G Protein ␤␥ Subunits Stimulate p114RhoGEF, a Guanine
Nucleotide Exchange Factor for RhoA and Rac1
Regulation of Cell Shape and Reactive Oxygen Species Production
Jiaxin Niu, Jasmina Profirovic, Haiyun Pan, Rita Vaiskunaite, Tatyana Voyno-Yasenetskaya
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Abstract—Rho GTPases integrate the intracellular signaling in a wide range of cellular processes. Activation of these G
proteins is tightly controlled by a number of guanine nucleotide exchange factors (GEFs). In this study, we addressed
the functional role of the recently identified p114RhoGEF in in vivo experiments. Activation of endogenous G
protein– coupled receptors with lysophosphatidic acid resulted in activation of a transcription factor, serum response
element (SRE), that was enhanced by p114RhoGEF. This stimulation was inhibited by the functional scavenger of G␤␥
subunits, transducin. We have determined that G␤␥ subunits but not G␣ subunits of heterotrimeric G proteins stimulated
p114RhoGEF– dependent SRE activity. Using coimmunoprecipitation assay, we have determined that G␤␥ subunits
interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, G␤␥ subunits stimulated SRE activity
induced by full-length and DH/PH domain of p114RhoGEF. Using in vivo pull-down assays and dominant-negative
mutants of Rho GTPases, we have determined that p114RhoGEF activated RhoA and Rac1 but not Cdc42 proteins.
Functional significance of RhoA activation was established by the ability of p114RhoGEF to induce actin stress fibers
and cell rounding. Functional significance of Rac1 activation was established by the ability of p114RhoGEF to induce
production of reactive oxygen species (ROS) followed by activation of NADPH oxidase enzyme complex. In summary,
our data showed that the novel guanine nucleotide exchange factor p114RhoGEF regulates the activity of RhoA and
Rac1, and that G␤␥ subunits of heterotrimeric G proteins are activators of p114RhoGEF under physiological conditions.
The findings help to explain the integrated effects of LPA and other G-protein receptor– coupled agonists on actin stress
fiber formation, cell shape change, and ROS production. (Circ Res. 2003;93:848-856.)
Key Words: Rho GTPases 䡲 guanine nucleotide exchange factor 䡲 actin cytoskeleton
䡲 serum response element 䡲 reactive oxygen species
G
uanine nucleotide exchange factors of the Dbl family are
multifunctional proteins that transduce diverse intracellular signals leading to the activation of Rho GTPases (see
review1). The tandem Dbl homology (DH) and pleckstrin
homology (PH) domains are shared by all members of this
family and represent a structural module responsible for the
catalyzing the GDP-GTP exchange of Rho proteins and,
therefore, their activation. Previous studies have shown that
the DH domain is responsible for the catalytic core of the
RhoGEF enzymatic activity,2 whereas the PH domain is
involved in intracellular targeting, lipid-binding, and protein
interaction.1,3 Furthermore, guanine nucleotide exchange factors (GEFs) may contain other conserved motifs such as RGS
(regulator of G-protein signaling) motif, SH3 (Src homology)
motif, and proline-rich SH3-binding motif,1 thereby supplying RhoGEFs with additional signaling functions.
Diverse upstream signals that stimulate RhoGEFs catalytic
activity include heterotrimeric G proteins, protein kinases,
adaptor molecules, and phospholipids.1 Lysophosphatidic
acid (LPA), bombesin, bradykinin, and other G-protein–
coupled receptor agonists can stimulate Rho pathways.4,5
Both G␣ and G␤␥ subunits have been implicated in activation of individual RhoGEFs. Thus, it was shown that G␣13
could activate several GEFs: p115RhoGEF, LARG, and
PDZ-RhoGEF.5–7 Similarly, G␤␥ subunits were shown to
activate Ras-GRF1/CDC25 and P-PEX1 exchange factors.8,9
Rho GTPases are members of Ras superfamily of monomeric 20 to 30 kDa GTP-binding proteins or small GTPases.4
The best-characterized Rho GTPases are RhoA, Rac1, and
Cdc42 play an important role in the regulation of actin
cytoskeleton.4
It is well established that Rac proteins play an important
role in the regulation of reactive oxygen species (ROS)
formation.10 –12 Multiprotein NADPH oxidase consisting of
p47phox, p67phox, gp91phox, and gp22phox is activated by Rac to
produce ROS in phagocytic leukocytes and other nonphagocytic cells.13
Original received April 14, 2003; revision received September 15, 2003; accepted September 16, 2003.
From the Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, Ill.
Correspondence to Tatyana Voyno-Yasenetskaya, University of Illinois, Department of Pharmacology (MC 868), 835 S Wolcott Ave, Chicago, IL
60612. E-mail [email protected]
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000097607.14733.0C
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p114RhoGEF Regulates Rho and Rac Function
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In this study, we report that p114RhoGEF (former KIAA
0521) is a novel guanine nucleotide exchange factor for
RhoA and Rac1 that can be activated by G␤␥ subunits of
heterotrimeric G proteins. In addition, we have shown that
p114RhoGEF is involved in the regulation of actin stress
fibers, cell shape, activation of NADPH oxidase, and ROS
formation.
Materials and Methods
Details concerning cDNA and reagents, Northern blot analysis,
RT-PCR, cell culture and transient transfection of cDNA, and crude
membrane preparation are described in the expanded Materials and
Methods section in the online data supplement available at http://
circresaha.org, along with further details for all procedures.
Immunoprecipitation and Western Blotting
Immunoprecipitation and Western blotting were performed as described previously.14
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Fluorescent Microscopy
Fluorescent microscopy was performed as described previously.14
In Vivo Rho GTPases Activation Assay
Activation of Rho proteins in vivo were determined by using recently
developed pull-down assays as described previously.15
Reporter Gene Assay
Serum response element (SRE)–mediated gene expression was
determined by SRE.L reporter system (Stratagene) as described
previously.15
Detection of ROS Generation by Fluorescence
Microscope and Flow Cytometry
Oxidant generation in NIH3T3 cells was measured as described.16
Results
Thrombin and LPA Stimulate p114RhoGEF via
G␤␥ Subunits
To determine the tissue distribution of p114RhoGEF, a
human multiple tissue Northern blot (Clontech) was hybridized with cDNA probe nt 90 to 683 of p114RhoGEF labeled
with [␣-32P]dCTP, as shown in Figure 1A. We have detected
the mRNA transcript of approximately 7.0 kb in every tissue
in the blot. Interestingly, an approximately 5.0-kb transcript
was detected in smooth muscle and possibly placenta, suggesting that splice variant of p114RhoGEF may exist. Because cDNA probe used for the Northern blotting was
targeted to the N-terminal domain of the protein (90 to 683
bp), one possibility could be that the splice variant may be
short of the C-terminal domain. This result was further
confirmed using RT-PCR analysis of total RNA preparations
isolated from 7 different cell lines (Figure 1B). We have
detected the expression of p114RhoGEF in 5 out of 7 cell
lines, which was consistent with the Northern blot analysis,
indicating that p114RhoGEF is a widely expressed in different tissues or cells.
Because p114RhoGEF could be activated by extracellular
signals acting via G protein– coupled receptors,17 we examined the identity of G proteins that mediate the p114RhoGEFdependent activation of SRE-mediated transcription of a
luciferase reporter gene.18 NIH3T3 cells were transiently
transfected with p114RhoGEF and SRE-driven luciferase
Figure 1. Distribution of p114RhoGEF in tissues and cell lines.
A, Northern blot analysis of a human multiple tissues blot. cDNA
nucleotide sequence 90 to 683 of p114RhoGEF was used as a
probe. B, RT-PCR analysis of different cell lines. Gene-specific
cDNA primers of p114RhoGEF or G␣13 were used as a probes
to detect expression of p114RhoGEF or G␣13. HL-60 indicates
promyelocytic cell line; THP-1, human monocyte cells; U-937,
histocytic lymphoma; NIH3T3, mouse fibroblasts; HLMVE,
human lung microvascular endothelial cells; HEK-293, human
kidney embryonic cells; and SMG, spermatogonia cell line.
reporter. To correct for variations in transfection efficiency,
an expression vector coding for ␤-galactosidase was cotransfected with the above constructs, and the expressed
␤-galactosidase activity was used for normalization of SRE
luciferase data. The data showed that stimulation of the cells
with LPA induced SRE activation (Figure 2A). Similarly,
p114RhoGEF transiently expressed in the cells induced
⬇2-fold higher SRE activation (Figure 2A). Importantly,
both LPA strongly potentiated SRE activity in the presence of
p114RhoGEF, suggesting that LPA can stimulate
p114RhoGEF.
Because G protein– coupled receptors activated by LPA
could activate multiple heterotrimeric G proteins, including
G␣12, G␣13, G␣q, and G␣i, we determined whether any of
the individual G protein subunits could activate
p114RhoGEF. In order to examine the involvement of the
specific G proteins in the p114RhoGEF signaling,
p114RhoGEF was cotransfected into NIH3T3 cells with
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constitutively active GTPase-deficient mutants of G␣13,
G␣12, G␣q, and G␣i2. Without p114RhoGEF transfection,
activated mutants of G␣13, G␣12, and G␣q induced SRE
activation (Figure 2B), which was consistent with previously
reported data.19 G␣i2 did not affect SRE activity. Cotransfection of p114RhoGEF with indicated G␣ subunits did not
result in additional potentiation of SRE activity (Figure 2B),
suggesting that involvement of G␣ subunits in the regulation
of p114RhoGEF is unlikely. In a positive control experiment
(Figure 2C), G␣13-induced SRE activation was strongly
potentiated in the cells expressing p115RhoGEF, a guanine
nucleotide exchange factor known to be activated by G␣13.5
This result demonstrates that G␣ subunit could activate a
suitable GEF.
Because receptor stimulation of heterotrimeric G proteins
results in a release of G␤␥ subunits that are capable of
stimulation of downstream signaling pathways, we have also
analyzed the effect of G␤1 and G␥2 subunits, the best
characterized G␤␥ combination20,21 on p114RhoGEFinduced SRE activation. Coexpression of plasmids encoding
G␤1 and G␥2 isoforms strongly enhanced the p114RhoGEFdependent activation of SRE (Figure 2B). However, neither
G␤1 nor G␥2 alone affected p114RhoGEF-dependent activation of SRE (data not shown). Further, the functional G␤␥
subunits scavenger, transducin,22 inhibited both LPAdependent and G␤1␥2-dependent potentiation of SRE activity induced by p114RhoGEF (Figure 2A). This result indicated that both exogenous and endogenous G␤␥ subunits
could activate p114RhoGEF.
G␤␥ Subunits Stimulate p114RhoGEF via
Interaction With Its DH/PH Domain
Because DH/PH domains are the hallmarks of RhoGEF
proteins1 and the G␤␥ subunits have been shown to interact
with PH domains of other signaling molecules,23 we hypothesized that G␤1␥2 subunits mediate their effect via interaction with DH/PH domain of p114RhoGEF. To test this
hypothesis, we have constructed the following p114RhoGEF
constructs tagged with Myc epitope: DH/PH domain (aa 1 to
492), extended DH/PH domain (aa 1 to 647), deletion of
proline cluster only (1 to 952), deletion of DH/PH domain
(C-terminal domain, aa 648 to 1015), and deletion of DH/PH
Figure 2. Agonist-induced activation of SRE via p114RhoGEF is
mediated by G␤1␥2 subunits of heterotrimeric G proteins. A,
Agonist-induced activation of SRE via p114RhoGEF. NIH3T3
cells seeded onto 24-well plates were transfected with 50 ng
per well of cDNAs encoding for pcDNA3 (vector), p114RhoGEF,
pSRE.L, and pCMV-␤gal. Some wells were transfected with 100
ng of transducin as indicated. Twenty-four hours after transfection, cells were serum-starved for 16 hours and stimulated with
1 ␮mol/L LPA or 1 U/mL thrombin for 6 hours. Thereafter, activity of SRE was determined as described in Materials and Methods. Presented data are the mean⫾SEM from three independent experiments. B, G␤␥ subunits are functionally coupled to
p114RhoGEF to induce SRE activation. 50 ng of each construct
(G␣13Q226L, G␣12Q229L, G␣qR145C, G␣i2Q205L, G␤1, G␥2,
and p114RhoGEF) was transfected for SRE measurement. Presented data are the mean⫾SEM from three independent experiments. C, SRE activation induced by G␣13Q226L is potentiated
by p115RhoGEF. NIH3T3 cells were transfected with 50 ng
pSRE.L, 50 ng pCMV-␤gal, and 100 ng of indicated constructs.
Samples from parallel transfection were used for Western blotting analysis with antibodies specific to G␣ subunits or with Myc
antibody to determine the expression of p114RhoGEF or p115
RhoGEF.
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Figure 3. G␤1␥2 subunits interacted with
p114RhoGEF. A, Schematic representation of
the domain structure of p114RhoGEF and its
deletion mutants. B, Coimmunoprecipitation of
p114RhoGEF with G␤1␥2 subunits. NIH3T3
cells grown in 10-cm2 dishes were transiently
transfected with vector alone or various Myctagged constructs of p114RhoGEF together
with Flag-tagged G␤1 subunit and G␥2 subunit.
Forty-eight hours after transfection, cells were
lysed and equal amount of cell lysates were
immunoprecipitated with agarose-conjugated
anti-Myc antibodies. Immunoprecipitates and
cell lysates were analyzed by Western blotting
using antibodies against Myc or Flag. C, G␤1␥2
subunits induced SRE activation via DH/PH
domain. 50 ng of each indicated construct was
transfected for SRE measurement. Presented
data are the mean⫾SEM from three independent experiments.
domain and proline cluster (aa 648 to 952) (Figure 3A).
NIH3T3 cells were transfected with p114RhoGEF constructs
together with Flag-tagged G ␤ 1 and G ␥ 2 subunits.
p114RhoGEF polypeptides were immunoprecipitated from
cell lysates using Myc antibody and presence of G␤ subunit
was determined using Flag antibody. Data showed that G␤1
subunit interacted with full-length p114RhoGEF, DH-PH
domain, and C-terminal domain (Figure 3B). Apparently,
lower G␤1 binding to full-length p114RhoGEF was due to
the lower expression of the full-length p114RhoGEF. We
concluded that G␤1 subunit may have two binding sites on
p114RhoGEF because it was able to interact with both
N-terminal and C-terminal regions of p114RhoGEF (Figure
3B). The function of a proline cluster remained unknown. We
then determined which G␤1-binding domain of p114RhoGEF
is required for G␤1␥2-mediated signaling. Data showed that
SRE activation by DH/PH domain of the p114RhoGEF was
further enhanced by G␤1␥2 subunits, whereas C-terminal
domains of p114RhoGEF did not affect SRE activity (Figure
3C).
p114RhoGEF Activates RhoA and Rac1 but not
Cdc42 Proteins
To evaluate the effect of p114RhoGEF on activation of
individual Rho GTPases, we used Rho-binding domain of
RhoA effector, rhotekin, to affinity precipitate active RhoA,
as a direct readout for RhoA activation. We have also used
Rac1- and Cdc42-binding domain of Rac1 and Cdc42 effector PAK to affinity precipitate active Rac1 and Cdc42 as a
direct readout for Rac1 and Cdc42 activation.
NIH3T3 cells were transfected with either full-length
p114RhoGEF, or the DH/PH domain of p114RhoGEF, constitutively active mutants of RhoA, Rac1, or Cdc42 were used
as a positive control when determining the activation of
appropriate Rho GTPase. Data showed that p114RhoGEF
induced a 3- to 4-fold increase in RhoA and Rac1 activity
(Figures 4A and 4B). Importantly, p114RhoGEF did not
activate Cdc42 (Figure 4C), suggesting the existence of
mechanism to regulate the specificity of p114RhoGEF toward different Rho GTPases. Equal protein expression of
full-length p114RhoGEF was controlled by Western blotting
(data not shown) to confirm that different effect of
p114RhoGEF on activation of Rho GTPases was not due to
difference in the amount of expressed protein. These data
provided the direct evidence of RhoA and Rac1 activation by
p114RhoGEF in mammalian cells.
To further support data of p114RhoGEF-dependent activation of RhoA and Rac1 proteins, we have determined the
involvement of specific Rho GTPases in p114RhoGEF-
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Figure 5. DH/PH domain of p114RhoGEF regulates actin organization. A, Actin stress fiber formation and cell rounding
induced by overexpression p114RhoGEF. NIH3T3 cells were
transfected with pcDNA3, RhoV14, full length, DH/PH, or
C-terminal domain (648 to 1015 aa) of p114RhoGEF and serumstarved for 16 hours before the experiment. Cells were fixed
with 4% paraformaldehyde, permeabilized with Triton X-100,
and double stained with Myc antibody to visualize expressed
proteins and rhodamine-phalloidin to visualize polymerized
actin. B, NIH3T3 cells were cotransfected with indicated constructs. Twenty-four hours after transfection, cells were fixed
and expression of the transfected constructs was determined by
staining with Myc antibody. Polymerized actin was determined
by staining with rhodamine-phalloidin. For each transfection, the
percentage of rounded and stress fiber– containing cells was
calculated from at least 400 Myc-positive cells counted. Presented data are the mean⫾SEM from three independent
experiments.
Figure 4. p114RhoGEF activates RhoA and Rac1 but not Cdc42.
NIH3T3 cells were transfected with 5 ␮g of RhoV14, RacV12,
Cdc42V12, full length, or DH/PH domain of p114RhoGEF as indicated and serum-starved for 24 hours. In vivo Rho GTPase activation assay was performed as described in Materials and Methods.
A, RhoA activity was determined as indicated by the amount of
RBD-bound RhoA (top) normalized to the amount of RhoA in
whole-cell lysates (bottom). Western blots from a representative
experiment showing activation of RhoA in response to
p114RhoGEF or RhoV14. B, Rac1 activity was determined as indicated by the amount of PBD-bound Rac1 (top) normalized to the
amount of Rac1 in whole-cell lysates (bottom). Western blots from
a representative experiment showing activation of Rac1 in
response to p114RhoGEF or RacV12. C, Cdc42 activity was determined as indicated by the amount of PBD-bound Cdc42 (top) normalized to the amount of Cdc42 in whole-cell lysates (bottom).
Western blots from a representative experiment showing activation
of Cdc42 in response to p114RhoGEF or Cdc42V12. D,
p114RhoGEF activates serum response element via RhoA and
Rac1 but not Cdc42 proteins. NIH3T3 cells seeded onto 24-well
plates were transfected with 50 ng pSRE.L, 50 ng pCMV-␤gal, and
100 ng of pcDNA3 vector, 100 ng of p114RhoGEF, and 200 ng
each of dominant-negative RhoA, Rac1, and Cdc42 as indicated.
Twenty-four hours after transfection, cells were serum-starved for
16 hours and activity of SRE was determined as described above.
Presented data are the mean⫾SEM from three independent
experiments.
dependent SRE activation. Because RhoA, Rac1, and Cdc42
are known to activate SRE,18 we have used dominantnegative mutants of these GTPases to inhibit p114RhoGEFinduced SRE activation. Importantly, dominant-negative mutants of RhoA and Rac1 but not Cdc42 inhibited SRE
activation induced by p114RhoGEF (Figure 4D). Thus, these
data further corroborated our finding that p114RhoGEF
regulated activity of RhoA and Rac1 but not Cdc42 proteins.
DH/PH Domain of p114RhoGEF Regulates
Actin Organization
As Rho protein is known to regulate actin stress fiber
formation and cell rounding,4 we analyzed the role of
p114RhoGEF and its domains in actin reorganization. To
perform these studies, NIH3T3 cells were transfected with
p114RhoGEF or its domains and stained with rhodaminephalloidin to determine the actin organization. To detect
changes in actin morphology and cell shape, cells expressing
Myc-tagged p114RhoGEF constructs were identified by detection with Myc antibody. Cells were either scored as
containing actin stress fibers without changes of the cell
shape or as rounded containing actin stress fibers. For each
transfection, the percentage of rounded and stress fiber–
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containing cells was calculated from at least 400 Mycpositive cells counted. In 40% of the cells expressing fulllength p114RhoGEF, actin stress fiber were detected; 50% of
the actin stress fiber-containing cells were also rounded
(Figures 5A and 5B). Importantly, DH/PH domain and
extended DH/PH domain induced actin cytoskeleton reorganization in almost 100% of the cells (Figures 5A and 5B),
suggesting that C-terminal domain may inhibit p114RhoGEF
function. Mutant with deleted proline cluster induced actin
cytoskeleton reorganization in ⬇80% of the transfected cells.
Finally, mutant with deleted DH/PH domain (C-terminal
domain) did not affect actin cytoskeleton (Figure 5A). Together, these data showed that DH/PH domain of
p114RhoGEF is involved in the cell rounding and actin stress
fiber formation.
p114RhoGEF Induces Reactive Oxygen Species
(ROS) Formation
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Activated Rac proteins are known to induce ROS formation
in nonphagocytic cells, such as NH 3T3 fibroblasts,10 via
activation of NADPH oxidase.11 To determine the physiological significance of p114RhoGEF-induced activation of Rac1,
we have analyzed if p114RhoGEF could induce ROS formation. We used the fluorescent redox-sensitive dye carboxy-H2
2⬘,7⬘-dichlorofluorescin diacetate (DCFDA) to determine if
p114RhoGEF could induce oxidant generation. To confirm
that NIH3T3 cells are capable of producing reactive oxygen
species, cells were challenged with tumor necrosis factor,
TNF-␣, a recognized ROS inducer24 for 15 minutes to allow
maximum oxidant accumulation during this period. Control
cells showed little fluorescence. In contrast, TNF-␣ induced
marked oxidant generation (Figure 6A). We then determined
if activated Rac (RacV12) could induce ROS formation in
NIH3T3 cells. NIH3T3 cells were transfected with constitutively active mutants of Rac1 and RhoA. Twenty-four hours
after transfection, cells were incubated with DCFDA for 20
minutes at 37°C, washed 3 times with cold phosphatebuffered saline (PBS), and observed under fluorescence
microscope. Data showed that the cells expressing activated
Rac1 also demonstrated the generation of ROS (Figure 6A),
which was in accordance with previously reported data.10 In
the cells expressing active RhoA, the ROS was not produced
(Figure 6B). Therefore, the experimental conditions used
allowed specific induction of ROS production by Rac1 but
not RhoA.
Finally, the generation of ROS was observed in the cells
expressing p114RhoGEF (Figure 6B). Importantly,
Figure 6. p114RhoGEF induces reactive oxygen species formation. A, TNF-␣–induced generation of ROS. NIH3T3 cells were
loaded with 10 ␮mol/L carboxy-H2DCFDA dye for 20 minutes.
Cells were washed and then stimulated with TNF-␣ for 15 minutes to yield maximum oxidant accumulation. Cells were analyzed by fluorescence microscopy as described in Materials and
Methods. B, NIH3T3 cells were transfected with RacV12,
RhoV14, or constructs of the full-length p114RhoGEF or
C-terminal domain of p114RhoGEF (648 to 1015 aa) as indicated. Twenty-four hours after transfection, cells were incubated
with 10 ␮mol/L fluorophore DCFDA for 20 minutes, and images
were analyzed with fluorescent microscope. Shown are representative image of three independent experiments. C, Flow
cytometric analysis of ROS production in NIH3T3 cells. NIH3T3
cells were seeded in 60-mm plates, transiently transfected with
either pcDNA3 or p114RhoGEF, 24 hours after transfection,
cells were incubated with 10 ␮mol/L of the oxidant sensitive
fluorophore DCFDA for 20 minutes, and the fluorescence intensity of the live cells was analyzed by flow cytometry using excitation and emission wavelength of 488 and 530 nm,
respectively.
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C-terminal peptide that lacked DH/PH domain did not induce
ROS generation. To reinforce the data obtained using fluorescence microscopy, we used flow cytometry to determine
the shift in the fluorescence intensity distribution of DCFDA
due to enhancement of ROS generation by p114RhoGEF
(Figure 6B). NIH 3T3 cells transiently transfected with either
pCDNA3 vector or full-length P114RhoGEF were analyzed
by flow cytometry Beckman Coulter Epics Elite ESP using
excitation and emission wavelength 488 or 530 nm, respectively. The mean DCFDA fluorescence intensity for ROS in
the control cells was 3.11, whereas the mean DCFDA
fluorescence intensity of the p114RhoGEF-expressing cells
was 4.35, showing that p114RhoGEF enhanced the production of ROS.
Production of ROS by the cell is a result of the activation
of NADPH oxidase, a multiprotein enzyme complex. As one
of the hallmarks of NADPH oxidase activation is a translocation of p67phox subunit from the cytoplasm to the membrane,13 we have determined if p114RhoGEF could induce
translocation of p67phox from the cytoplasm to the membrane.
We have constructed the GFP-tagged p67phox subunit and
transiently expressed it in NIH3T3 fibroblasts. In unstimulated cells, GFP-p67phox subunit was localized throughout the
cytoplasm (Figure 7A), verifying that the localization of
GFP-p67phox subunit was similar to the localization of endogenous p67phox subunit. Stimulation of the cells with 1 ␮mol/L
LPA for 10 minutes resulted in translocation of the GFPp67phox subunit to the plasma membrane (Figure 7A). We then
analyzed distribution of p67phox subunit in the cells transfected
with domains of p114RhoGEF. NIH3T3 cells were cotransfected with GFP- p67phox subunit and Myc-tagged constructs
of p114RhoGEF. Localization of p67phox subunit in a Mycpositive cells showed that both full-length p114RhoGEF and
its DH/PH domain induced translocation of the p67phox subunit to the plasma membrane (Figure 7B). Importantly,
C-terminal domain did not affect the intracellular localization
of p67phox subunit.
We next determined the association of p67phox with membrane fraction using Western blotting analysis of total cell
Figure 7. p114RhoGEF induced translocation of p67phox. A,
NIH3T3 (80 000) cells were seeded on the gelatin-precoated
coverslips in 12-well plates and were transiently transfected with
pEGFP-p67phox; 8 hours after transfection, cells were starved for
12 to 16 hours and stimulated with 1 ␮mol/L LPA for 10 minutes. B, NIH3T3 (80 000) cells were seed on the gelatinprecoated coverslips in 12-well plates and were transiently
cotransfected with either 5 ␮g of pCDNA3, the full length, or the
DH/PH domain of p114RhoGEF together with pEGFP-p67phox as
indicated; 8 hours after transfection, cells were starved for 12 to
16 hours. Expression of the DH/PH domain and the full-length
P114RhoGEF was detected by anti-Myc antibodies. Shown is
representative image of three independent experiments. C, Both
DH/PH domain and full-length p114RhoGEF enhanced p67phox
membrane translocation. NIH 3T3 cells seeded onto10-cm
plates were transiently transfected with 5 ␮g pcDNA3, pcDNA3p67phox, Myc-tagged DH/PH domain, Myc-tagged full-length
p114RhoGEF, or HA-tagged RacV12. Twenty-four hours after
transfection, cells were either lysed in RIPA buffer to prepare the
whole-cell lysates or collected in PBS to prepare membrane
fraction as described in detail in Materials and Methods. Crude
membrane and total lysate samples were analyzed by Western
blot using antibodies against p67phox. Bottom panel shows
expression of the DH/PH domain and the full-length
P114RhoGEF detected by anti-Myc antibodies, and the expression of RacV12 detected by HA antibody. E, p114RhoGEF
induced association of p67phox with activated Rac1. NIH 3T3
cells seeded onto10-cm plates were transiently transfected with
5 ␮g pcDNA3, pcDNA3- p67phox, Myc-tagged DH/PH domain,
Myc-tagged full-length p114RhoGEF, or HA-tagged RacV12.
Rac1 activation was determined by the amount of PBD-bound
Rac1 as described previously. The same complexes were
probed with p67phox antibody to determine the association of
p67phox with activated Rac. Shown representative Western blot
of three independent experiments.
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lysates and membrane fractions in the cells expressing vector,
DH-PH domain or full-length p114RhoGEF. Data showed
that both DH-PH domain and full-length p114RhoGEF induced membrane translocation of p67phox (Figure 7C). Importantly, in a positive control experiment, RacV12 also induced
translocation of p67phox subunit to the plasma membrane
fraction.
As binding of Rac to p67phox is a crucial step in the
activation of NADPH oxidase and the interaction of Rac with
p67 phox is strictly GTP-dependent, 12 we analyzed if
p114RhoGEF-induced activation of Rac would result in the
p67phox association with activated fraction of Rac. In this
experiment, lysates of the cells transfected with vector,
DH-PH domain of full-length p114RhoGEF were incubated
with
glutathione-Sepharose
bead-bound
glutathione
S-transferase (GST) fusion protein of the Rac1-binding domain of Rac1 effector PAK. Thereafter, presence of Rac or
p67phox in complex with Rac1-binding domain was analyzed
using Rac1 or p67phox antibodies, respectively (Figure 7D).
Data showed that both DH-PH domain and full-length
p114RhoGEF induced association of activated Rac with the
Rac1-binding domain. In addition, p67phox was also associated
with Rac/Rac1-binding domain complex in the cells stimulated with DH-PH domain and full-length p114RhoGEF
(Figure 7D). Importantly, in a positive control experiment,
RacV12 was also found in association with p67phox subunit.
Taken together, these data demonstrated that p114RhoGEF
could induce ROS formation likely via activation of NADPH
oxidase.
Discussion
In the present study, we have demonstrated for the first time
that G␤␥ subunits of heterotrimeric G proteins could activate
recently identified guanine nucleotide exchange factor
p114RhoGEF17 (Figure 8). We have also determined that
p114RhoGEF could activate RhoA and Rac1 but not Cdc42
GTPases. The functional importance of RhoA activation was
supported by p114RhoGEF-induced actin stress fiber formation and cell rounding. We have also found that DH/PH
domain of the protein is needed for the actin cytoskeleton
reorganization. Functional significance of Rac1 activation
was indicated by p114RhoGEF-induced reactive oxygen
species formation and activation of NADPH oxidase, the
enzyme complex responsible for ROS generation.
G␤␥ subunits have been implicated in the regulation of
Rho GTPase– driven cellular events. Thus, it was reported
that G␤␥ subunits could induce stress fiber formation and
focal adhesion assembly in a Rho-dependent manner.25 One
of the possible mechanisms for such G␤␥-dependent activation of Rho GTPases is activation of guanine nucleotide
exchange factors. Two GEFs, Ras-GRF1 and P-Pex1, that
could be activated by G␤␥ subunits have been recently
identified.8,9 Interestingly, both proteins are Rac-specific
guanine nucleotide exchange factors. Because G␤␥ subunits
could be released on stimulation of all G protein– coupled
receptors, recognition that G␤␥ subunits can stimulate some
of the guanine nucleotide exchange factors may lead to
improved understanding of the regulation of small G proteins
via G protein– coupled receptors.
p114RhoGEF Regulates Rho and Rac Function
855
Figure 8. Proposed model for the ligand-dependent activation
of p114RhoGEF. Stimulation of G protein– coupled receptors
resulted in release of G␤␥ subunits, which in turn activate
p114RhoGEF and downstream RhoA and Rac1 small G proteins. Functionally, stimulation of RhoA and Rac1 proteins
results in the cell shape regulation, actin cytoskeleton changes,
and generation of reactive oxygen species.
G␤␥ subunits are known to interact with PH domains of
several signaling proteins. Thus, G␤␥ subunits interact with
PH domains of ␤-adrenergic receptor kinase, ␤ARK (see
review23), Bruton tyrosine kinase,3 and phospholipase ␤C226
and phospholipase ␤C3.27 It is believed that the interaction of
G␤␥ with the PH domains results in the activation of these
enzymes. Because p114RhoGEF contains the PH domain, it
is possible that G␤␥ subunits activate p114RhoGEF via
interaction with its PH domain. It is our future challenge to
address the molecular mechanism of G␤␥-induced activation
of p114RhoGEF.
p114RhoGEF was initially identified by searching protein
databases using DH domain and described as a Rho-specific
GEF.17 Our data show that p114RhoGEF could activate Rac1
in addition to RhoA protein. As two different approaches to
measure GTPases’ activation were used, it can explain the
apparent discrepancy of the data. In the present work, we used
the approach that allowed us to assess the GTP binding to
Rho GTPases in vivo on coexpression with p114RhoGEF.
Detection of GTP-bound cellular Rho GTPases provided
direct evidence for activation of the specific Rho GTPases.
Basal state of GEFs activity that ranges from inactive to
partially active is regulated by four possible regulatory modes
that involve intra- and intermolecular rearrangements.1 Thus,
GEF activity could be inhibited by interaction between DH
and PH domains, interaction of a regulatory domain with
DH/PH domains, oligomerization of DH domains, and direct
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October 31, 2003
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protein-protein interactions with regulatory proteins. Using
deletion mutants of p114RhoGEF, we have shown that
DH/PH domain produced the most pronounced changes in the
cell shape and actin cytoskeleton. Thus, in DH/PH domain
expressing cells, almost 100% displayed changes in the cell
shape and actin cytoskeleton, whereas full-length
p114RhoGEF induced changes in the cell shape and actin
cytoskeleton in only 40% of the cells. This observation also
suggested the existence of the regulatory mechanism that will
be addressed in the future studies. Using C-terminal of
p114RhoGEF as a bait in yeast two-hybrid assay, we are
currently screening human lung microvascular endothelial
cell library in search for a potential interacting proteins that
might help to further elucidate the mechanism of
p114RhoGEF regulation.
Reactive oxygen species generated during metabolism can
enter into reactions that, when uncontrolled, can affect certain
processes leading to clinical manifestations (for review see28).
NADPH oxidase complex is found in a variety of phagocytic
and nonphagocytic cells and plays a crucial role in hostdefense mechanism during phagocytosis. NADPH oxidase is
a highly regulated membrane-bound enzyme complex, which
catalyzes the production of superoxide by one-electron reduction of oxygen using NADPH as electron donor. The core
enzyme consists of five subunits: p40phox, p47phox, p67phox,
p22phox, and gp91phox. In the basal state, p40phox, p47phox, and
p67phox exist in the cytosol as a complex, whereas p22phox and
gp91phox are located in membranes of secretory vesicles in
neutrophils and other cells such as fibroblasts.13 Rac proteins
activate NADPH oxidase by participating in the electron
transfer reactions.11 Our data showed that p114RhoGEF
could activate Rac1 proteins in in vivo pull-down assay,
induce generation of ROS and translocation of p67phox to the
membrane, and promote interaction between activated Rac1
and p67phox.
In conclusion, the results presented here suggested that
ligand stimulation of the novel guanine nucleotide exchange
factor p114RhoGEF might activate RhoA- and Rac1mediated signaling pathways and results in physiologically
significant cellular events.
Acknowledgments
These studies were supported by NIH grants GM56159 and
GM65160 and by grant from American Heart Association to T.V.Y.,
predoctoral fellowship 0110205Z from the American Heart Association (AHA) to J.N., and predoctoral fellowship 0310030Z from the
AHA (to J.P.). T.V.Y. is an Established Investigator of the AHA. We
thank Dr Steven Vogel for critical reading and editing of this
manuscript.
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25.
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G Protein βγ Subunits Stimulate p114RhoGEF, a Guanine Nucleotide Exchange Factor for
RhoA and Rac1: Regulation of Cell Shape and Reactive Oxygen Species Production
Jiaxin Niu, Jasmina Profirovic, Haiyun Pan, Rita Vaiskunaite and Tatyana Voyno-Yasenetskaya
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Circ Res. 2003;93:848-856; originally published online September 25, 2003;
doi: 10.1161/01.RES.0000097607.14733.0C
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