FORUM REVIEW ARTICLE
Downstream Targets and Intracellular
Compartmentalization in Nox Signaling
Kai Chen, Siobhan E. Craige, and John F. Keaney, Jr.
Reactive oxygen species (ROS) have become recognized for their role as second messengers in a multitude of
physiologic responses. Emerging evidence points to the importance of the NADPH oxidase family of ROS-
producing enzymes in mediating redox-sensitive signal transduction. However, a clear paradox exists between
the specificity required for signaling and the nature of ROS as both diffusible and highly reactive molecules. We
seek to understand the targets and compartmentalization of the NADPH oxidase signaling to determine how
NADPH oxidase–derived ROS fit into established signaling paradigms. Herein we review recent data that link
cellular NADPH oxidase enzymes to ROS signaling, with a particular focus on the mechanism(s) involved in
achieving signaling specificity. Antioxid. Redox Signal. 11, 2467–2480.
example, growth-factor signals are integrated with internal-
state information to produce decisions on cell growth, dif-
ferentiation, or proliferation (47, 88, 99). The mechanisms
whereby cells coordinate this information have been the
subject of study for more than a century.
In one paradigm of classic receptor-mediated signaling,
ligand engagement is followed by the production of a dif-
fusible second messenger that interacts with a target to effect
the signal. This arrangement supports both signal conduction
over space and signal amplification, because most second
messengers are produced via an enzymatic process. Typically,
second messengers are small, diffusible molecules that rap-
idly activate effector proteins (i.e., protein kinases, protein
phosphatases, ion channels) within the cell through binding
Well-studied second messengers include cyclic nucleotides
(cAMP, cGMP), modified lipids (inositol phosphates, eicosa-
noids), and sequestered ions (Ca2þ). Generally, second mes-
sengers are specific for their effector targets and are often
restricted to their organelle(s) of generation.
The discovery of nitric oxide (?NO) as an intracellular sig-
naling molecule posed a problem for the classic signaling
paradigms. Unlike other signaling molecules,?NO was both
freely diffusible across membranes and (comparatively)
highly reactive. Whereas Ca2þor cyclic nucleotides are rela-
tively specific for their targets,?NO was able to react with a
number of intracellular species at considerable distances from
he mammalian cell constantly receives signals from its
its site of synthesis. Thus, the discovery of?NO as a signaling
regard to the properties and behavior of second messengers.
The notion that an authentic second messenger may have
promiscuous reactivity and broad diffusibility is in keeping
2?) and hydrogen peroxide (H2O2) may also
considered a by-product of oxidative metabolism with no
specific function. However, with the discovery that many
signaling pathways are influenced by ROS (21, 106, 121), it
became clear that ROS production must be considered within
the context of how cells respond to environmental stimuli and
process information. Because the reactivity of many ROS is
potentially damaging to important cellular components, such
as lipids, proteins, and nucleic acids, it follows that cells must
have mechanisms for promoting ROS-signaling specificity
and the limitation of collateral damage. The purpose of this
review is to examine the literature concerning ROS-signaling
specificity, with a particular focus on NADPH oxidase–
derived ROS and their downstream targets.
The NADPH oxidases are a family of multimeric enzymes
specifically designed to translocate electrons (from NADPH)
ROS. Central to this function is a catalytic subunit (officially
known as the Nox or Duox gene products) with a conserved
general domain structure (Fig. 1) that consists of six trans-
membrane helices, binding sites for NADPH and FAD, and
Division of Cardiovascular Medicine, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts.
ANTIOXIDANTS & REDOX SIGNALING
Volume 11, Number 10, 2009
ª Mary Ann Liebert, Inc.
heme-coordinating histidine residues. Five isoforms (Nox 1
to 5) function strictly as oxidases, and two isoforms (Duox 1
and 2) contain peroxidase homology domains, although the
functional capacity of these domains is not clear.
For the last several years, multiple Nox genes have been
described, with the gene products demonstrating wide-
ranging tissue distribution, suggesting that ROS production
may have broad implications for the cellular phenotype (23,
61) (Table 1). The literature regarding the Nox and Duox genes
and the NADPH oxidase enzyme family is ambiguous. Many
published reports refer to ‘‘NADPH oxidase’’ without regard
requirements for the catalytic activity of different Nox gene
products (Table 1) is not yet complete. In this review, there-
fore, we identify specific NADPH oxidases by their Nox gene
product. The term ‘‘NADPH oxidase’’ will be reserved for
instances in which the specific gene product is not known.
concert with multicellular organization (60), suggesting that
they contribute to the distinction among the functions of dif-
ferent cell types. Moreover, observations in primitive organ-
isms suggest that NADPH oxidases may be important for
stress responses. In the slime mold, Dictyostelium discoideum,
nutrient stress induces individual amoebae to aggregate into a
slug that can produce a spore-bearing fruiting body. Deletions
that disable NADPH oxidase function interrupts the formation
of a fruiting body (64). Thus, the NADPH oxidase family is
evolutionary conserved and affects stress-induced behavior.
The first-identified and best-studied Nox isoform is known
as Nox2 (originally termed gp91phox), which was identified in
phagocytes for its role in antimicrobial ROS production (3).
Nox 2 requires another integral membraneprotein, p22phoxfor
protein stabilization, but the activation of Nox2 is dependent
on the translocation of several other cytosolic regulatory
subunits, specifically p47phox, p67phox, p40phox, and Rac. As
indicated in Table 1, NADPH oxidase family members have
distinct regulatory patterns that do not strictly mirror those of
Nox2. For example, Nox1 is found in cells that do not express
p47 or p67. In such cells, Nox1 catalytic activity is supported
by Nox organizer 1 (NoxO1), and Nox activator 1 (NoxA1).
WithregardtoNox4,constitutive activity andnorequirement
of cytosolic factors for activity seem to exist. Thus, it is not
surprising that NADPH oxidase activity has been implicated
in a host of varied cellular functions.
Understanding how NADPH oxidase–derived ROS are
able to achieve target selectivity and specificity is paramount
(21, 70). Conceptually, the target selectivity of these may exist
at the level of the chemical characteristics of the oxidants or,
perhaps, intracellular antioxidants. Alternatively, selectivity
of NADPH oxidase–derived ROS could also involve their
specific reactivity with putative targets in the cytosol or pro-
teins. Finally, one could consider the intracellular localization
or compartmentalization or both of NADPH oxidase catalytic
activity as a means of specifying ROS signaling. In the re-
related to specificity of cell signaling.
Chemical Aspects of ROS Signaling Specificity
Reactive oxygen species
Although multiple ROS can be generated from the
metabolism=excitation of oxygen, these species are not all
equally reactive with respect to prospective targets. Many of
these species have very short half-lives, leading to little rele-
vance in terms of signaling. For example, the hydroxyl radical
(?OH) is the most unstable ROS, with a half-life of 10?9s (92),
indicating its limited ability to transmit signals across any
significant distance. Two major ROS that result from NADPH
oxidase enzymes are superoxide (O?
oxide (H2O2). These two species have the most favorable
chemical profiles as signaling molecules.
Superoxide, a negatively charged molecule, has limited
mobility across biologic membranes. Its diffusion is depen-
dent on anion channels in the membrane. Thus, one aspect of
its spatial specificity is related to its potential confinement
within organelles such as mitochondria, endosomes, and,
classically, phagosomes. This confinement is a limitation for
reactivity, superoxide can work both as an oxidant and as a
reductant. It is the protonated form of superoxide (HO2?) that
acts as an oxidant, but only a small fraction of superoxide is
superoxide at physiologic pH is that of a reductant; it reduces
iron and reacts with Fe-S centers. It is difficult to estimate the
half-life of O?
2?, as it is very dependent on the local concen-
trations of superoxide dismutase (SOD); however, it is clear
that dismutation to H2O2is its major mechanism of elimina-
2?) and hydrogen per-
translocate electrons (from NADPH) across
a membrane, which results in the formation
of ROS (predominantly O?
NADPH oxidase family members (Nox 1 to 5
and Duox 1 and 2) share conserved features,
including six transmembrane domains, where
transmembrane domains III and V contain
four heme-binding histidines. The carboxy
terminus consists of an FAD-binding domain
followed by an NADPH-binding domain.
Nox5 includes an additional
calmodulin-like Ca2þ-binding domain. Duox
1 and 2 also include peroxidase homology
NADPH oxidase family members
2?). The seven
2468CHEN ET AL.
Peroxide is a two-electron oxidant that acts as an electro-
phile and can react with protein thiol moieties to produce a
variety of sulfur oxidation states including disulfides, sulfenic
(?SOH), sulfinic (?SO2H), or sulfonic (?SO3H) acid products
(25, 30). This reactivity affords (mostly) reversible posttrans-
lational modification of proteins that would be important for
cellsignaling. Moreover,therelatively longerhalf-lifeofH2O2
compared with other ROS in biologic systems affords better
activity as an autocrine and paracrine messenger (21). An
important question that also must be addressed is how to
control the oxidizing activity of ROS and limit the potential
damage to biologic molecules. One mechanism to limit excess
oxidation is through cellular antioxidant defenses.
The intracellular environment is generally thought to be
replete with antioxidant activity. This activity is the result of
both low-molecular-weight antioxidants and antioxidant
proteins. Glutathione (GSH) is a tripeptide made of gluta-
mate, cysteine (Cys), and glycine that is a ubiquitous intra-
cellular antioxidant important for cellular protection against
ROS, electrophiles, and xenobiotics. Glutathione also is im-
portant for maintaining ascorbate, itself a potent intracellular
antioxidant, in a reduced state. With regard to proteins, the
formation of a mixed GSH-protein disulfide (glutathionyla-
tion) has been shown to protect proteins against irreversible
oxidation, as the mixed disulfide can be reduced by en-
zyme systems such as glutaredoxin (Grx). The intracellular
space typically contains GSH in the range of 3 to 10mM and
active against O?
2?, and GSH will attenuate, but not abro-
gate, the reactivity of H2O2. Thus, any ROS production within
the cellular cytosol is likely to encounter considerable low-
molecular-weight antioxidant activity.
Along with direct-acting antioxidants, many antioxidant
enzymes function to reduce oxidants. In mammals, three
isoforms of superoxide dismutase are known: cytoplasmic
SOD, which is a copper=zinc dismutase (SOD1); mitochon-
drial manganese SOD (SOD2); and extracellular Cu=Zn SOD
(SOD3; ec-SOD). The primary function of the SODs is to cat-
alyze the dismutation of superoxide to hydrogen peroxide,
which can then either function in signaling reactions or be
further reduced towater bycatalaseora peroxidase. Based on
the mechanism of enzyme action, it is generally believed that
the principal product of the NADPH oxidase enzymes is su-
peroxide. However, some NADPH oxidase isoforms release
H2O2(11), and a considerable amount of NADPH oxidase
signaling has been linked to H2O2. The specific determinants
The glutathione peroxidases (GPxs) reduce peroxides by
transferring electrons from GSH with the generation of oxi-
dized glutathione (GSSG). Catalase is found primarily in the
peroxisomes and efficiently converts H2O2to water and ox-
ygen, hence ‘‘quenching’’ signaling. However, it is likely that
Table 1. NADPH Oxidase Family Members Exhibit a Wide Spectrum of Diversity Including Differences
in Terms of Amino Acid Sequence Identity (here Compared to the Prototypical Nox2), Required Subunits
for Activation, Intracellular Localization, Tissue Distribution, and Cellular Function
Nox family member;
% sequence identity
to Nox2Necessary subunitsLocalizationTissue distribution
Nox 1; 60% NOXO1, NOXA1,
colon, smooth muscle,
wide distribution; strong
expression in phagocytes
maintenance of blood
activation of microglia
host defense and
Nox 3; 56% NOXO1, NOXA1,
inner ear, fetal kidney,
fetal spleen, skull bone,
hematopoietic stem cells,
testis, spleen, lymph nodes,
vascular smooth muscle,
bone marrow, pancreas,
placenta, ovary, uterus,
thyroid, airway epithelia,
development of inner
ear vestibular system
Nox 4; 39%
migration, oxygen sensor
channel, cell proliferation
Nox 5; 27%none ND
Duox 1=2; 50%p22phox, DUOXA1,
biosynthesis of thyroid
References: Nox1 (2, 4, 6, 27, 59, 65, 104, 107), Nox2 (15, 39, 46, 68, 84, 105, 108), Nox 3 (5, 111, 112), Nox 4 (20, 26, 35, 45, 50, 90, 114, 125),
Nox 5 (7, 8, 23), Duox 1=2 (28, 36, 38).
*These subunits are interchangeable for NoxO1 and NoxA1.
ND, not determined.
TARGETS AND COMPARTAMENTALIZATION IN NOX SIGNALING 2469
the main intracellular H2O2scavengers are the peroxiredox-
ins. The peroxiredoxins (Prxs; see later) are a ubiquitous class
of efficient H2O2scavengers with much higher binding af-
finities than other H2O2scavengers (116). Peroxiredoxins re-
move H2O2 to yield water and form an intermolecular
disulfide bond, which then can be reduced by thioredoxin
(Trx). Hence, Trx is not directly involved in removal of ROS,
but indirectly through a supportive role to the Prxs. Oxidized
Trx is then reduced by thioredoxin reductase by using
NADPH as an electron donor. As discussed later, many of
these antioxidant enzymes are located in close proximity to
the NADPH oxidase enzymes, conceptually allowing man-
agement of the ROS response. The specific role(s) of anti-
oxidant enzymes in NADPH oxidase signaling is(are) a
relatively underdeveloped area, and many gaps exist in our
Increasing evidence indicates that redox-dependent pro-
tein modification is an important mechanism in signal trans-
duction that parallels, in part, the body of knowledge relating
to classic phosphorylation. The availability and accessibility
of protein targets for reaction with ROS is one mechanism for
specificity in redox-sensitive signal transduction. In the fol-
lowing section, we discuss protein moieties typically subject
to redox modification, as well as specific classes of proteins
and protein complexes.
Protein Moieties Subject to Redox Modification
Iron-sulfide (Fe-S) centers
The charged molecule of O?
centers are involved in a variety of reactions because of their
highly studied reactions is their involvement in electron
transfer, as in the mitochondrial electron-transport chain,
which contains the biggest multi–Fe-S protein known, NADH
dehydrogenase. However, Fe-S proteins are also involved in
non–electron-transfer functions, such as substrate binding
surrounding groups and hence serve as active sites of en-
zymes (12). One example of the effect O?
is the protein aconitase, in which destabilization of the Fe-S
cluster by superoxide inhibits enzyme activity, thereby lim-
iting mitochondrial respiration (32, 33). The predilection of
superoxide for Fe-S centers has been exploited by Escherichia
coli in the form of the SoxR protein. This is a [2Fe-2S] protein
that undergoes univalent oxidation of the Fe-S centers by
superoxide (29). Oxidation of SoxR renders it able to bind to
its target genes, known collectively assoxRS,that coordinate a
complex stress response, including synthesis of proteins such
as SOD (29). The SoxR protein is specific for one-electron
oxidations, responding to superoxide and nitric oxide, but not
to hydrogen peroxide or hydroxyl radical (34, 44). Although
no orthologues have been described in higher organisms,
multiple proteins contain Fe-S centers and may thus be sus-
ceptible to influence by superoxide.
2?is attracted to Fe-S. These
2?has on Fe-S centers
An attractive mechanism for the conversion of peroxide
into cellular signals is through cysteine (Cys) oxidation. Al-
most all proteins contain Cys residues that are subject to ox-
idation. The inherent reactivity of these residues is due to the
fact that they contain sulfur, which exists stably in multiple
oxidation states, making it a versatile component in biologic
systems. The most highly active and most reduced form of
sulfur inbiomoleculesis the thiol(R-SH), presentinCys.Most
protein Cys residues are protonated at physiologic pH, with a
pKa of *8.5. The protonated thiol is not very reactive with
ROS, which allows the basis of ROS specificity in signaling in
anions (RS?). The most reactive thiolates are characterized by
pKa values in the range of 5.0 and are typically produced
when the Cys residue is surrounded by basic amino acids (44,
72, 127). Cysteine thiolates are oxidized by ROS to a sul-
fenic acid (RSOH) intermediate, which can rapidly react
with neighboring groups such as thiols [forming intra=
intermolecular disulfides (RSSRs)], nitrogens [forming sulfe-
namides (RSNRs)], or GSH (forming S-glutathionylated
mixed disulfides), all of which participate in altering protein
acid by ROS can lead to formation of sulfinic (RSO2H) and
sulfonic (RSO3H) acid, which reactions are generally consid-
ered to be irreversible (Fig. 2) (98). The exception to this is the
reactivation of some peroxiredoxins by sulfiredoxin (13, 51).
Interconversions of methionine (Met) residues may serve a
similar function, in which the sulfur in Met is present as a
thioether (–CH2–S–CH3) and can be oxidized to a sulfoxide.
However, Met is inherently less reactive than the thiol moiety,
and therefore, this review primarily focuses on the oxidation
of Cys residues, which encompasses the majority of what we
know about ROS regulation of signaling targets.
Direct Protein Targets
Protein tyrosine phosphatases
The best-documented targets for ROS are protein tyrosine
phosphatases (PTPs), whose enzymatic activity is abolished
by oxidation of a Cys residue in their active site. Protein ty-
rosine phosphatases are a superfamily of genes consisting of
*100 members, which function to remove the phosphate
group from proteins phosphorylated on tyrosine residues by
protein tyrosine kinases (PTKs). Thus, one can promote ty-
rosine phosphorylation through either stimulation of the ki-
equation that is germane for redox regulation of tyro-
sine phosphorylation. AllPTPs arecharacterizedbyanactive-
site motif that consists of Cys and arginine (Arg) separated by
five residues (I=V-C-XX-G-X-X-R-S=T), where X is any amino
acid (10). The proximity of the basic Arg residue creates a
microenvironment amenable to thiolate formation (pKa¼
4.7–5.4) that facilitates the Cys to function as a nucleophile
and abstract a proton. In the presence of an electrophile such
as H2O2, the active-site Cys residue is oxidized to a sulfenic
acid intermediate followed by rapid intraprotein conversion
to a cysteine sulfenyl-amide (Fig. 3). This chemistry produces
an active-site conformational change that inhibits substrate
binding (18, 30, 67, 98).
The reversible oxidation of PTP cysteine residues lends it-
self to a tonic level of control, as outlined in Fig. 3. As ROS
levels increase, a relative shift occurs in any local population
of PTPs from the reduced to the oxidized form of the enzyme.
This shift leaves the PTP unavailable for modulation of sig-
2470 CHEN ET AL.
naling and has the effect of accentuating the local activity of
PTKs. In contrast, a reducing environment (such as a relative
antioxidant abundance or lack of ROS) would tend to drive
the population of PTPs to the reduced state, increasing en-
zyme activity and attenuating the activity of PTKs. Thus, the
relative oxidizing tone, particularly in a localized environ-
ment, has the potential to modulate signaling tonically. This
concept is in keeping with the notion that redox signaling
tends to modulate pathways rather than to function as a strict
binary (yes=no) proposition (52).
The proposed paradigm fits well with our understanding
of NADPH oxidase enzymes and their impact on tyrosine
phosphorylation and PTPs. The first indication of NADPH
oxidase regulation of PTPs came from studies of the Nox2 in
phagocytes, in which it was speculated that this Nox enzyme
inactivated PTPs in the respiratory burst (128). Consistent
with this notion, treatment of macrophages with the NADPH
oxidase inhibitor DPI was associated with reduced ROS for-
mation, increased PTP activity, and decreased PTK activation
(128). The potential link between Nox2 and neutrophil PTP
activitywasrefined bythestudies demonstratingthatTNF-a–
mediated neutrophil activation resulted in a rapid distribu-
tion of NADPH oxidase components (Nox2, p22phox, p47phox,
(122). Similar experiments in adherent neutrophils from
Nox2-incompentent patients demonstrated impaired activa-
tion and tyrosine phosphorylation of Src-family kinases (fgr
and lyn) in the cytoskeletal fraction (121). Of interest, the lack
of Nox2 did not completely abrogate fgr=lyn tyrosine phos-
phorylation, further supporting the notion that ROS tend to
play a modulatory role in signaling.
Evidence indicating that NADPH oxidase regulates PTP
activity also extends beyond phagocytes. For example, Nox4
is highly expressed in insulin-sensitive adipocytes (73), and
insulin signaling is sensitive to PTP activity (41). This
knowledge prompted Mahedev and colleagues (73) to in-
vestigate the relation between Nox4 and insulin signaling.
They found that Nox4 was important in regulation of insulin-
receptor signal transduction, as the Nox4-derived ROS pro-
duced by receptor-ligand engagement led to inactivation of
PTP1B, enhancing downstream signal transduction. This
paradigm was recently broadened to include IL-4 receptor
activation that promoted downstream signaling through
activation of PI3K and subsequent activation of Nox1 and
Nox5L, which in turn inhibited PTP1B (101).
Other PTPs also have been implicated in NADPH oxidase–
mediated regulation. In vascular smooth muscle cells,
angiotensin II activation of Nox1 was shown to inhibit the Src
homology protein tyrosine phosphatase-2 (PTP SHP-2), thus
enhancing Akt activation. Nox4 also has been found to en-
hance growth factor–induced antiapoptotic action through
inactivation of low-molecular-weight protein tyrosine phos-
phatase (LMW-PTP), thus promoting JAK2 activity and pro-
moting survival in pancreatic cancer cells (66). In endothelial
migration, the cytosolic component of the Nox2 complex,
p47phox, was shown to be tethered to Hic5 (a scaffolding
protein) by the small adaptor protein TRAF4, thus creating
localized signaling that inactivates PTP-PEST, which also is
found associated with Hic-5 (117).
dues are fundamental in ROS signal
transduction. (A) Protein Cys residues
with the pKa lower than intracellular pH
are readily deprotonated, leading to
formation of the more-reactive thiolate
anion (RS?). (B) ROS reaction with
thiolate anions leads to formation of
sulfenic acid (RSOH), which is highly
reactive. (C) Sulfenic acid can form
reversible modifications with surround-
ing thiols, nitrogens, or GSH to form
disulfides, sulfenamides, or protein S-
glutathionylation, leading to changes in
protein function. (D) Reversible Cys
modifications are reduced by antioxi-
dant systems such as glutaredoxin (Grx)
and thioredoxin (Trx) (E) Sulfenamides
can also be further oxidized to sulfinic
(RSO2H) or sulfonic (RSO3H) acid, which
is generally considered irreversible.
Protein cysteine (Cys) resi-
to oxidant-induced signaling. (A) In the presence of an
electrophile such as H2O2, the active-site cysteine (R-S?)
residue is oxidized to a sulfenic acid (R-SOH) intermediate.
(B) This is followed by rapid intraprotein conversion to a
cysteine sulfenyl-amide, which produces an active-site con-
formational change that inhibits substrate binding. (C) The
activity of the PTPs is restored by antioxidant=antioxidant
systems such as glutathione (GSH)=glutaredoxin (Grx).
Protein tyrosine phosphatases (PTPs) are subject
TARGETS AND COMPARTAMENTALIZATION IN NOX SIGNALING2471
Although PTP oxidation has been well documented, many
gaps exist in our understanding of the mechanisms whereby
this occurs and moreover how this oxidation allows specific-
ity. As one can appreciate from the data outlined earlier, a
number of ligands have been linked to activation of one or
several NADPH oxidase isoforms. In addition, multiple PTPs
have been identified as targets of NADPH oxidase activation.
However, we do not know much about the molecular re-
quirements that dictate signaling specificity. For example,
PDGF-receptor ligand engagement is known to be specific for
SHP-2 (79) inactivation, whereas blanket treatment of cul-
tured cells with H2O2leads to inactivation of multiple PTPs
(79). Thus, the requirements for signaling specificity are not
Recent observations from our group have begun to shed
light on this issue in which endothelial Nox4 was shown to be
localized to the endoplasmic reticulum (ER), and this finding
was a key feature of Nox4 regulation of PTP1B, another ER-
resident protein. Nox4-dependent oxidative modification of
PTP1B required both proteins to be in the ER, as a mutant
form of PTP1B located in the cytosol was no longer oxidized
by Nox4. This was fundamentally important in the regulation
of EGF signaling in which Nox4-mediated PTP1B oxidation
was associated with reduced dephosphorylation of EGF re-
ceptor in proximity to the ER, resulting in accentuated EGFR-
dependent signaling and cell proliferation (Fig. 4) (20).
Protein tyrosine kinases
In addition to the aforementioned effects of NADPH
oxidase–derived ROS on PTPs, evidence suggests that kinases
themselves are downstream targets for ROS. The MAP ki-
nases have been implicated in many redox-dependent sig-
naling events; however, it is unclear whether they are direct
targets of ROS or are solely activated through inactivation of
upstream phosphatases or as downstream targets of protein
tyrosine kinases (PTKs) (76). The PTKs themselves are po-
tential direct targets of ROS in which *80% of known PTKs
contain a conserved C-terminal CXXXXXXXMXXCW motif
(where X is any residue), and *96% contain the MXXCW
portion of the motif (83). These data suggest that PTKs should
demonstrate direct redox regulation. UV light induces ROS
production that facilitated the dimerization and activation of
the receptor tyrosine kinase Ret (55, 56). This effect was due to
the oxidation of C-terminal Cys residues that facilitated Ret
dimerization and activation by autophosphorylation (55, 56).
Given that similar Cys residues are conserved in other protein
that these kinases also are subject to oxidation-induced acti-
is of particular interest. Those investigators found that cell
adhesion and integrin ligand engagement was associated with
residues at positions 245 and 487. Mutating these residues in-
hibited ROS-mediated Src activation resulting from integrin
ligand engagement. This paradigm has also been specifically
linked to Nox4activation. Block and colleagues (14) found that
angiotensin II treatment of mesangial cells resulted in Nox4
upregulation and ROS production, which resulted in the oxi-
dation-mediated activation of Src, a critical step in angiotensin
II–induced fibronectin expression (14).
These data linking Nox4 to Src oxidation are consistent
with some of the information on intracellular Nox4 localiza-
tion. In smooth muscle cells, Nox4 is found closely associated
with focal adhesion complexes (45), where Src becomes lo-
calized on cell-surface integrin ligand engagement (95). Thus,
one could certainly envision the targeted oxidation of Src in
focal adhesions that result from the integrin-induced ROS
signal. Thus, available data support the concept that ROS
have a dual role in modulating tyrosine phoshorylation via
promotion of tyrosine phosphorylation and inhibition of tyro-
activity. (A)Vesiculation of inter-
nalized EGFR trafficks phosphor-
ylated EGFR near the endoplasmic
reticulum (ER). (B) Nox 4 is local-
ized to the ER in close proximity to
PTP1b, thus influencing PTP1b
activity. (C) ROS emanating from
ER Nox4 are then able to mediate
EGFR signaling through deactiva-
tion of PTP1b, leading to increased
EGFR phosphorylation and re-
cycling of phosphorylated (active)
receptor to the plasma membrane.
Nox 4 mediates EGFR
2472 CHEN ET AL.
Small G proteins
Small GTPases are proteins (20 to 25kDa) that bind to
guanosine triphosphate (GTP) in their active state and are
inactive when bound to GDP. Small GTPases are known to
regulate a wide variety of processes, such as cell proliferation,
differentiation, movement, and lipid vesicle transport (9). The
Ras family of proteins are among the small GTPases, and one
important mechanism of Ras protein modification involves
Cys residues that are conserved in almost all Ras family
members. The carboxy-terminal Cys 186 is part of the CAAX
motif site for isoprenylation, but at least four other surface-
exposed Cys residues (Cys 118, 181, 184, and 186) have been
identified that are likely subjects to oxidation=modification.
Cys 118 has been shown to interact with?NO, leading to in-
creased nucleotide exchange and Ras activation (62), and all
four sites can be modified by oxidants (74). With regard to
ROS, the Cys 118 site is known to be targeted by O?
facilitates GDP dissociation from Ras and replacement with
GTP (42), thus leading to increased GTPase activity (63).
Angiotensin II–induced activation of NADPH oxidase and
subsequent ROS production facilitates Ras activity through
glutathionylation (S-SG) of Cys118, where this modification is
necessary for angiotensin II–induced smooth muscle cell hy-
pertrophy (1) (Fig. 5). Glutathionylation of Ras also occurs in
cardiac myocytes, where mechanical strain induces NADPH
oxidase–mediated glutathionylation ofCys118,leading toErk
activation and increased protein synthesis (91). Taken to-
gether, these data implicate Ras as a downstream target for
NADPH oxidase–derived ROS. The precise mechanisms
whereby NADPH oxidase catalytic activity is targeted to Ras,
however, are not known.
Protein disulfide isomerases
The protein disulfide isomerases (PDIs) are found pre-
dominantly in the ER and are involved in protein processing,
in which they catalyze the formation of Cys disulfide bonds
through thiol=disulfide exchange, allowing correct protein
folding. These proteins are active in the oxidized state; thus,
the maintenance of an oxidized subcellular ER compartment
is necessary for proper protein folding. Studies with PDI
members have demonstrated that these proteins are involved
in NADPH oxidase activation (50) and as downstream targets
of NADPH oxidase signaling (22). Forced overexpression of
active Nox1 led to the oxidation of ERp72, an ER protein and
PDI family member, on Cys residues in a thioredoxin domain
(22). These Cys modifications were associated with attenua-
tion of Erp72 reductase activity, an effect that also was seen
with cell incubation with EGF.
The peroxiredoxin (Prx) family of peroxidases catalyze the
reduction of peroxides with the aid of reducing equivalents
from thiol-containing proteins. They are very conserved from
an evolutionary standpoint, and all Prxs exist as homodimers
(94). A conserved cysteine residue is present in the NH2-
terminal region that is the primary site of peroxide-induced
oxidation. The six mammalian Prx isoforms are divided into
three subgroups, designated 2-Cys, atypical 2-Cys, and 1-Cys
[for review, see (24)]. The best-characterized Prx isoforms are
the 2-Cys types, which have been identified in the cytosol
(I and II), the mitochondria (III), and the endoplasmic retic-
The catalytic cycle of the 2-Cys Prxs is well understood
and involves H2O2-mediated oxidation of the conserved N-
terminal to a sulfenic acid (RSOH), prompting its subsequent
reaction with the C-terminal Cys of the other homodimer to
form an intermolecular disulfide. The fully reduced enzyme
is reconstituted through thioredoxin-mediated reduction.
Under conditions of high H2O2stress, the Cys residues can
become further oxidized tothe sulfinic form (RSO2H), leading
to the formation of Prx aggregates. The ATP-dependent re-
duction of ‘‘hyperoxidized’’ Prx is accomplished by sulfir-
edoxin, and leads to the clearing of the Prx aggregates. Recent
evidence indicates that hyperoxidation of Prx and oligomer
formation is associated with cell-cycle arrest, which can be
reversed by clearing these aggregates via Prx reduction (89).
These data suggest that the oxidation state of Prxs themselves
may represent a signaling loop.
Evidence indicates that Prxs are involved in the control of
NADPH oxidase signaling, principally as a means of attenu-
ating the ROS signal. Endotoxic shock by LPS is known to be
mediated, in part, by NADPH oxidase, and mice deficient in
with peptide growth factors, such as insulin, PDGF, and EGF,
is associated with transient NADPH oxidase–mediated inac-
Recently, Choi and colleagues (24) demonstrated that Prx II is
recruited to PDGF receptors on PDGF stimulation and at-
tenuates the ROS-mediated PTP inactivation. These findings
attenuated the arterial neointimal thickening after injury—a
process known to involve PDGF signaling (77, 77). Thus, Prxs
are a downstream target of NADPH oxidase, primarily as a
Cys residues. In this paradigm, Cys118 is glutathionylated
(S-SG) under oxidizing conditions. Glutathionylation of
Cys118 stimulates nucleotide exchange (GDP to GTP), lead-
ing to enhanced Ras activation.
Ras proteins undergo modification of conserved
TARGETS AND COMPARTAMENTALIZATION IN NOX SIGNALING2473
modulating influence on the extent of PTP inhibition and
Among the more intricate means of regulating redox sig-
naling is through the formation of signaling complexes to
colocalize either ROS production or ROS targets with appro-
priate effector proteins. This concept is in keeping with other
known methods of signaling (e.g., phosphorylation, ubiqui-
tination, acetylation), in which protein complex formation is
important for the specificity of signaling.
Transcription factor Nrf2 regulates the inducible expres-
sion of cytoprotective genes via cis-acting antioxidant-
responsive elements or electrophile response elements (57).
The expression and activity of Nrf2 is under tight control by
its negative regulator, Keap1. Redox-sensitive cysteine resi-
dues in Keap1 are responsible for determining whether the
local ‘‘oxidant tone’’ warrants the Nrf2-driven genetic pro-
gram. In the normal highly reducing environment of the cell,
Keap1 forms a complex with Nrf2 that facilitates its targeting
by cullin family ubiquitin E3 ligases for ubiquitination and
proteasomal degradation (58, 126). Oxidation of the Cys res-
idue in Keap1 triggers its dissociation from Nrf2, preventing
Nrf2 degradation. As a result, free Nrf2 is thus translocated
study revealed the physiological significance of each reactive
cysteine residue of Keap1 by using the transgene comple-
mentation analysis in vivo. Cys273 and Cys288 seemed to be
keys to the constitutive repression of Nrf2 activity, whereas
Cys151 was obligatory for the full activation of Nrf2 (120). By
using the NADPH oxidase inhibitor, diphenyleneiodonium
(DPI), NADPH oxidase was identified as acting upstream of
Keap1=Nrf2, whereas Keap1 was a downstream effector for
oxidase activity (100). Similarly, results were seen by using
hyperoxia-induced Nrf2-dependent transcription that was
blocked by diphenylene iodonium, implicating NADPH oxi-
dase enzymes as mediators of Nrf2 activation (86).
The regulation of apoptosis-signaling kinase-1 (ASK-1) is
somewhat analogous to that of Nrf2. Reduced thioredoxin 1
(Trx1) associates with ASK-1, and this complex results in in-
hibition of ASK-1 activity (97, 109). The oxidation of Trx1
leads to its dissociation from ASK-1, thereby freeing its en-
zymatic activity for activation of its downstream targets,
such as JNK (48, 97). NADPH oxidase–mediated generation
of H2O2plays a critical role in this process, as the ADP-
stimulated respiratory burst in alveolar macrophages leads to
transient and localized oxidation of Trx, affording ASK1 ac-
tivation that leads to an inflammatory response through
MKK4-JNK1=c-Jun signaling (71).
The redox control of NF-kB signaling is complex, wherein,
cytoplasmic oxidation of NF-kB subunits can activate gene
transcription, but nuclear NF-kB oxidation impairs DNA
binding (54). However, one component of ROS regulation of
NF-kB is reminiscent of the ROS-sensitive protein sequestra-
tion seen with Nrf2=Keap1. Normally, NF-kB is sequestered
in the cytosol by IkB, which, when phosphorylated, is tar-
geted for degradation and releases NF-kB to translocate to the
nucleus. It has recently been demonstrated that cytosolic ROS
can promote NF-kB nuclear translocation through control of
the NF-kB=IkB interaction. The dynein light chain LC8 asso-
ciates with IkB and inhibits its phosphorylation by IkB kinase
(IKK). However, on TNF-a stimulation, LC8 becomes oxi-
dized by NADPH oxidase and dissociates from IkB, allowing
its IKK-dependent phosphorylation and release of NF-kB to
the nucleus (53). The reduced state of LC8 is maintained
by thioredoxin-related protein 14 (TRP14), and thus, TRP14
is a counterregulatory component of redox-sensitive NF-kB
The preceding paragraphs demonstrated the importance of
locating redox-sensitive NADPH oxidase targets in proximity
with their effector molecules. However, this type of protein–
protein interaction does not explain how NADPH oxidase–
derived ROS might affect one particular target (e.g., LC8 or
Trx) in preference to another. In the following paragraphs, we
focus on examples of locating ROS production in proximity to
the specific ROS-sensitive targets.
Protein Localization in the Control of NOX Signaling
The localization of signaling molecules near their sites of
of multicomponent pathway-specific complexes is accom-
plished, in part, through protein scaffolds that afford greater
specificity than simple diffusion of the same limited number
of components. The ability of ROS-generating enzymes to fit
within this paradigm depends on the specific localization of
Keap1. Redox-sensitive cysteine residues in Keap1 are im-
portant in Nrf2 association with Keap1. Keap1 forms a
complex with Nrf2 that facilitates its targeting by cullin
family ubiquitin E3 ligases for ubiquitination and proteaso-
mal degradation. However, oxidation of Cys residues in
Keap1 triggers its dissociation from Nrf2, allowing Nrf2 to
translocate into the nucleus and activate stress-response
Nrf2 is under the control of its negative regulator,
2474 CHEN ET AL.
ROS production and the limitation of ROS dissemination. The
former may be accomplished through subcellular localization
of Nox isoforms and their adaptor proteins. The latter is im-
portant to minimize extraneous and unintended ROS signal-
ing. We address these two concepts in turn, realizing that
considerably more data exist for localization of ROS produc-
tion than for focal ROS scavenging. In this regard, we now
turn our attention to mechanisms whereby ROS generation is
localized to its site of signaling.
Activation of Nox2 requires its association with adaptor
proteins such as p47phoxand p67phox,commensurate with their
incorporation into the cytoskeletal fraction. These data, com-
bined with observations that leading-edge membrane ruffles
are enriched with Nox2 components, indicate that oxidant
production is localized (43). Consistent with these observa-
tions, cytoskeletal association of p47phoxhas been observed in
endothelial cells (40, 68), and NADPH oxidase activity ap-
pears important for endothelial cell migration (81) and an-
giogenesis (113). The link between NADPH oxidase activity
and cell migration is critically dependent on p47phox(81). The
phosphorylation of this NADPH oxidase subunit is among
the earliest features of Nox2 activation, and p47phoxbinds
moesin (115) and WAVE1(117), two proteins that are preva-
lent within leading-edge lamellipodia. Thus, it appears that
edge lamellipodia, with a resultant localized burst of ROS
activity in the leading edges of cells. This contention is sup-
ported by observations that disrupting p47 association with
WAVE1, or inhibiting NADPH oxidase activity, impairs
membrane ruffle formation in endothelial cells (117). The
notion that NADPH oxidase activity is linked to actin dy-
namics is supported by observations in smooth muscle cells
that cortactin, another actin-binding protein, coordinates
p47phoxlocalization to F-actin in response to angiotensin II,
thereby facilitating NADPH oxidase–mediated activation of
p38 MAP kinase and Akt (110). If one considers that cyto-
skeletal rearrangement is also highly dependent on tyrosine
phosphorylation, it is not surprising that NADPH oxidase–
mediated ROS production plays a critical role in this process.
Focal-adhesion behavior is coordinated by the Rho family
of small GTPases (85, 96) that includes Rac1 (Ras-related C3
botulinum toxin substrate 1). Nox2 has been shown to me-
diate endothelial cell migration in a Rac1-dependent manner
(81, 113). The coordination of Nox2 with Rac1 involves a
scaffolding protein known as IQGAP1 (49). IQGAP1 is in-
volved in microtubule stabilization via binding of CLIP-170 at
microtubule caps present in cell cortical regions (16, 17, 75).
This scaffold is critical for directional migration and serves to
coordinate Nox2 and Rac1 targeting to the leading edges of
TNF receptor–associated factor 4 is an orphan adaptor pro-
tein that binds to the focal contact scaffold Hic-5. This adaptor
protein is critical for cell migration, as TRAF4-deficient mice
and flies display migration defects, such as impaired neural
tube closure in mice and incomplete dorsal closure in flies (19,
93). One role of TRAF-4 in migration relates to its binding to
p47phoxand association with Hic-5 that leads to p47phoxlocali-
zation in focal complexes. This localization targets NADPH
oxidase activity to lamellipodia and supports cell migration as
an interruption of this process using siRNA (TRAF4 or Hic-5)
or oxidant-scavenging impaired migration (118). The final
target appears to be PTP-PEST, as it was oxidatively modified
by TRAF4 activation and independent PTP-PEST inhibition
enhanced membrane ruffling (118). The interaction of TRAF4
with p47phoxis also thought to be involved in the specific ox-
idative activation of MAPK8=JNK (119). Thus, available data
indicate that TRAF4 is an important scaffold for the targeting
of NADPH oxidase–derived ROS that contribute to endo-
thelial cell migration and membrane-ruffle formation.
Lipid rafts, first proposed by Simons and Ikonen (102), are
cholesterol- and sphingolipid-rich plasma-membrane do-
mains that also contain caveolae microdomains, the latter
consisting of flask-shaped membrane invaginations that
use caveolin as a scaffolding protein (87). Caveolae function
as signaling complexes that contain molecules such as G
kinases, and protein tyrosine kinases. These lipid rafts are
thought to provide for spatial organization of specific signal-
ing pathways and, as one might expect, have been implicated
muscle cells is associated with caveolae incorporation of the
angiotensin II, type I receptor (AT1R) (129) that is accompa-
nied by Rac1 recruitment and NADPH oxidase–mediated
EGF receptor transactivation, which is Src-dependent (78).
The involvement of lipid rafts has also been extended to en-
dothelial cells. Endothelial cell treatment with TNF-a results
in p47phoxrecruitment into raft domains that helps co-localize
NADPH oxidase with eNOS to support the generation of
(123). Thus, it appears that existing paradigms for lipid raft–
mediated signaling compartmentalization also apply to ROS-
mediated signal transduction.
Another paradigm of cell signaling involves the formation
among cellular compartments. One classic example of this
pathway is the cycling of EGF receptors from the cell surface
to the ER for PTP1b-mediated dephosphorylation, thereby
providing for regulation of growth-factor signaling. As
shown in Fig. 4, this particular process is subject to regulation
by Nox4 (20). As an intriguing twist on this paradigm, Li et al.
(69) demonstrated that formation of an active interleukin-1
(IL-1) receptor complex in the endosomal compartment re-
quired Nox2-derived ROS (69), perhaps via endosomal an-
ionic transporters that facilitate superoxide release (82).
Consistent with these observations, Miller and colleagues (80)
recently found that TNF-a– and IL-1b–induced Nox1 activa-
tion occurs in early endosomes, where Nox1 is colocalized
with CIC-3, an anion channel that affords charge neutraliza-
tion from the electron flow of superoxide generation (80). The
activation of NF-kB in this system was dependent on en-
dosomal activation of Nox1.
TARGETS AND COMPARTAMENTALIZATION IN NOX SIGNALING 2475
Conclusions and Knowledge Gaps
Available data indicate that ROS signaling is a local process,
consistent with existing paradigms for phosphorylation-based
signaling. The specificity of ROS signaling appears to involve
both the localization of ROS production and the limitation of
extraneous ROS dissemination. The preceding paragraphs re-
viewed the evidence that ROS production can be localized via
the characteristics of specific ROS, the protein targets for
modification, and the adaptor proteins for NADPH oxidase
localization. The mechanisms for the prevention of ROS
‘‘leaking’’ away from these redox microdomains, however, re-
main unclear. The cytosol may constrain ROS ‘‘leaking’’ from
their intended targets, although this phenomenon would be
very difficult to test because of the presence of multiple anti-
oxidant species. Thus, it seems clear that more study will be
site of action. Recent studies indicating that peroxiredoxin lo-
calization to the PDGF receptor is important for the limitation
of PDGF signaling (24) may provide clues for future investi-
gation. It seems likely that we will find ‘‘antioxidant scaffolds’’
that could restrict the ROS flux in a manner that would maxi-
mize its signal-to-noise ratio. Ultimately, a clearer under-
standing of ROS localization will help us unravel the cellular
mechanism(s) for processing of information.
K. Chen is the recipient of a Scientist Development Grant
form the American Heart Association, and this work was
partially supported by NIH grants AG027081; HL67266;
HL081587 and HL68758 to J. F. Keaney Jr.
1. Adachi T, Pimentel DR, Heibeck T, Hou X, Lee YJ, Jiang B,
Ido Y, and Cohen RA. S-glutathiolation of Ras mediates
redox-sensitive signaling by angiotensin II in vascular
smooth muscle cells. J Biol Chem 279: 29857–29862, 2004.
2. Ago T, Kitazono T, Kuroda J, Kumai Y, Kamouchi M,
Ooboshi H, Wakisaka M, Kawahara T, Rokutan K, Ibayashi
S, and Iida M. NAD(P)H oxidases in rat basilar arterial
endothelial cells. Stroke 36: 1040–1046, 2005.
3. Babior BM, Kipnes RS, and Curnutte JT. Biological defense
mechanisms: the production by leukocytes of superoxide, a
potential bactericidal agent. J Clin Invest 52: 741–744, 1973.
4. Banfi B, Clark RA, Steger K, and Krause KH. Two novel
proteins activate superoxide generation by the NADPH
oxidase NOX1. J Biol Chem 278: 3510–3513, 2003.
5. Banfi B, Malgrange B, Knisz J, Steger K, Dubois-Dauphin M,
and Krause KH. NOX3, a superoxide-generating NADPH
oxidase of the inner ear. J Biol Chem 279: 46065–46072, 2004.
6. BanfiB,Maturana A,JaconiS,ArnaudeauS,Laforge T, Sinha
B, Ligeti E, Demaurex N, and Krause KH. A mammalian Hþ
channel generated through alternative splicing of the
7. Banfi B, Molnar G, Maturana A, Steger K, Hegedus B,
Demaurex N, and Krause KH. A Ca(2þ)-activated NADPH
oxidase in testis, spleen, and lymph nodes. J Biol Chem 276:
8. Banfi B, Tirone F, Durussel I, Knisz J, Moskwa P, Molnar
GZ, Krause KH, and Cox JA. Mechanism of Ca2þactivation
of the NADPH oxidase 5 (NOX5). J Biol Chem 279: 18583–
9. Bar-Sagi D and Hall A. Ras and Rho GTPases: a family
reunion. Cell 103: 227–238, 2000.
10. Barford D, Jia Z, and Tonks NK. Protein tyrosine phos-
phatases take off. Nat Struct Biol 2: 1043–1053, 1995.
11. Bedard K and Krause KH. The NOX family of ROS-
generating NADPH oxidases: physiology and pathophysi-
ology. Physiol Rev 87: 245–313, 2007.
12. Beinert H. Iron-sulfur proteins: ancient structures, still full
of surprises. J Biol Inorg Chem 5: 2–15, 2000.
13. Biteau B, Labarre J, and Toledano MB. ATP-dependent
reduction of cysteine-sulphinic acid by S. cerevisiae sul-
phiredoxin. Nature 425: 980–984, 2003.
14. Block K, Eid A, Griendling KK, Lee DY, Wittrant Y, and
Gorin Y. Nox4 NAD(P)H oxidase mediates Src-dependent
tyrosine phosphorylation of PDK-1 in response to angio-
tensin II: role in mesangial cell hypertrophy and fibronectin
expression. J Biol Chem 283: 24061–24076, 2008.
15. Borregaard N, Heiple JM, Simons ER, and Clark RA. Sub-
cellular localization of the b-cytochrome component of the
human neutrophil microbicidal oxidase: translocation
during activation. J Cell Biol 97: 52–61, 1983.
16. Brandt DT and Grosse R. Get to grips: steering local actin
dynamics with IQGAPs. EMBO Rep 8: 1019–1023, 2007.
17. Briggs MW and Sacks DB. IQGAP proteins are integral
components of cytoskeletal regulation. EMBO Rep 4: 571–
18. Caselli A, Marzocchini R, Camici G, Manao G, Moneti G,
Pieraccini G, and Ramponi G. The inactivation mechanism
of low molecular weight phosphotyrosine-protein phos-
phatase by H2O2. J Biol Chem 273: 32554–32560, 1998.
19. Cha GH, Cho KS, Lee JH, Kim M, Kim E, Park J, Lee SB,
and Chung J. Discrete functions of TRAF1 and TRAF2 in
Drosophila melanogaster mediated by c-Jun N-terminal ki-
nase and NF-kappaB-dependent signaling pathways. Mol
Cell Biol 23: 7982–7991, 2003.
20. Chen K, Kirber MT, Xiao H, Yang Y, and Keaney JF Jr.
Regulation of ROS signal transduction by NADPH oxidase
4 localization. J Cell Biol 181: 1129–1139, 2008.
21. Chen K, Thomas SR, and Keaney JF Jr. Beyond LDL oxi-
dation: ROS in vascular signal transduction. Free Radic Biol
Med 35: 117–132, 2003.
22. Chen W, Shang WH, Adachi Y, Hirose K, Ferrari DM, and
Kamata T. A possible biochemical link between NADPH
oxidase (Nox) 1 redox-signalling and ERp72. Biochem J 416:
23. Cheng G, Cao Z, Xu X, van Meir EG, and Lambeth JD.
Homologs of gp91phox: cloning and tissue expression of
Nox3, Nox4, and Nox5. Gene 269: 131–140, 2001.
24. Choi MH, Lee IK, Kim GW, Kim BU, Han YH, Yu DY,
Park HS, Kim KY, Lee JS, Choi C, Bae YS, Lee BI, Rhee SG,
and Kang SW. Regulation of PDGF signalling and vascu-
lar remodelling by peroxiredoxin II. Nature 435: 347–353,
25. Claiborne A, Miller H, Parsonage D, and Ross RP. Protein-
sulfenic acid stabilization and function in enzyme catalysis
and gene regulation. FASEB J 7: 1483–1490, 1993.
26. Cucoranu I, Clempus R, Dikalova A, Phelan PJ, Ariyan S,
Dikalov S, and Sorescu D. NAD(P)H oxidase 4 mediates
transforming growth factor-beta1-induced differentiation
of cardiac fibroblasts into myofibroblasts. Circ Res 97: 900–
27. Cui XL, Brockman D, Campos B, and Myatt L. Expression
of NADPH oxidase isoform 1 (Nox1) in human placenta:
involvement in preeclampsia. Placenta 27: 422–431, 2006.
2476CHEN ET AL.
28. De DX, Wang D, Many MC, Costagliola S, Libert F, Vassart
G, Dumont JE, and Miot F. Cloning of two human thyroid
cDNAs encoding new members of the NADPH oxidase
family. J Biol Chem 275: 23227–23233, 2000.
29. Demple B, Hidalgo E, and Ding H. Transcriptional regu-
lation via redox-sensitive iron-sulphur centres in an oxi-
dative stress response. Biochem Soc Symp 64: 119–128, 1999.
30. Denu JM and Tanner KG. Specific and reversible inactiva-
evidence for a sulfenic acid intermediate and implications
for redox regulation. Biochemistry 37: 5633–5642, 1998.
31. Frei B. Reactiv oxygen species and antioxidant vitamins:
mechanisms of action. Am J Med 97(suppl 3A): 3A, 1994.
32. Gardner PR, Nguyen DD, and White CW. Aconitase is a
sensitive and critical target of oxygen poisoning in cultured
mammalian cells and in rat lungs. Proc Natl Acad Sci U S A
91: 12248–12252, 1994.
33. Gardner PR, Raineri I, Epstein LB, and White CW. Super-
oxide radical and iron modulate aconitase activity in
mammalian cells. J Biol Chem 270: 13399–13405, 1995.
34. Gaudu P and Weiss B. SoxR, a [2Fe-2S] transcription factor,
is active only in its oxidized form. Proc Natl Acad Sci U S A
93: 10094–10098, 1996.
35. Geiszt M, Kopp JB, Varnai P, and Leto TL. Identification of
renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci
U S A 97: 8010–8014, 2000.
36. Geiszt M, Witta J, Baffi J, Lekstrom K, and Leto TL. Dual
oxidases represent novel hydrogen peroxide sources sup-
porting mucosal surface host defense. FASEB J 17: 1502–
37. Giannoni E, Buricchi F, Raugei G, Ramponi G, and Chiar-
ugi P. Intracellular reactive oxygen species activate Src
tyrosine kinase during cell adhesion and anchorage-
dependent cell growth. Mol Cell Biol 25: 6391–6403, 2005.
38. Grasberger H and Refetoff S. Identification of the matura-
tion factor for dual oxidase: evolution of an eukaryotic
operon equivalent. J Biol Chem 281: 18269–18272, 2006.
39. Groemping Y and Rittinger K. Activation and assembly of
the NADPH oxidase: a structural perspective. Biochem J 386:
40. Gu Y, Xu YC, Wu RF, Souza RF, Nwariaku FE, and Terada
LS. TNFalpha activates c-Jun amino terminal kinase
through p47(phox). Exp Cell Res 272: 62–74, 2002.
41. Haj FG, Zabolotny JM, Kim YB, Kahn BB, and Neel BG.
Liver-specific protein-tyrosine phosphatase 1B (PTP1B) re-
expression alters glucose homeostasis of PTP1B-=- mice.
J Biol Chem 280: 15038–15046, 2005.
42. Heo J and Campbell SL. Superoxide anion radical modu-
lates the activity of Ras and Ras-related GTPases by a
radical-based mechanism similar to that of nitric oxide.
J Biol Chem 280: 12438–12445, 2005.
43. Heyworth PG, Robinson JM, Ding J, Ellis BA, and Badwey
JA. Cofilin undergoes rapid dephosphorylation in stimu-
lated neutrophils and translocates to ruffled membranes
enriched in products of the NADPH oxidase complex: ev-
idence for a novel cycle of phosphorylation and dephos-
phorylation. Histochem Cell Biol 108: 221–233, 1997.
44. Hidalgo E, Ding H, and Demple B. Redox signal trans-
duction via iron-sulfur clusters in the SoxR transcription
activator. Trends Biochem Sci 22: 207–210, 1997.
45. Hilenski LL, Clempus RE, Quinn MT, Lambeth JD, and
Griendling KK. Distinct subcellular localizations of Nox1
and Nox4 in vascular smooth muscle cells. Arterioscler
Thromb Vasc Biol 24: 677–683, 2004.
46. Huang J, Hitt ND, and Kleinberg ME. Stoichiometry of p22-
phox and gp91-phox in phagocyte cytochrome b558. Bio-
chemistry 34: 16753–16757, 1995.
47. Hunter T. Signaling: 2000 and beyond. Cell 100: 113–127,
48. Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi
T, Takagi M, Matsumoto K, Miyazono K, and Gotoh Y.
Induction of apoptosis by ASK1, a mammalian MAPKKK
that activates SAPK=JNK and p38 signaling pathways.
Science 275: 90–94, 1997.
49. Ikeda S, Yamaoka-Tojo M, Hilenski L, Patrushev NA,
Anwar GM, Quinn MT, and Ushio-Fukai M. IQGAP1
regulates reactive oxygen species-dependent endothelial
cell migration through interacting with Nox2. Arterioscler
Thromb Vasc Biol 25: 2295–2300, 2005.
50. Janiszewski M, Lopes LR, Carmo AO, Pedro MA, Brandes
RP, Santos CX, and Laurindo FR. Regulation of NAD(P)H
oxidase by associated protein disulfide isomerase in vas-
cular smooth muscle cells. J Biol Chem 280: 40813–40819,
51. Jeong W, Park SJ, Chang TS, Lee DY, and Rhee SG. Mole-
cular mechanism of the reduction of cysteine sulfinic acid
of peroxiredoxin to cysteine by mammalian sulfiredoxin.
J Biol Chem 281: 14400–14407, 2006.
52. Jones DP. Radical-free biology of oxidative stress. Am J
Physiol Cell Physiol 295: C849–C868, 2008.
53. Jung Y, Kim H, Min SH, Rhee SG, and Jeong W. Dynein
light chain LC8 negatively regulates NF-kappaB through
the redox-dependent interaction with IkappaBalpha. J Biol
Chem 283: 23863–23871, 2008.
54. Kabe Y, Ando K, Hirao S, Yoshida M, and Handa H. Redox
regulation of NF-kappaB activation: distinct redox regula-
tion between the cytoplasm and the nucleus. Antioxid Redox
Signal 7: 395–403, 2005.
55. Kato M, Iwashita T, Akhand AA, Liu W, Takeda K, Ta-
keuchi K, Yoshihara M, Hossain K, Wu J, Du J, Oh C, Ka-
wamoto Y, Suzuki H, Takahashi M, and Nakashima I.
Molecular mechanism of activation and superactivation of
Ret tyrosine kinases by ultraviolet light irradiation. Antioxid
Redox Signal 2: 841–849, 2000.
56. Kato M, Iwashita T, Takeda K, Akhand AA, Liu W,
Yoshihara M, Asai N, Suzuki H, Takahashi M, and Naka-
shima I. Ultraviolet light induces redox reaction-mediated
dimerization and superactivation of oncogenic Ret tyrosine
kinases. Mol Biol Cell 11: 93–101, 2000.
57. Katsuoka F, Motohashi H, Ishii T, Aburatani H, Engel JD,
and Yamamoto M. Genetic evidence that small maf pro-
teins are essential for the activation of antioxidant response
element-dependent genes. Mol Cell Biol 25: 8044–8051, 2005.
58. Kobayashi A, Kang MI, Okawa H, Ohtsuji M, Zenke Y,
Chiba T, Igarashi K, and Yamamoto M. Oxidative stress
sensor Keap1 functions as an adaptor for Cul3-based E3
ligase to regulate proteasomal degradation of Nrf2. Mol Cell
Biol 24: 7130–7139, 2004.
59. Kobayashi S, Nojima Y, Shibuya M, and Maru Y. Nox1
regulates apoptosis and potentially stimulates branching
morphogenesis in sinusoidal endothelial cells. Exp Cell Res
300: 455–462, 2004.
60. Lalucque H and Silar P. NADPH oxidase: an enzyme for
multicellularity? Trends Microbiol 11: 9–12, 2003.
61. Lambeth JD. NOX enzymes and the biology of reactive
oxygen. Nat Rev Immunol 4: 181–189, 2004.
62. Lander HM, Hajjar DP, Hempstead BL, Mirza UA, Chait
BT, Campbell S, and Quilliam LA. A molecular redox
TARGETS AND COMPARTAMENTALIZATION IN NOX SIGNALING2477
switch on p21(ras): structural basis for the nitric oxide-
p21(ras) interaction. J Biol Chem 272: 4323–4326, 1997.
63. Lander HM, Ogiste JS, Pearce SF, Levi R, and Novogrodsky
A. Nitric oxide-stimulated guanine nucleotide exchange on
p21ras. J Biol Chem 270: 7017–7020, 1995.
64. Lardy B, Bof M, Aubry L, Paclet MH, Morel F, Satre M, and
Klein G. NADPH oxidase homologs are required for nor-
mal cell differentiation and morphogenesis in Dictyostelium
discoideum. Biochim Biophys Acta 1744: 199–212, 2005.
65. Lassegue B, Sorescu D, Szocs K, Yin Q, Akers M, Zhang Y,
Grant SL, Lambeth JD, and Griendling KK. Novel
gp91(phox) homologues in vascular smooth muscle cells:
nox1 mediates angiotensin II-induced superoxide formation
and redox-sensitive signaling pathways. Circ Res 88: 888–
66. Lee JK, Edderkaoui M, Truong P, Ohno I, Jang KT, Berti A,
Pandol SJ, and Gukovskaya AS. NADPH oxidase promotes
pancreatic cancer cell survival via inhibiting JAK2 de-
phosphorylation by tyrosine phosphatases. Gastroenterology
133: 1637–1648, 2007.
67. Lee SR, Kwon KS, Kim SR, and Rhee SG. Reversible inac-
tivation of protein-tyrosine phosphatase 1B in A431 cells
stimulated with epidermal growth factor. J Biol Chem 273:
68. Li JM and Shah AM. Intracellular localization and pre-
assembly of the NADPH oxidase complex in cultured en-
dothelial cells. J Biol Chem 277: 19952–19960, 2002.
69. Li Q, Harraz MM, Zhou W, Zhang LN, Ding W, Zhang Y,
Eggleston T, Yeaman C, Banfi B, and Engelhardt JF. Nox2
and Rac1 regulate H2O2-dependent recruitment of TRAF6
to endosomal interleukin-1 receptor complexes. Mol Cell
Biol 26: 140–154, 2006.
70. Linnane AW, Kios M, and Vitetta L. Healthy aging: regu-
lation of the metabolome by cellular redox modulation and
prooxidant signaling systems: the essential roles of super-
oxide anion and hydrogen peroxide. Biogerontology 8: 445–
71. Liu H, Zhang H, Iles KE, Rinna A, Merrill G, Yodoi J,
Torres M, and Forman HJ. The ADP-stimulated NADPH
oxidase activates the ASK-1=MKK4=JNK pathway in alve-
olar macrophages. Free Radic Res 40: 865–874, 2006.
72. Lohse DL, Denu JM, Santoro N, and Dixon JE. Roles of
aspartic acid-181 and serine-222 in intermediate forma-
tion and hydrolysis of the mammalian protein-tyrosine-
phosphatase PTP1. Biochemistry 36: 4568–4575, 1997.
73. Mahadev K, Motoshima H, Wu X, Ruddy JM, Arnold RS,
Cheng G, Lambeth JD, and Goldstein BJ. The NAD(P)H
oxidase homolog Nox4 modulates insulin-stimulated gen-
eration of H2O2and plays an integral role in insulin signal
transduction. Mol Cell Biol 24: 1844–1854, 2004.
74. Mallis RJ, Buss JE, and Thomas JA. Oxidative modification
of H-ras: S-thiolation and S-nitrosylation of reactive cyste-
ines. Biochem J 355: 145–153, 2001.
75. Mateer SC, Wang N, and Bloom GS. IQGAPs: integrators of
the cytoskeleton, cell adhesion machinery, and signaling
networks. Cell Motil Cytoskeleton 55: 147–155, 2003.
76. Matsuzawa A and Ichijo H. Redox control of cell fate by
MAP kinase: physiological roles of ASK1-MAP kinase
pathway in stress signaling. Biochim Biophys Acta 1780:
77. Mehta D, George SJ, Jeremy JY, Izzat MB, Southgate KM,
Bryan AJ, Newby AC, and Angelini GD. External stent-
ing reduces long-term medial and neointimal thickening
and platelet derived growth factor expression in a pig
model of arteriovenous bypass grafting. Nat Med 4: 235–
78. Mehta PK and Griendling KK. Angiotensin II cell signaling:
physiological and pathological effects in the cardiovascular
system. Am J Physiol Cell Physiol 292: C82–C97, 2007.
79. Meng TC, Fukada T, and Tonks NK. Reversible oxidation
and inactivation of protein tyrosine phosphatases in vivo.
Mol Cell 9: 387–399, 2002.
80. Miller FJ Jr, Filali M, Huss GJ, Stanic B, Chamseddine A,
Barna TJ, and Lamb FS. Cytokine activation of nuclear
factor kappa B in vascular smooth muscle cells requires
signaling endosomes containing Nox1 and ClC-3. Circ Res
101: 663–671, 2007.
81. Moldovan L, Moldovan NI, Sohn RH, Parikh SA, and
Goldschmidt-Clermont PJ. Redox changes of cultured en-
dothelial cells and actin dynamics. Circ Res 86: 549–557,
82. Mumbengegwi DR, Li Q, Li C, Bear CE, and Engelhardt JF.
Evidence for a superoxide permeability pathway in en-
dosomal membranes. Mol Cell Biol 28: 3700–3712, 2008.
83. Nakashima I, Takeda K, Kawamoto Y, Okuno Y, Kato M,
and Suzuki H. Redox control of catalytic activities of
membrane-associated protein tyrosine kinases. Arch Bio-
chem Biophys 434: 3–10, 2005.
84. Nauseef WM. Assembly of the phagocyte NADPH oxidase.
Histochem Cell Biol 122: 277–291, 2004.
85. Nobes CD and Hall A. Rho, rac, and cdc42 GTPases reg-
ulate the assembly of multimolecular focal complexes
associated with actin stress fibers, lamellipodia, and filo-
podia. Cell 81: 53–62, 1995.
86. Papaiahgari S, Kleeberger SR, Cho HY, Kalvakolanu DV,
and Reddy SP. NADPH oxidase and ERK signaling regu-
lates hyperoxia-induced Nrf2-ARE transcriptional response
in pulmonary epithelial cells. J Biol Chem 279: 42302–42312,
87. Parton RG and Simons K. The multiple faces of caveolae.
Nat Rev Mol Cell Biol 8: 185–194, 2007.
88. Pawson T and Nash P. Assembly of cell regulatory systems
through protein interaction domains. Science 300: 445–452,
89. Phalen TJ, Weirather K, Deming PB, Anathy V, Howe AK,
van der Vliet A, Jonsson TJ, Poole LB, and Heintz NH.
Oxidation state governs structural transitions in peroxi-
redoxin II that correlate with cell cycle arrest and recovery.
J Cell Biol 175: 779–789, 2006.
90. Piccoli C, Ria R, Scrima R, Cela O, D’Aprile A, Boffoli D,
Falzetti F, Tabilio A, and Capitanio N. Characterization of
mitochondrial and extra-mitochondrial oxygen consuming
reactions in human hematopoietic stem cells: novel evi-
dence of the occurrence of NAD(P)H oxidase activity. J Biol
Chem 280: 26467–26476, 2005.
91. Pimentel DR, Adachi T, Ido Y, Heibeck T, Jiang B, Lee Y,
Melendez JA, Cohen RA, and Colucci WS. Strain-
stimulated hypertrophy in cardiac myocytes is mediated by
reactive oxygen species-dependent Ras S-glutathiolation.
J Mol Cell Cardiol 41: 613–622, 2006.
92. Pryor WA. Oxy-radicals and related species: their forma-
tion, lifetimes, and reactions. Annu Rev Physiol 48: 657–667,
93. Regnier CH, Masson R, Kedinger V, Textoris J, Stoll I,
Chenard MP, Dierich A, Tomasetto C, and Rio MC. Im-
paired neural tube closure, axial skeleton malformations,
and tracheal ring disruption in TRAF4-deficient mice. Proc
Natl Acad Sci U S A 99: 5585–5590, 2002.
2478 CHEN ET AL.
94. Rhee SG Kang SW, Jeong W, Chang TS, Yang KS, and Woo
HA. Intracellular messenger function of hydrogen peroxide
and its regulation by peroxiredoxins. Curr Opin Cell Biol 17:
95. Roach T, Slater S, Koval M, White L, Cahir McFarland ED,
Okumura M, Thomas M, and Brown E. CD45 regulates
Src family member kinase activity associated with macro-
phage integrin-mediated adhesion. Curr Biol 7: 408–417,
96. Rottner K, Hall A, and Small JV. Interplay between Rac and
Rho in the control of substrate contact dynamics. Curr Biol
9: 640–648, 1999.
97. Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K,
Sawada Y, Kawabata M, Miyazono K, and Ichijo H.
Mammalian thioredoxin is a direct inhibitor of apoptosis
signal-regulating kinase (ASK) 1. EMBO J 17: 2596–2606,
98. Salmeen A, Andersen JN, Myers MP, Meng TC, Hinks JA,
Tonks NK, and Barford D. Redox regulation of protein
tyrosine phosphatase 1B involves a sulphenyl-amide in-
termediate. Nature 423: 769–773, 2003.
99. Schlessinger J. Cell signaling by receptor tyrosine kinases.
Cell 103: 211–225, 2000.
100. Sekhar KR, Crooks PA, Sonar VN, Friedman DB, Chan JY,
Meredith MJ, Starnes JH, Kelton KR, Summar SR, Sasi S,
and Freeman ML. NADPH oxidase activity is essential for
Keap1=Nrf2-mediated induction of GCLC in response to
101. Sharma P, Chakraborty R, Wang L, Min B, Tremblay ML,
Kawahara T, Lambeth JD, and Haque SJ. Redox regu-
lation of interleukin-4 signaling. Immunity 29: 551–564,
102. Simons K and Ikonen E. Functional rafts in cell membranes.
Nature 387: 569–572, 1997.
103. Stocker R and Keaney JF Jr. Role of oxidative modifications
in atherosclerosis. Physiol Rev 84: 1381–1478, 2004.
104. Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D,
Chung AB, Griendling KK, and Lambeth JD. Cell trans-
formation by the superoxide-generating oxidase Mox1.
Nature 401: 79–82, 1999.
105. Sumimoto H, Miyano K, and Takeya R. Molecular com-
position and regulation of the Nox family NAD(P)H oxi-
dases. Biochem Biophys Res Commun 338: 677–686, 2005.
106. Sundaresan M, Yu ZX, Ferrans VJ, Irani K, and Finkel T.
Requirement for generation of H2O2for platelet-derived
growth factor signal transduction. Science 270: 296–299,
107. Szanto I, Rubbia-Brandt L, Kiss P, Steger K, Banfi B, Kovari
E, Herrmann F, Hadengue A, and Krause KH. Expression
of NOX1, a superoxide-generating NADPH oxidase, in
colon cancer and inflammatory bowel disease. J Pathol 207:
108. Tejada-Simon MV, Serrano F, Villasana LE, Kanterewicz BI,
Wu GY, Quinn MT, and Klann E. Synaptic localization of a
functional NADPH oxidase in the mouse hippocampus.
Mol Cell Neurosci 29: 97–106, 2005.
109. Tobiume K, Saitoh M, and Ichijo H. Activation of apoptosis
signal-regulating kinase 1 by the stress-induced activating
phosphorylation of pre-formed oligomer. J Cell Physiol 191:
110. Touyz RM, Yao G, Quinn MT, Pagano PJ, and Schiffrin EL.
p47phox associates with the cytoskeleton through cortactin
in human vascular smooth muscle cells: role in NAD(P)H
oxidase regulation by angiotensin II. Arterioscler Thromb
Vasc Biol 25: 512–518, 2005.
111. Ueno N, Takeya R, Miyano K, Kikuchi H, and Sumimoto
H. The NADPH oxidase Nox3 constitutively produces su-
peroxide in a p22phox-dependent manner: its regulation by
oxidase organizers and activators. J Biol Chem 280: 23328–
112. Ueyama T, Geiszt M, and Leto TL. Involvement of Rac1 in
activation of multicomponent Nox1- and Nox3-based
NADPH oxidases. Mol Cell Biol 26: 2160–2174, 2006.
113. Ushio-Fukai M, Tang Y, Fukai T, Dikalov SI, Ma Y, Fuji-
moto M, Quinn MT, Pagano PJ, Johnson C, and Alexander
RW. Novel role of gp91(phox)-containing NAD(P)H oxi-
dase in vascular endothelial growth factor-induced signal-
ing and angiogenesis. Circ Res 91: 1160–1167, 2002.
114. Vallet P, Charnay Y, Steger K, Ogier-Denis E, Kovari E,
Herrmann F, Michel JP, and Szanto I. Neuronal expression
of the NADPH oxidase NOX4, and its regulation in mouse
experimental brain ischemia. Neuroscience 132: 233–238,
115. Wientjes FB, Reeves EP, Soskic V, Furthmayr H, and Segal
AW. The NADPH oxidase components p47(phox) and
p40(phox) bind to moesin through their PX domain. Bio-
chem Biophys Res Commun 289: 382–388, 2001.
116. Wood ZA, Poole LB, and Karplus PA. Peroxiredoxin evo-
lution and the regulation of hydrogen peroxide signaling.
Science 300: 650–653, 2003.
117. Wu RF, Gu Y, Xu YC, Nwariaku FE, and Terada LS. Vas-
cular endothelial growth factor causes translocation of
p47phox to membrane ruffles through WAVE1. J Biol Chem
278: 36830–36840, 2003.
118. Wu RF, Xu YC, Ma Z, Nwariaku FE, Sarosi GA Jr, and
Terada LS. Subcellular targeting of oxidants during endo-
thelial cell migration. J Cell Biol 171: 893–904, 2005.
119. Xu YC, Wu RF, Gu Y, Yang YS, Yang MC, Nwariaku FE,
and Terada LS. Involvement of TRAF4 in oxidative acti-
vation of c-Jun N-terminal kinase. J Biol Chem 277: 28051–
120. Yamamoto T, Suzuki T, Kobayashi A, Wakabayashi J,
Maher J, Motohashi H, and Yamamoto M. Physiological
significance of reactive cysteine residues of Keap1 in de-
termining Nrf2 activity. Mol Cell Biol 28: 2758–2770, 2008.
121. Yan SR and Berton G. Regulation of Src family tyrosine ki-
nase activities in adherent human neutrophils: evidence that
reactive oxygen intermediates produced by adherent neu-
trophils increase the activity of the p58c-fgr and p53=56lyn
tyrosine kinases. J Biol Chem 271: 23464–23471, 1996.
122. Yan SR, Fumagalli L, Dusi S, and Berton G. Tumor necrosis
factor triggers redistribution to a Triton X-100-insoluble,
cytoskeletal fraction of beta 2 integrins, NADPH oxidase
components, tyrosine phosphorylated proteins, and the
protein tyrosine kinase p58fgr in human neutrophils ad-
herent to fibrinogen. J Leukoc Biol 58: 595–606, 1995.
123. Yang B and Rizzo V. TNF-alpha potentiates protein-
tyrosine nitration through activation of NADPH oxidase
and eNOS localized in membrane rafts and caveolae of
bovine aortic endothelial cells. Am J Physiol Heart Circ
Physiol 292: H954–H962, 2007.
124. Yang CS, Lee DS, Song CH, An SJ, Li S, Kim JM, Kim CS,
Yoo DG, Jeon BH, Yang HY, Lee TH, Lee ZW, El-Benna J,
Yu DY, and Jo EK. Roles of peroxiredoxin II in the regu-
lation of proinflammatory responses to LPS and protection
against endotoxin-induced lethal shock. J Exp Med 204:
TARGETS AND COMPARTAMENTALIZATION IN NOX SIGNALING2479
125. Yang S, Madyastha P, Bingel S, Ries W, and Key L. A new Download full-text
superoxide-generating oxidase in murine osteoclasts. J Biol
Chem 276: 5452–5458, 2001.
126. Zhang DD, Lo SC, Cross JV, Templeton DJ, and Hannink
M. Keap1 is a redox-regulated substrate adaptor protein for
a Cul3-dependent ubiquitin ligase complex. Mol Cell Biol
24: 10941–10953, 2004.
127. Zhang ZY and Dixon JE. Active site labeling of the Yersinia
protein tyrosine phosphatase: the determination of the pKa
of the active site cysteine and the function of the conserved
histidine 402. Biochemistry 32: 9340–9345, 1993.
128. Zor U, Ferber E, Gergely P, Szucs K, Dombradi V, and
Goldman R. Reactive oxygen species mediate phorbol
ester-regulated tyrosine phosphorylation and phospholi-
pase A2 activation: potentiation by vanadate. Biochem J 295:
129. Zuo L, Ushio-Fukai M, Ikeda S, Hilenski L, Patrushev N,
and Alexander RW. Caveolin-1 is essential for activation of
Rac1 and NAD(P)H oxidase after angiotensin II type 1 re-
ceptor stimulation in vascular smooth muscle cells: role in
redox signaling and vascular hypertrophy. Arterioscler
Thromb Vasc Biol 25: 1824–1830, 2005.
Address correspondence to:
University of Massachusetts Medical School
Department of Medicine=Cardiovascular
381 Plantation St.
Biotech 5, Suite 200
Worcester, MA 01605
Date of first submission to ARS Central, March 19, 2009; date
of acceptance, March 22, 2009.
Abl¼Abelson tyrosine kinase
ASK-1¼apoptosis signaling kinase-1
AT1R¼angiotensin II receptor type I
cAMP¼cyclic adenosine monophosphate
cGMP¼cyclic guanosine monophosphate
CLIP-170¼CAP-Gly domain-containing linker
EGF¼epidermal growth factor
Hic5¼hydrogen peroxide-induced clone 5
IQGAP1¼IQ motif containing GTPase-
activating protein 1
JAK2¼Janus kinase 2
Keap1¼Kelch-like ECH-associated protein 1
Lck¼lymphocyte-specific protein tyrosine
NF-kB¼nuclear factor kB
NoxA1¼Nox activator 1
NoxO1¼Nox organizer 1
PDGF¼platelet-derived growth factor
PDIs¼protein disulfide isomerases
PTKs¼protein tyrosine kinases
PTPs¼protein tyrosine phosphatases
PTP SHP-2¼Src homology protein tyrosine
Rac1¼Ras-related C3 botulinum toxin
ROS¼reactive oxygen species
TNF¼tumor necrosis factor
TRAF4¼TNF receptor–associated factor
TRP14¼thioredoxin-related protein 14
2480 CHEN ET AL.