, 5 min). Block (3% BSA + 5% donkey serum, 1h). Co-stain: (a) anti-paxillin (primary marker for FAs) + phalloidin (stress fibers) + DAPI; (b) anti-phospho-FAK Y397 + anti-vinculin; (c) anti-phospho-paxillin Y118 + anti-talin. Use spectrally separated fluorescent secondaries.

4. Acquire Z-stack images (confocal, 63× oil, NA 1.4). Image FA region in single Z-plane (TIRF or confocal slice at basal membrane). Acquire F-actin in separate channel.

5. FA quantification (ImageJ/CellProfiler): Threshold paxillin channel; identify individual FA structures as discrete objects (size filter: 0.2–5 μm²); measure per-cell: (a) number of FAs; (b) mean FA area; (c) FA elongation (aspect ratio); (d) paxillin and phospho-FAK intensity per FA; (e) stress fiber density (phalloidin mean intensity × cell area fraction with fibers).

6. Collocalization: Compare paxillin vs. phospho-FAK Y397 colocalization (Pearson's coefficient). Defactinib treatment: paxillin-positive FAs persist (structural) but phospho-FAK Y397-positive FAs disappear (signaling component).

Expected outcomes: Control cells: elongated paxillin+ FAs at stress fiber termini; high phospho-FAK Y397 colocalization with paxillin at FAs; F-actin stress fibers traversing cell. Defactinib → loss of phospho-FAK Y397 at FAs; nascent adhesions may persist but mature FAs reduced in size/number. PP2 → loss of phospho-paxillin Y118; disruption of CRK-DOCK180-Rac1 (reduced lamellipodia). Blebbistatin → FAs disassemble to focal complexes (smaller, more numerous, less elongated). Y-27632 → loss of stress fibers; FAs disassemble or fail to mature.

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Protocol 3: Anoikis Assay — ECM Deprivation-Induced Apoptosis

Objective: Quantify anoikis sensitivity and assess whether FAK/SRC/PI3K signaling is required for suspension survival.

Materials: Low-attachment plates (polyHEMA-coated or ultra-low-attachment tissue culture treated), defactinib, PI3K inhibitor (BKM120 or GDC-0941), annexin V/PI flow cytometry kit, caspase-3/7 activity assay (Caspase-Glo), anti-phospho-AKT S473, anti-AKT, anti-cleaved caspase-3.

Procedure:

1. PolyHEMA coating: Dissolve poly-2-hydroxyethyl methacrylate (polyHEMA) in 95% ethanol (12 mg/mL). Coat 6-well plates (0.5 mL/well), allow to air-dry completely (48h in hood). Recoat and dry twice more. Rinse with PBS before use. This creates a non-adhesive surface preventing ECM attachment.

2. Plate equal numbers of cells (2×10⁵/well) on: (a) fibronectin-coated (adherent, survival control), (b) polyHEMA-coated (suspension, anoikis), (c) polyHEMA + defactinib (1 μM), (d) polyHEMA + PI3K inhibitor (GDC-0941, 1 μM), (e) polyHEMA + vehicle.

3. At 24h and 48h: (a) collect all cells (adherent: trypsinize; suspension: centrifuge); (b) Annexin V/PI staining (flow cytometry): early apoptosis = annexin V+/PI-; late apoptosis/necrosis = annexin V+/PI+. (c) Caspase-3/7 activity (Caspase-Glo 3/7): measure luminescence as caspase activity proxy.

4. Signaling readout: Collect parallel cell samples at 6h for Western blot. Probe for phospho-AKT S473 (survival signal: should drop in suspension but remain in adherent cells), phospho-FAK Y397 (should drop in suspension), cleaved caspase-3 (p17, apoptosis marker: should increase in suspension).

5. Cancer cell comparison: Include an anoikis-resistant cancer cell line (e.g., MDA-MB-231 or H1299) alongside normal epithelial cells. Anoikis-resistant cells should maintain phospho-AKT and survive in suspension; defactinib may sensitize them.

Expected outcomes: Normal epithelial cells: >50% annexin V+ at 48h in suspension (anoikis); adherent cells <5% apoptosis. Anoikis-resistant cancer cells: <20% annexin V+ in suspension; maintain partial phospho-FAK Y397 and phospho-AKT even in suspension. Defactinib or GDC-0941 treatment in suspension: increases apoptosis further in normal cells; sensitizes anoikis-resistant cells to suspension-induced death.

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Protocol 4: FAK-SRC Co-Immunoprecipitation

Objective: Confirm FAK-SRC complex formation in an adhesion-dependent and inhibitor-sensitive manner.

Materials: Anti-FAK (IP grade), anti-SRC (IP grade), anti-phospho-FAK Y397 (detection), anti-phospho-SRC Y416 (detection), protein A/G beads, non-denaturing lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, protease + phosphatase inhibitors).

Procedure:

1. Plate cells on fibronectin (adherent, FAK-SRC complex expected) vs. polyHEMA (suspension, minimal complex expected). At 1h, add defactinib (1 μM) to some fibronectin wells. Lyse cells at 2h under non-denaturing conditions.

2. Pre-clear lysate (500 μg total protein) with protein A/G beads (30 min, 4°C). Reserve 5% as input.

3. IP: Add anti-FAK (5 μg) to pre-cleared lysate overnight at 4°C. Add protein A/G beads (2h). Wash 5× co-IP buffer. Elute with Laemmli sample buffer (95°C, 5 min).

4. Western blot input + IP eluate: probe for (a) SRC total (co-IP detection; should be enriched in FAK IP from fibronectin cells vs. suspension); (b) phospho-FAK Y397 (confirm FAK activation in IP); (c) phospho-SRC Y416 (SRC activity in complex); (d) FAK total (IP efficiency control). Probe IgG IP (isotype control) as specificity control.

5. Quantification: Calculate co-IP efficiency = (SRC signal in FAK IP)/(SRC signal in input × %IP). Compare fibronectin adherent vs. suspension vs. defactinib-treated.

Expected outcomes: Fibronectin adherent cells: robust SRC co-IP with FAK; high phospho-FAK Y397 and phospho-SRC Y416 in the complex. Suspension cells: 3–5× reduction in SRC co-IP with FAK; reduced phospho-Y397. Defactinib-treated adherent cells: FAK IP efficiency maintained (FAK still localizes to FAs) but phospho-FAK Y397 abolished and SRC co-IP reduced (pY397-SH2 interaction impaired), confirming that Y397 is required for efficient SRC recruitment in trans.

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Protocol 5: Migration and Invasion Assay — FAK-SRC Pathway in Cell Motility

Objective: Quantify the contribution of FAK-SRC signaling to 2D migration (scratch assay) and 3D invasion (Matrigel transwell).

Materials: Fibronectin, Matrigel (growth factor-reduced), transwell inserts (8 μm pore), defactinib, PP2, CK-666 (Arp2/3 inhibitor), SMIFH2 (formin inhibitor), scratch/wound healing assay system or IncuCyte live imaging platform.

Procedure:

1. 2D scratch/wound healing: Plate cells on fibronectin-coated 6-well plates to confluence. Scratch with 200 μL pipette tip using the IncuCyte WoundMaker or manual scratch tool. Wash debris. Add media with: vehicle, defactinib (1 μM), PP2 (10 μM), or CK-666 (50 μM). Acquire images every 2h for 24–48h. Measure wound closure area (IncuCyte software or ImageJ).

2. 3D Matrigel invasion: Coat transwell inserts (8 μm pore) with Matrigel (1 mg/mL, 30 μL/well, polymerize at 37°C, 1h). Add cells to upper chamber in serum-free medium + inhibitors. Add 10% FBS or specific chemoattractant (fibronectin 20 μg/mL) to lower chamber. Incubate 24–48h. Fix invaded cells on lower membrane face with 4% PFA (10 min), stain with crystal violet or DAPI. Count invaded cells (5 random fields/insert, ×10 objective).

3. Inhibitor panel: Include: defactinib (FAK), PP2 (SRC), NSC 23766 (RAC1/TIAM1), Y-27632 (ROCK), CK-666 (Arp2/3), SMIFH2 (formin). These dissect different actin assembly mechanisms downstream of FAK-SRC.

4. FAK re-expression rescue: Use FAK-null MEFs (FAK-/- MEFs available from David Schlaepfer lab) transfected with: vector only, FAK-WT, FAK-Y397F (autophosphorylation-deficient), FAK-R454 (kinase-dead). Confirm that FAK-Y397F-expressing cells show reduced migration and invasion vs. FAK-WT, equivalent to FAK-null cells.

Expected outcomes: Control cells: ~70–90% wound closure at 24h on fibronectin. Defactinib → reduced closure to ~30–50%; PP2 similar effect. CK-666 → reduced lamellipodia and closure. Matrigel invasion: defactinib + PP2 combination synergistically reduces invasion. FAK-Y397F rescue: migration intermediate between FAK-null and FAK-WT (some kinase-independent FAK functions remain). Formin inhibition (SMIFH2) reduces stress-fiber-dependent directed migration but less effect on random migration.

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Pathway Integration

FAK-GF Receptor Cross-Talk

Growth factor receptors (EGFR, PDGFR, VEGFR2, IGF-1R) activate SRC, which then phosphorylates FAK Y576/577 and paxillin Y31/118 independently of integrin engagement. Conversely, integrin-FAK signaling recruits EGFR and PDGFR to focal adhesions and enhances their ligand-independent activation. This bidirectional crosstalk creates synergy between matrix adhesion and growth factor signaling.

FAK-p53 Interaction

FAK's FERM domain directly interacts with p53, sequestering it at focal adhesions and promoting its MDM2-mediated degradation — providing a FAK-specific mechanism of p53 suppression in adherent cells. FAK inhibition in cancer cells can partially restore p53 nuclear function independently of genotoxic stress.

FAK-Mechanosensing

FAK is a central mechanosensor: applied force (via actomyosin or AFM) increases FAK Y397 phosphorylation and downstream signaling in a tension-dependent, ROCK-dependent manner. FAK integrates both biochemical (integrin ligand identity) and biophysical (matrix stiffness, applied force) cues to calibrate downstream signaling output.

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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.