Redox Biology 80 (2025) 103514

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Redox Biology
journal homepage: www.elsevier.com/locate/redox

Redox regulation of focal adhesions

Gianmarco Matrullo a , Giuseppe Filomeni a,b , Salvatore Rizza b,*
a
Department of Biology, University of Rome “Tor Vergata”, 00100, Rome, Italy
b
Redox Biology Group, Danish Cancer Institute, 2100, Copenhagen, Denmark

A R T I C L E I N F O A B S T R A C T

Keywords: Focal adhesions (FAs), multi-protein complexes that link the extracellular matrix to the intracellular cytoskel­
Focal adhesion eton, are key mediators of cell adhesion, migration, and proliferation. These dynamic structures act as me­
Redox regulation chanical sensors, transmitting stimuli from the extracellular to intracellular environment activating in this way
S-nitrosylation
signaling pathways and enabling cells to adapt to environmental changes. As such, FAs are critical for tissue
Integrins
Anoikis
organization and serve as hubs governing cell spatial arrangement within the organism.
Cell migration The assembly, reactivity, and functional regulation of FAs are tightly controlled by post-translational modi­
fications, including redox modulation by reactive oxygen and nitrogen species. Increasing evidence suggests that
redox signaling plays a pivotal role in both the physiological and pathological functions of FAs and their
downstream processes. Redox regulation affects various components of the FA complex, including integrins, focal
adhesion kinase 1 (FAK1), SRC, adapter proteins, and cytoskeletal elements.
In this review, we provide an updated overview of the complex interplay between redox signaling and post-
translational modifications in FAs. We explore how redox reactions influence the structure, dynamics, and
function of FAs, shedding light on their broader implications in health and disease.

Reactive oxygen species (ROS), as well as nitric oxide (NO), and its
derivatives, commonly known as reactive nitrogen species (RNS), have
1. Introduction long been recognized for their oxidant properties, which can damage all
classes of biomolecules [5]. However, under normal physiological con­
Cell adhesion is a fundamental process that supports the organiza­ ditions, these reactive molecules are integral components of a tightly
tion, function, and integrity of multicellular organisms. Through the regulated network of redox reactions (e.g., oxidation and reduction) that
action of various adhesion molecules, cells attach to the extracellular influence a wide range of cellular functions and regulatory pathways,
matrix (ECM) or to other cells, facilitating communication, migration, including cell adhesion [6,7].
maintenance of tissue homeostasis, and response to changes in the In this review, we provide the latest insights into the redox regula­
extracellular environment [1]. Cell adhesion is mediated by a special­ tion of key focal adhesion components.
ized group of surface proteins known as cell adhesion molecules (CAMs),
including integrins, cadherins, selectins, and immunoglobulin-like 2. Focal adhesions structure and function
adhesion molecules [2]. Cell adhesion encompasses both cell-cell and
cell-ECM interactions, which are regulated by the assembly of dynamic 2.1. Focal adhesion structure
multi-protein complexes called focal adhesions (FAs). Focal adhesions
play a critical role in organizing tissue structure, mediating cell in­ Focal adhesions are dynamic multi-protein structures that link the
teractions, and regulating signal transduction processes such as cell cytoskeleton to the ECM [1,3]. They consist of clusters of trans­
proliferation, differentiation, and apoptosis [3]. Since cell adhesion is membrane integrins that anchor to intracellular actin filaments via
essential for maintaining cellular homeostasis, defects in this process can adaptor proteins such as talin, vinculin, and paxillin, while connecting
lead to pathogenic outcomes. One prominent example is metastatic to ECM proteins like fibronectin [8]. FAs are typically organized in a
cancer, which results from impaired regulation of cell attachment to the three-layered structure [9]: the bottom integrin signaling layer (ISL), the
ECM, allowing primary cancer cells to survive and metastasize to distant middle force transduction layer (FTL), and the top actin-regulatory layer
tissues [3,4].

* Corresponding author.
E-mail address: rizza@cancer.dk (S. Rizza).

https://doi.org/10.1016/j.redox.2025.103514
Received 4 November 2024; Received in revised form 7 January 2025; Accepted 23 January 2025
Available online 24 January 2025
2213-2317/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-
nc/4.0/).
G. Matrullo et al. Redox Biology 80 (2025) 103514

Abbreviations Grb2 Growth Factor Receptor-bound Protein 2

GSK3β Glycogen Synthase Kinase-3 Beta
AKT Protein Kinase B ILK Integrin-Linked Kinase
ARL Actin-Regulatory Layer iNOS Inducible Nitric Oxide Synthase
BCAR1 Breast Cancer Anti-estrogen Resistance Protein 1 ISL Integrin Signaling Layer
Bcl-2 B-cell lymphoma 2 protein family LOX Lysyl Oxidase
CAMs Cell Adhesion Molecules LMW-PTP Low Molecular Weight Protein Tyrosine Phosphatase
CDC25c Cell Division Cycle 25c MAPK Mitogen-Activated Protein Kinase
CDK1 Cyclin-Dependent Kinase 1 NO Nitric Oxide
CDK4/6 Cyclin-Dependent Kinases 4 and 6 NOS Nitric Oxide Synthase
cGMP Cyclic Guanosine Monophosphate NOX NADPH Oxidase
CKIs Cyclin-Dependent Kinase Inhibitors PI3K Phosphoinositide 3-Kinase
ECM Extracellular Matrix PKG Protein Kinase G
EGFR Epidermal Growth Factor Receptor PLK1 Polo-Like Kinase 1
ERKs Extracellular Signal-Regulated Kinases RNS Reactive Nitrogen Species
FA Focal Adhesion ROS Reactive Oxygen Species
FAK1 Focal Adhesion Kinase 1 SH Src Homology
FERM 4.1, Ezrin, Radixin, Moesin Homology SNO S-Nitrosylated
FTL Force Transduction Layer Src Proto-Oncogene Tyrosine-Protein Kinase
GAPDH Glyceraldehyde-3-phosphate dehydrogenase VASP Vasodilator-Stimulated Phosphoprotein
GEF Guanine Exchange Factor WASP Wiskott-Aldrich Syndrome Protein

(ARL) [10] (Fig. 1). These layers are distinct in their temporal regulation 2.2. Biological functions of focal adhesions
and functional contributions.
The ISL is the first interface to form as integrins bind to the ECM and FAs are critical junctions that physically connect the intracellular
initiate clustering. Upon integrin clustering, a series of adaptor proteins cytoskeleton to the ECM, enabling cells to sense and respond to their
are recruited by the cytoplasmic tails of integrins, providing a platform microenvironment. As dynamic structures, FAs undergo continuous as­
for other intracellular FA components to assemble. This process connects sembly, disassembly, and remodeling in response to extracellular and
integrins to actin filaments and establishes a mechanosensor apparatus intracellular signals, serving as mechanosensors. Mechanosensing is the
[11]. The formation of this complex activates downstream signaling process by which cells translate mechanical stimuli into biological sig­
cascades that ultimately regulate cell migration, survival, and differen­ nals that ultimately control migration, survival, and proliferation [22].
tiation in response to mechanical and biochemical cues. The mechanosensing machinery begins to assemble when integrins bind
Integrins are a large family of cell surface glycoprotein receptors that to the ECM substrate, forming a physical connection to the actin/myosin
bind to various ECM components such as collagen and laminin. Paxillin cytoskeleton via the spatially and temporally regulated assembly of FA
is one of the main adaptor and scaffold proteins of FAs. Paxillin directly components [22]. This dynamic machinery underpins the concept of the
binds integrins and numerous interacting partners, contributing to the “molecular clutch” in force transmission and cell motility, first proposed
FA signaling cascade [12,13]. Among its interactors, focal adhesion ki­ by Mitchison and Kirschner in 1988 [23]. The ability of cells to migrate
nase 1 (FAK1) plays a key regulatory role in FA signaling. FAK1 is a on a substrate relies on the contractility of the actin/myosin cytoskel­
non-receptor tyrosine kinase that is directly recruited by paxillin during eton, which generates a pulling force through the molecular clutch. In
FA formation and contributes to signal transduction through its kinase this model, the mechanosensing apparatus engages and disengages the
activity, by phosphorylating itself and other target proteins. Addition­ ECM, acting as an anchor. This pulling force is generated by myosin,
ally, FAK1 acts as a binding platform for other signaling proteins, such as which contracts actin filaments, transmitting force through the entire FA
members of the proto-oncogene tyrosine-protein kinase (Src) family structure via proteins that directly link actin to integrins (e.g., talin) and
[14]. Talin is another large adaptor protein that, like paxillin, binds indirectly through other adaptor and effector proteins, such as vinculin,
directly to integrins and extends across all three FA layers. Talin in­ FAK1, and paxillin [22,23]. This process is described as a “load-and-fail”
teracts with paxillin, FAK1, and additional FA components, such as cycle of the molecular clutch, where integrins bind to the substrate,
vinculin and actin. Talin plays an important role in mechanosensing by myosin generates force, and the clutch eventually disengages, causing
transducing intracellular mechanical forces into biochemical signals the cycle to restart and pushing the cell forward [23,24].
[15]. The interaction between talin and vinculin is crucial for vinculin The mechanical function of FAs is also crucial in adherent, static
activation [16,17]. Vinculin is a key scaffold protein of the FTL, acting as cells. Most mammalian cells exhibit “anchorage-dependent prolifera­
a hub that interacts with numerous partners. Unlike paxillin and talin, tion”, in which cell adhesion is required for progression through the cell
vinculin does not directly bind integrins and is recruited to FAs with cycle [25,26]. The regulation of proliferation by FAs is
slower kinetics. It plays a key role in linking FAs to actin filaments [18]. well-documented, primarily occurring during the G1/S phase transition
Vinculin has a unique head-tail structure, facilitating the transition be­ of the cell cycle, where cells decide whether to replicate their DNA and
tween its closed and open-active states. In its active conformation, proceed to mitosis or enter a quiescent state [26,27]. The central player
vinculin displays multiple protein-binding sites, allowing interaction in this process is focal adhesion kinase 1 (FAK1), which controls
with partners from both the bottom layer, such as paxillin and talin, and downstream pathways such as the phosphoinositide 3-kinase (PI3K)/­
the top layer, such as vasodilator-stimulated phosphoprotein (VASP) protein kinase B (AKT) and the mitogen-activated protein kinase
and zyxin [16,19]. VASP and zyxin are typically recruited together in (MAPK)/extracellular signal-regulated kinases (ERKs) signaling cas­
the ARL and localize to actin filaments, where they regulate actin cades [28]. Both signaling axes promote cell cycle progression through
polymerization in a co-dependent manner and stimulate Rho GTPase cyclin D accumulation and degradation of cyclin-dependent kinase in­
activity [20,21]. hibitors (CKIs) [29,30]. Specifically, the MAPK/ERK pathway upregu­
lates cyclin D expression, while PI3K/AKT activation inhibits glycogen

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Fig. 1. Schematic representation of a focal adhesion.

The extracellular matrix (ECM) connects with integrin receptors at the integrin signaling layer. Integrins span the plasma membrane, linking the ECM to intracellular
signaling molecules. In the middle force transduction layer, proteins such as paxillin, ILK (integrin-linked kinase), talin, FAK1 (focal adhesion kinase 1), SRC, vinculin,
and (vasodilator-stimulated phosphoprotein) VASP play critical roles in signal transduction and stabilization of the complex. At the actin-regulatory layer, the actin
cytoskeleton anchors to the complex, facilitating cellular responses to mechanical signals from the ECM. This multilayered structure integrates extracellular and
intracellular biochemical and mechanical signals to regulate cell behavior. The figure was partially created with Biorender. Filomeni, G. (2024) https://BioRender.
com/t00b078.

synthase kinase-3β (GSK3β), leading to cyclin D stabilization [31]. protein sites, promoting FA disassembly and potentially influencing
Cyclin D then activates cyclin-dependent kinases 4 and 6 (CDK4/6), both cell cycle progression and migration [34,35]. CDK1 has also been
promoting the transition from G1 to S phase and subsequent mitosis. The shown to interact directly with talin, phosphorylating it on S1589 in the
G2/M phase transition is also suggested to be regulated by R8 domain, providing a mechanism by which CDK1 localizes to FAs and
integrin-adhesion signaling. This transition is finely controlled by a phosphorylates its substrates within the adhesion complex [36].
balance of activating and inactivating phosphorylation of CDK1, the In addition to their roles in adhesion, motility, and proliferation, FAs
main effector of mitosis. The activities of the nuclear kinase Wee1 and are also critical regulators of cell survival and apoptosis. The specific
the phosphatase Cell Division Cycle 25c (CDC25c), both of which form of apoptosis triggered by inappropriate cell adhesion or detach­
regulate CDK1, are controlled by integrin-mediated signaling through ment from the ECM is termed anoikis [37]. Anoikis is crucial for
the stimulation of polo-like kinase 1 (PLK1) [32,33]. maintaining tissue architecture by preventing reattachment to inap­
Conclusively, FAs increase during the G1/S phase as integrin-based propriate substrates and suppressing the survival of displaced cells,
downstream signaling drives cells into the S phase; conversely, during thereby inhibiting metastatic growth. Anoikis is regulated through both
the G2/M transition, cell adhesion decreases as FAs rapidly disassemble, intrinsic (mitochondrial) and extrinsic (death receptor-mediated) path­
allowing the extensive morphological and cytoskeletal rearrangement ways, culminating in the activation of caspases 8 or 9, DNA fragmen­
necessary for mitosis [26,34]. tation, and programmed cell death [38,39]. FAK1 plays a fundamental
Recent findings have identified CDK1 as a key player in the interplay role in preventing anoikis through the activation of anti-apoptotic fac­
between FAs and the cell cycle. CDK1 phosphorylates over 100 adhesion tors, such as the Bcl-2 protein family, and inhibition of pro-apoptotic

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proteins [40]. Through the PI3K/AKT pathway, FAK1 mediates the while glutaredoxins, thioredoxins, S-nitrosoglutathione reductase, and
phosphorylation and inactivation of the pro-apoptotic factor Bad, SNO-CoA reductase reduce oxidized and S-nitrosylated cysteines
thereby limiting cytochrome c release from mitochondria and inhibiting [65–68]. The integrated activities of these enzymes are essential for
the apoptotic cascade [41,42]. Simultaneously, FAK1-mediated activa­ maintaining proper biological functions, and due to the rapid kinetics of
tion of the MAPK/ERK pathway leads to the degradation of redox reactions, they ensure that cells can promptly respond and adapt
pro-apoptotic proteins Bim and Bad through phosphorylation-mediated to stimuli.
ubiquitination [43,44].
Interestingly, ECM engagement also inhibits the extrinsic apoptotic 4. Redox regulation of focal adhesions
pathway, although the detailed mechanism remains unclear and needs
further investigation [45,46]. Focal adhesions are particularly subjected to the effects of ROS and
RNS due to their positioning at the cell membrane and, possibly due to
3. Redox signaling their association with cellular organelles, such as mitochondria [69].
ROS and RNS, through reversible modifications of FA components, in­
A redox reaction is a chemical process characterized by the transfer fluence FA dynamics, thereby impacting cell adhesion, migration, and
of electrons, where one substance undergoes oxidation (electron loss) downstream signal transduction. The regulatory role of ROS in cell
and another undergoes reduction (electron gain). ROS and RNS are key migration was initially documented in studies showing that ROS scav­
cellular signaling molecules that regulate numerous processes through enging suppressed chemotaxis and migration [6]. One of the early
redox reactions, including cell adhesion [47]. models based on these observations proposed that NOX enzymes are the
In biological systems, the most abundant oxygen-containing reactive main source of ROS at the leading edge of migrating cells, facilitating
species are the superoxide radical anion (O•- 2 ) and hydroxyl radical cell migration by inhibiting specific phosphatases [6,70,71].
(•OH), as well as the reactive non-radical hydrogen peroxide (H2O2). In recent years, our understanding of the redox regulation of FAs and
These molecules are produced by membrane-associated NADPH oxi­ their downstream biological functions has significantly advanced,
dases (NOXs), mainly in vascular and white blood cells, where they play revealing a complex network of regulatory mechanisms that operate at
roles in maintaining vascular homeostasis and mediating immune re­ multiple levels within the FA, affecting numerous components of the
sponses, respectively [48,49]. Increased NOXs expression is a feature complex. In the next section, we will provide a detailed overview of how
observed in human cancer [50] and it has been correlated to both and where redox reactions regulate focal adhesion signaling by modu­
initiation and tumor progression, owing to the regulation of lating key redox-sensitive components.
redox-sensitive oncogenic signaling pathways [51]. NOX-produced ROS
influence several hallmarks of cancer, including genomic instability, cell 4.1. Integrins
growth and survival, angiogenesis, invasion and metastasis [51,52].
Consequently, NOXs have been tested as drug targets in cancer therapy, Integrins, the primary adhesion receptors on which FAs assemble,
with significant success achieved through compounds that specifically represent the first line of connection between the cell and the ECM.
target NOX isoforms expressed in cancer cells [53]. ROS are also pro­ Integrins are expressed as heterodimers composed of α and β subunits. In
duced physiologically in the mitochondrial electron transport chain humans, 18 α subunits and 8 β subunits have been identified, which
during cellular respiration and within peroxisomes, where they combine to form 24 different heterodimers [72,73].
contribute to metabolic function [54,55]. Structurally, each integrin subunit comprises three key domains: i) a
Nitric oxide (•NO), the precursor of several reactive nitrogen species transmembrane domain; ii) a long extracellular domain facing the ECM;
(RNS), is a gaseous radical produced enzymatically by three isoforms of and iii) two short cytoplasmic regions responsible for binding to scaffold
nitric oxide synthase (NOS) [56–58], which are expressed in most and adaptor proteins [73]. The high structural heterogeneity of integrins
human tissues. The interaction of NO with reactive oxygen species (ROS) allows them to recognize a wide variety of ECM ligands, resulting in
leads to the formation of highly reactive molecules, including perox­ different subunit pairs exhibiting affinities for collagen, fibronectin,
ynitrite (ONOO⁻), nitrogen dioxide (•NO2), and dinitrogen trioxide laminin, and other ECM components [73]. Furthermore, distinct pat­
(N2O3). These species can modify biomolecules through reactions such terns of integrin expression are typically observed between different cell
as peroxidation and nitration, often resulting in cellular damage [59]. types, contributing to the diversity of integrin complexes across tissues
However, within physiological concentrations, both ROS and RNS [72–74]. The 24 integrin heterodimers are typically grouped based on
mediate reversible protein modifications, with cysteine residues being a their ligand affinity and subunit composition. Despite their heteroge­
primary target [60]. neity, α subunits share a common feature: an extracellular β-propeller
Cysteines can readily undergo oxidation when situated in an structure, referred to as the head. This domain is connected to the
appropriate chemical environment that allows deprotonation, reacting intracellular region by a leg composed of a “thigh” domain and two
with hydrogen peroxide (H2O2) or NO-derived species (such as NO+, “calf” domains. Ligand binding occurs through the α-I domain, which is
N2O3 and •NO2). These reactions result in the formation of S-hydrox­ inserted in the β-propeller and contains a Mg2⁺ ion [73]. The β chains, in
ylated (SOH) or S-nitrosylated (SNO) adducts, which may further contrast, contain β-I domain with regulatory functions. While the α
resolve into disulfides if another sulfhydryl group is in close proximity. chain’s β-propeller is central to ligand specificity, the β-I domain mod­
Specifically, S-nitrosylation of proteins primarily occurs through the ulates ligand binding through the effects of Ca2⁺ (inhibitory) or Mn2⁺
reaction of proteins with species generated from the interaction between (activating) ions, which ultimately induce the conformational changes
NO and ROS. Notable examples include the S-nitrosylation of the protein required for integrin activation [73,75].
p21 Ras, which involves the direct reaction of Cys118 with •NO2 [61], Several integrin subunits are targets for redox modifications that
and the S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase tune their function and downstream signaling. Integrin domains are
(GAPDH), catalyzed by N₂O₃ formed during cell exposure to characterized by the presence of multiple cysteine residues, some of
NO-releasing drugs [62]. These post-translational modifications signif­ which are engaged in disulfide bridges [76,77]. Researchers have
icantly influence protein function, localization, and turnover, thereby identified at least 56 cysteine residues in β chains and more than 19 in α
modulating various signaling pathways [63]. chains [78,79]. Disulfide bridge formation induced by ROS and RNS has
To maintain redox homeostasis and keep ROS and RNS within been reported to regulate β1 integrins, activating the α7/β1 and α4/β1
physiological limits, cells express a range of highly efficient enzymatic heterodimers [80,81]. Similar results have been observed for β2 and β3
systems. Antioxidant enzymes such as superoxide dismutase, catalase, integrins, which are activated by NOX-dependent production of H2O2
glutathione peroxidase and peroxiredoxins neutralize excess ROS [64], [82]. Integrin activation and ROS production are linked in a positive

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feedback loop. Integrin clustering upon ECM engagement stimulates correlated with NO levels in in vivo models of atherosclerosis, suggest­
ROS production by activating Ras-related C3 botulinum toxin substrate ing a role for RNS in regulating ILK stability [87]. Coherently, ILK mRNA
1 (Rac-1), a small GTPase that promotes ROS production via NOX and levels are downregulated by NO [88]. NO-mediated degradation of ILK
5-lipoxygenase (5-LOX) [83]. These ROS, in turn, further enhance negatively impacts GSK-3 phosphorylation and paxillin protein levels,
integrin activation (Fig. 2A). which are known to interact with ILK at the FA site [86] (Fig. 2C).
RNS are also emerging as potent modulators of integrin signaling. Conversely, ILK overexpression has been correlated with elevated
Isaac and colleagues identified Cys86 in the α6 integrin subunit as a endothelial NOS levels, highlighting an additional layer of regulation
potential target for S-nitrosylation [84]. This modification enhances α6 that merits further exploration [89].
heterodimerization with β1 and promotes cell migration in prostate
cancer cell lines [84] (Fig. 2B). Another example of how integrin
S-nitrosylation affects cell physiology deals with macrophages. Upon 4.2. FAK1
activation, they upregulate inducible NOS (iNOS) and produce large
amounts of NO which is required to nitrosylate β1 integrins on Cys555. FAK1 is a key kinase involved in regulating most FA functions. FAK1
This modification prevents integrin degradation via ubiquitination and is often overexpressed and activated in malignant cancers, functioning
ultimately enhances inflammatory response [85] (Fig. 2B). as an oncogene by promoting cell motility, proliferation, and survival.
In addition to integrins, integrin-associated proteins are also targets FAK1 is a ubiquitously expressed protein consisting of three main do­
of redox modifications. Notably, integrin-linked kinase (ILK), a regula­ mains: a protein 4.1, ezrin, radixin, moesin homology (FERM) domain, a
tory protein interacting with the cytoplasmic tail of integrins, plays a central kinase domain, and a focal adhesion targeting (FAT) domain
key role in regulating downstream integrin pathways. However, it re­ [90]. The N-terminal FERM domain mediates interactions with various
mains unclear whether ILK acts primarily through its kinase or scaf­ receptors, such as the epidermal growth factor receptor (EGFR) and
folding activity [86]. Interestingly, ILK levels have been inversely platelet-derived growth factor receptor (PDGFR). It also facilitates
intramolecular interactions with the kinase domain, resulting in a closed

Fig. 2. Redox regulation of integrins. (A) Integrins interaction with the extracellular matrix (ECM) activates Ras-related C3 botulinum toxin substrate 1 (Rac-1), a
small GTPase that promotes ROS production via NADPH oxidase (NOX) and 5-lipoxygenase (5-LOX). These ROS stimulate the formation of disulfide bridges (CS-SC)
enhancing integrin activation, thus promoting cell motility, survival, and proliferation. (B) Nitric oxide (NO) produced by nitric oxide synthase (NOS), influences
integrin function through S-nitrosylation (-SNO) of specific cysteine residues (C86 and C555), affecting integrin heterodimerization (the first) and turnover (the
second). (C) NO can negatively impact on the activity and stability of integrin-linked kinase (ILK). ILK inactivation leads to a drop in glycogen synthase kinase-3 beta
(GSK3-β) phosphorylation, this promoting cell motility and proliferation. Partially created in BioRender. Filomeni, G. (2024) https://BioRender.com/t00b078.

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inactive conformation [91]. The FAT domain is required for FAK1 phosphorylation at Tyr397 in uterine serous carcinoma, an aggressive
recruitment to FAs, through binding to paxillin and talin. form of endometrial cancer [97]. In melanoma, NOX4 silencing or in­
Autophosphorylation at Tyr397 within the kinase domain is the hibition leads to a ROS-dependent decrease in cell viability, which is
primary activation event, which occurs when FAK1 undergoes a linked to reduced FAK1 phosphorylation and impaired FAK1/Src
conformational change to an open state following its interaction with interaction – both events contributing to anoikis-mediated cell death
clustered integrins. This initial phosphorylation is crucial for recruiting [98].
Src family members and forming a transient complex, facilitated by in­ FAK1 functions and regulation are also influenced by RNS. Recent
teractions between paxillin/talin and the FAT domain [91]. Src kinases findings indicate that FAK1 undergoes S-nitrosylation at different
subsequently phosphorylate additional tyrosine residues, including cysteine residues, Cys658 and Cys459, in tumor models of hep­
Tyr576/577, which sustains FAK1 kinase activity, and Tyr925, which is atocarcinoma [99]. Notably, S-nitrosylation at Cys658 was found to
required to recruit the adaptor protein growth factor receptor-bound enhance FAK1 activation, sustaining autophosphorylation at Tyr397
protein 2 (Grb2) and stimulate the mitogen-activated protein kinase and promoting FAK1 pro-tumorigenic activity through a
(MAPK) cascade [90]. Another target of Src is Tyr861, whose phos­ Src-independent mechanism [99] (Fig. 3A). On the contrary, S-nitro­
phorylation promotes the association of paxillin with breast cancer sylation of Cys459 did not show any effects on anoikis resistance but
anti-estrogen resistance protein 1 (BCAR1, also known as P130Cas), altered tumor spheroid size, this suggesting a multilayered regulation of
thereby activating signaling pathways that enhance cell motility and FAK1 by S-nitrosylation [99].
invasion [92,93]. Paxillin itself is phosphorylated by the FAK1-Src Another significant factor in the regulation of FAK1 is the low mo­
complex at multiple residues, which is a key event in controlling FA lecular weight protein tyrosine phosphatase (LMW-PTP), which controls
dynamics and vinculin recruitment [94]. the phosphorylation status of FAK1 [99,100]. LMW-PTP is a target of
FAK1 regulation through phosphorylation is complemented syner­ redox regulation, with both ROS and RNS reported to inhibit its activity
gistically by redox modifications, which further influence the various (Fig. 3B). Chiarugi and colleagues [47] proposed a model in which
roles of FAK1. ROS have been shown to positively regulate FAK1 adhesion-dependent ROS production sustains FAK1 and its downstream
phosphorylation in several experimental models [95]. Rac1-dependent effectors by inhibiting LMW-PTP. It is well documented that LMW-PTP
ROS production, following integrin clustering, enhances both integrin undergoes inactivation by ROS and RNS, both in vitro and in vivo
activation and FAK1 signaling, ultimately promoting anoikis resistance [101–103]. The inactivation mechanism of LMW-PTP involves the for­
and anchorage-independent growth in liver cancer cells [96]. Similarly, mation of a reversible disulfide bridge between Cys12 and Cys17 caused
treatment with H2O2 has been reported to increase FAK1 by both species. This allows the generation of a reactive dithiol in which

Fig. 3. Interplay between redox modification and phosphorylation of FAK1, SRC and Paxillin. (A) Reactive oxygen species (ROS) generated by integrin clustering, or
nitric oxide (NO)-mediated S-nitrosylation (-SNO) of C658, lead to increased autophosphorylation of focal adhesion kinase 1 (FAK1) at Y397. This event promotes
FAK1 activation and phosphorylation of target proteins such as Paxillin, on Y88, and proto-oncogene tyrosine-protein kinase (SRC), on Y416. SRC activation, in turn,
triggers phosphorylation of FAK (on Y861 and Y576/7) and Paxillin (on Y31 and Y18). (B) Low molecular weight protein tyrosine phosphatase (LMW-PTP), which
controls the phosphorylation status of FAK1, undergoes inactivation upon oxidation by ROS or RNS (reactive nitrogen species) of two vicinal cysteines (C12 and C17)
with the formation of a disulfide bridge. (C) ROS can directly activate SRC by modifying the redox-sensitive C245 and C487, or by inducing a disulfide bridge
between C122 and C164 that disrupts the autoinhibitory conformation. (D) SRC activity is positively regulated by NO thought S-nitrosylation of C498. (E) The overall
balance of ROS/RNS signaling converging on FAK/SRC complex impacts on downstream adaptor and effector proteins such as Paxillin, and ultimately regulates cell
adhesion and migration. Partially created in BioRender. Filomeni, G. (2024) https://BioRender.com/t00b078.

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Cys12 serves as the redox-sensitive cysteine, while Cys17 acts as the activation both directly and indirectly, through FAK1. SRC is also
resolving cysteine, preventing irreversible oxidation of Cys12 during involved in the integrin-iNOS signaling axis. It has been proposed that
prolonged exposure to ROS and RNS [101]. α9/β1 integrin regulates cell migration in an SRC- and iNOS-dependent
The redox regulation of FAK1 acquires an additional layer of manner, as their inhibition reduces integrin-mediated cell migration
complexity if we include indirect pathways (i.e. the redox regulation of [114]. SRC activation through α9/β1 integrin activity has been proposed
protein involved in FAK1 turnover, transcription and translation), pos­ to stimulate both iNOS (via a FAK-independent mechanism) and Rac-1,
itive and negative feedback loops (e.g. SRC activation by FAK1 which which promotes ROS production through NOX4 and LOX, as previously
stimulates FAK1 autoactivation in a positive loop), and interactions discussed. The redox activation of the signaling axis iNOS/SRC/FAK is a
between FA components and enzymes involved in redox signaling. An critical mechanism allowing macrophage migration during infection
example of the latter comes from studies in neutrophils showing that [115]. This has been observed in macrophages exposed to lipopolysac­
FAK1 directly interacts with iNOS to enhance NO production, which charide (LPS), a major constituent of Gram-negative bacteria known to
subsequently inhibits β2 integrin clustering in a self-regulatory loop. stimulate NO production by iNOS [116]. Collectively, all these findings
Sustained iNOS activity also leads to the S-nitrosylation of actin fila­ indicate an elaborate regulatory network of redox reactions that finely
ments, causing FAK1 to dissociate from the cytoskeleton. This event tune integrin and FA signaling.
ultimately turns off the signal as it triggers a reduction in iNOS activity
allowing integrin function to be restored [104]. 4.4. Paxillin, talin, vinculin and cytoskeleton

4.3. c-Src Scaffolding proteins, such as paxillin, talin, and vinculin, are essen­
tial components of FAs, serving as docking platforms for recruiting other
c-Src, commonly referred to as SRC, is the most well-known member proteins within the FA multilayer structure, and bridging actin filaments
of the non-receptor Src tyrosine kinase family, to which FAK1 also be­ to integrins and the plasma membrane.
longs. SRC and FAK1 share high sequence homology in the kinase Paxillin is a 68 kDa protein initially identified as a phosphorylation
domain and frequently act in a coordinated manner. SRC is considered target of v-Src [117]. It contains several protein-binding domains,
an oncogene, primarily due to its role in driving cell proliferation and including five leucine-rich sequences (LD motifs) in the N-terminus and
survival [105]. Similar to other members of the Src kinase family, SRC four zinc-finger LIM domains in the C-terminal region [117,118]. The LD
consists of six structural domains: i) the Src homology 4 (SH4) domain, domains mediate protein-protein interactions, enabling paxillin to
which contains a myristoylation and membrane-localization signal; ii) a function as a docking hub that links integrins to other FA-associated
unique region of 50–70 residues that is not conserved among family proteins such as SRC, FAK1, ILK, vinculin, and talin. The LIM domains
members; iii) the SH3 domain; iv) the SH2 domain; v) the catalytic are responsible for anchoring paxillin to the plasma membrane and the
domain; and vi) a short regulatory tail [106,107]. Like FAK1, SRC is integrin cytoplasmic tail, either directly or indirectly [118,119]. Pax­
maintained in a closed and inactive conformation through interactions illin’s docking ability is strictly regulated by phosphorylation at multiple
involving their SH2 and SH3 domains. Full activation requires the residues, primarily catalyzed by the FAK/SRC complex. Phosphorylation
release of these intramolecular interactions, followed by autophos­ of tyrosine residues Tyr31, Tyr88, and Tyr118 by SRC and FAK allows
phorylation at Tyr416 within the activation loop. Conversely, inhibitory for the recruitment of downstream effectors (Fig. 3E). Additionally,
phosphorylation of Tyr527 in the C-terminal region promotes interac­ phosphorylation of Ser130 and Ser126, mediated by GSK-3 and the
tion with the catalytic domain and stabilizes the closed, inactive MAPK cascade, respectively, are important regulatory switches in cell
conformation. Dephosphorylation of this residue is required for SRC migration [120,121]. The list of phosphorylation sites includes addi­
activation [106,107]. Notably, the viral ortholog of SRC, v-Src, lacks the tional residues located within the LIM domains, which affect paxillin
C-terminal region and is thus constitutively active, functioning as a recruitment to FAs [118,122]. There is limited evidence regarding the
potent oncodriver [108]. direct influence of ROS on paxillin functions. The hydroxyl radical
The activity of SRC and, reasonably, of the other Src family members, (•HO) has been suggested to enhance paxillin phosphorylation [22,118,
is modulated by redox modifications. As previously mentioned, ROS and 123], although the exact regulatory mechanism remains unclear. It is
RNS produced during integrin clustering enhance cell adhesion and likely that this effect is indirectly mediated by other redox-sensitive
anoikis resistance through upregulation of FAK1 activity. However, players, with FAK1 being a primary candidate.
these phenotypes may not be solely dependent on FAK1 hyperactivation, Talin is a large, 270 kDa protein with a complex structure composed
as substantial evidence has accumulated over the years regarding ROS of three major domains: a globular head in the N-terminal region, a
and RNS effects on SRC. ROS can directly activate SRC by modifying central domain containing 13 α-helix bundles (referred to as rods), and a
redox-sensitive cysteines, such as Cys245 and Cys487, which impacts dimerization domain at the C-terminal region [124]. The FERM domain
cell survival [83], or by inducing a disulfide bridge between Cys122 and within the globular head mediates talin binding to integrin tails [125].
Cys164 within the SH2 domain that disrupts the autoinhibitory The central rod domain contains multiple vinculin-binding sites and a
conformation [109] (Fig. 3C). well-characterized actin-binding site [126]. Talin regulation occurs at
Initially documented in 1999 [110], NO donors stimulate the different levels. It can exist in either a globular or extended conforma­
auto-phosphorylation of SRC on Tyr416. SRC undergoes S-nitrosylation tion. The F3 subdomain of the FERM domain can bind to the rod domain,
on Cys498 within the catalytic domain, a residue conserved among other resulting in an autoinhibitory conformation where F3 is no longer able
Src family members (Fig. 3D). This redox modification stimulates SRC to interact with the integrin β tails. This autoinhibition is thought to be
kinase activity, ultimately enhancing cancer cell motility and invasive disrupted by F3 affinity for phosphatidylinositol 4,5-bisphosphate,
potential [111]. Similarly, in mouse embryonic fibroblasts, it has been which regulates talin conformation [124,125]. Talin activation and
observed that SRC S-nitrosylation, phosphorylation and activation are localization are also regulated by vinculin binding to the rod domains, a
stimulated by NO-releasing compounds and fetal bovine serum (which mechanism that influences the membrane targeting of activated talin
induces iNOS expression), thereby altering the proliferation and [126].
migration of these cells [112]. SRC activation through S-nitrosylation Studies conducted in muscle fibers have shown that NO alters talin
has also been linked to anoikis resistance via the downregulation of the mRNA expression via a cGMP-dependent protein kinase (PKG) mecha­
pro-apoptotic protein Bim and inhibition of caspase-3 cleavage [113]. nism, while direct S-nitrosylation of talin inhibits its proteasomal
Interestingly, NO donors stimulate FAK1 activation even in cells degradation [127,128] (Fig. 4A). More recently, it was shown that NO
genetically ablated for key members of the Src family [111], while FAK1 donors reduce talin stabilizing phosphorylation at Ser425 via the soluble
silencing reduces SRC activation [99], suggesting that NO mediates SRC guanylate cyclase and PKG pathways [129].

7
G. Matrullo et al. Redox Biology 80 (2025) 103514

Fig. 4. Redox regulation of Talin, Vinculin and actin

Schematic representation of redox regulations occurring at the level of focal adhesion’s adaptor proteins and cytoskeleton. (A) Talin direct S-nitrosylation (-SNO) on
an unknown residue triggered by nitric oxide (NO), inhibits talin proteasomal degradation. However, the same modification reduces the stabilizing phosphorylation
of talin on S425. (B) Under NO fluxes, vinculin undergoes S-nitrosylation on C328, although the functional role of this modification is unknown. (C) Reactive oxygen
species (ROS) produced by NADPH oxidase (NOX) upon integrin activation, stimulate the oxidation of actin C272 and C374 to form a disulfide bridge, which is
critical for vinculin recruitment to actin filaments. (D) ROS stimulate actin glutathionylation (GS-) and cysteine oxidation (on C217 and C257), whereas NO promotes
actin S-nitrosylation (on C374). All these actin redox modifications impair actin polymerization and filaments elongation. (E) NO-induced S-nitrosylation of fila­
mentous actin on four cysteine residues near the C-terminal end of β-actin increases the affinity with vasodilator-stimulated phosphoprotein (VASP), this enhancing
actin polymerization. Partially created in BioRender. Filomeni, G. (2024) https://BioRender.com/t00b078.

Vinculin, another major scaffold protein of FAs, acts as an important Actin is a family of globular proteins that form microfilaments and
connector between actin filaments and FAs. Vinculin is a 116 kDa pro­ thin filaments, the principal components of the cytoskeleton. Actin is
tein organized in a multi-domain structure, including a head, a proline- extensively regulated by redox modifications, both reversibly and irre­
rich linker, and a tail at the C-terminus [130]. Like talin, vinculin tail versibly, which can target cysteine residues, tyrosines, methionines, and
and head can bind in a closed and inactive conformation. Upon activa­ histidines [136]. H2O2 induces oxidation of several actin cysteines,
tion, vinculin undergoes a conformational change that exposes several resulting in the formation of disulfide bridges, glutathionylation, or
protein-binding sites, allowing it to interact with various partners. The sulfenic acid. Nitric oxide and related species cause reversible S-nitro­
vinculin-actin connection is crucial for FA function, as it facilitates the sylation of cysteine thiols, while increased nitrosative stress from per­
recruitment of active regulators of actin polymerization, such as VASP oxynitrite leads to irreversible tyrosine nitration. Methionine, in turn,
and zyxin. Zyxin contains actin-binding domains in its N-terminus, while can be oxidized to sulfoxide and subsequently sulfone derivative [136].
VASP contains a WASP homology 1 (WH1) domain that enables the The main effects of redox modifications on actin include dynamics
binding to Zyxin, the interaction to vinculin, and VASP recruitment to (polymerization and depolymerization), mechanical properties, and
actin filaments [9,21]. Together, these proteins regulate actin poly­ interactions with actin-binding proteins. In the context of FA signaling,
merization in a co-dependent manner by interacting with guanine ex­ an example is the NOX4-dependent oxidation of actin at Cys272 and
change factors (GEFs) and stimulating Rho GTPase activity [9,131]. Cys374 upon integrin activation, which is critical for vinculin recruit­
High-throughput studies conducted in uterine smooth muscle and pe­ ment to actin filaments [137] (Fig. 4C). Glutathionylation or S-nitro­
ripheral blood mononuclear cells revealed that vinculin undergoes sylation of Cys374 has been suggested to impair actin elongation and
S-nitrosylation, presumably at Cys328 [132–134] (Fig. 4B). This finding polymerization [138] (Fig. 4D). Similar effects have been observed upon
is in line with an earlier study demonstrating that high-glucose treat­ oxidation of Cys217 and Cys257 [139]. A study conducted in neutro­
ment reduces vinculin S-nitrosylation (alongside other structural pro­ phils showed that VASP has a high affinity for S-nitrosylated filamentous
teins) in endothelial cells [135]. However, none of these studies actin, which involves four cysteine residues near the C-terminal end of
explored the functional role of vinculin redox modifications. β-actin [140] (Fig. 4E). This interaction enhances actin polymerization

8
G. Matrullo et al. Redox Biology 80 (2025) 103514

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