From the Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794-5222
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Rac GTP-binding protein controls signal transduction pathways that are critical for mitogenesis and oncogenesis (1, 2). The biochemical nature of these signaling pathways is presently unknown. Here we report that a region in Rac1 (residues 124-135), previously defined as the insert region (3), is essential for its mitogenic activity. Deletion of this region does not interfere with the ability of Rac1 to induce cytoskeletal changes or to activate the Jun kinase mitogen-activated protein kinase cascade but abrogates Rac1-induced stimulation of DNA synthesis and Rac1-mediated superoxide production in quiescent fibroblasts. Treatment of cells with agents that abolish superoxide generation inhibits specifically the mitogenic effect of Rac1. Our results identify an effector site in Rac1 that is necessary for mitogenic signaling and implicate superoxide generation as a candidate effector pathway of Rac1-dependent cell growth.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rac proteins have been shown to play a fundamental role in signaling pathways controlling actin polymerization, transcriptional activation, cellular proliferation, and superoxide generation (1, 4-7). Genetic and biochemical studies have indicated that the signaling activities of Rac are mediated by distinct target molecules (8-10). A number of Rac targets have been identified to date, based on their ability to interact preferentially with the GTP-bound form of Rac. These include the p65 Ser/Thr protein kinase PAK,1 the Ser/Thr kinase p160ROCK, a cytoplasmic component of the NADPH oxidase complex p67PHOX, and the Rac-binding protein POR1 (reviewed in Ref. 11). Using chimeras made between Rac and the related GTPases Rho and Cdc42 as well as site-specific mutations, it has been shown that Rac interacts with its target proteins through multiple effector sites. Rac has an N-terminal effector-binding domain encompassing amino acids 26-40 that is essential for the induction of actin polymerization as well as the interaction with the target enzymes PAK and the NADPH oxidase (12-14). A second C-terminal effector region with the same target specificity has been identified at amino acids 143-175 (12). More recently, a third effector site at residues 124-135, called the insert region, has been identified and shown to be necessary for the activation of NADPH oxidase in phagocytes (3, 14, 15).
In the present study we investigated the role of the insert region in Rac-dependent signaling in fibroblasts. Using a Rac mutant lacking the insert region, we demonstrate that this region is critical for the ability of Rac to promote cell cycle progression. Furthermore, we show that the insert region contributes to Rac-induced mitogenesis specifically by controlling the generation of superoxide. Our findings suggest that Rac-mediated superoxide generation might play an important role in the control of cell growth.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Microinjection and Immunofluorescence-- For microinjection experiments, COS1 or REF-52 cells were plated onto gridded glass coverslips and cultured in DMEM supplemented with FBS (10%). The cells were grown to confluence and then placed in DMEM with 0.5% FBS for 24 h before microinjection. A plasmid mixture containing indicated plasmids in microinjection buffer (50 mM Hepes (pH 7.2), 100 mM KCl, and 5 mM NaPO4) was microinjected into cell nuclei. For monitoring membrane ruffles and protein expression, cells were fixed in 3.7% formaldehyde in PBS for 30 min at room temperature. The coverslips were incubated for 1 h at 37 °C with mouse antibody to T7 epitope (Novagen) in PBS containing albumin (2 mg/ml) and then with a mixture of fluorescein-conjugated goat antibody to mouse immunoglobulin G and rhodamine-labeled phalloidin (0.01 mg/ml) (Molecular Probes). The cells were photographed with a Zeiss Axiphot fluorescence microscope.
DNA Synthesis--
For monitoring DNA synthesis, REF-52 cells
were plated on gridded coverslips and serum-starved for 24 h.
Cells were injected with 2.5 µg/ml of each of the indicated
constructs, and BrdUrd (10 µM) was added to the cell
culture medium at a 2 h after injection. After 30 h, cells
were fixed in acid alcohol (ethanol:water:acetic acid 90:5:5) for
1 h at 20 °C and immunostained with mouse monoclonal antibody
to BrdUrd (Sigma) as described (16). At least 100 injected cells were
scored in each assay for quantitation. Chemical inhibitors affecting
superoxide or other reactive oxidants (300 units/ml superoxide
dismutase (Sigma), 20 mM
N-acetyl-L-cysteine (Sigma), and 5 µM diphenylene iodonium chloride (Toronto Research)) were added immediately after injection.
Superoxide Production-- COS1 cells were plated onto glass coverslips, grown to confluence, and then either microinjected or transfected with the indicated expression vectors. For microinjection, cells were injected with 50 µg/ml of expression plasmids. After 10 h, the cells were incubated with either 300 units/ml SOD or vehicle (DMEM). The medium was then replaced with DMEM containing 0.5% nitro blue tetrazolium (Sigma) with or without SOD. The cells were incubated for 1 h at 37 °C before being fixed and stained with anti-T7 monoclonal antibodies. For quantitation, COS1 cells on coverslips were placed in 10-cm tissue culture dishes and transfected with 10 µg of the indicated expression vector using the CaPO4 method. pCMV-GFP was used as a negative control. After 12 h of incubation with the DNA-CaPO4 precipitate, cells were washed three times with PBS and allowed to recover in DMEM containing 5% fetal calf serum for 12 h. The serum containing medium was then replaced with DMEM for an additional 12 h. The coverslips were incubated in 0.5% NBT in DMEM for 1 h. Cells were stained using either anti-Raf (Transduction Laboratories) or T7 monoclonal antibodies, and those that expressed the exogenous protein were then scored for the presence of the reduced form of NBT, blue formazan. Measurement of intracellular reactive oxygen species generation by the DCFDA loading method was carried out as described (17).
Jun Kinase (JNK) Activity--
For monitoring JNK activation,
cells were cotransfected with 10 µg of FLAG-tagged JNK1 and 10 µg
of expression vectors containing no insert, RacV12, or RacV12,Ins
using the CaPO4 method. After a 12-h incubation with the
DNA-CaPO4 precipitates, cells were incubated in medium
containing FBS (5%) for 6 h and then incubated for 12 h in
serum-free medium. JNK1 immunocomplex kinase assay was carried out as
described (8). The reaction products were analyzed by
SDS-polyacrylamide gel electrophoresis and visualized by
autoradiography. Fold activation was determined using the Storm 860 PhosphorImager in combination with Image Quant v1.1 software (Molecular
Dynamics).
Expression Plasmids--
Plasmid pCGT, which is derived from
pCGN with a replacement of the HA epitope by the T7 epitope, was used
as as a mammalian expression vector to express the various Rac mutants.
pCGT RacV12,H40 and pCGT RacV12,L37 were created as described (8) and
ligated into the XbaI-BamHI sites of pCGT. pCGT
RacV12,Ins was created as described (3) except that the
Cys189 to Ser mutation originally made to increase protein
stability was not incorporated. The CMV-GFP vector was constructed by
fusing the cytomegalovirus promoter to the GFP reporter.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate the significance of the insert region for the
biological effects of Rac, we examined the signaling activities of a
mutant form of activated Rac (RacV12) containing a complete deletion of
the insert region between residues 124-135 (RacV12,Ins). Earlier
microinjection studies have established that Rac-mediated changes in
the actin cytoskeleton lead to the formation of membrane ruffles (4).
Therefore, the role of the insert region in Rac-induced actin
polymerization was examined by microinjection of serum-starved COS1
cells with expression plasmids encoding T7 epitope-tagged versions of
Rac mutants. We found that RacV12,
Ins was as effective as RacV12 in
inducing actin polymerization and membrane ruffling, as judged by
filamentous actin staining with rhodamine phalloidin (Fig.
1a). Immunofluorescence
staining of the injected cells confirmed that both Rac mutants were
expressed to the same extent and displayed an overall similar
subcellular distribution pattern (not shown). These results indicate
that the insert region is not necessary for effector interactions that
control actin cytoskeleton rearrangements. It has been shown that Rac
can stimulate kinase cascades leading to the activation of the JNK
mitogen-activated protein kinase cascade (5, 6). This activation is
presumably mediated by the activation of the Rac target PAK (18-20).
To test the Rac insert region mutant for its ability to activate the
JNK mitogen-activated protein kinase cascade, COS1 cells were
cotransfected with expression plasmids encoding the Rac mutants and a
plasmid encoding FLAG-tagged version of JNK1. JNK activity was assayed
by immunocomplex kinase assay using glutathione
S-transferase-c-Jun as a substrate. RacV12,
Ins and RacV12
stimulated JNK activity to a similar extent, indicating that the insert
region is not required for Rac-induced JNK activation (Fig.
1b). These results are consistent with the observation that Rac-induced PAK activation is not affected by insert region mutations (3). We conclude that Rac insert region is dispensable for two major
biological activities of Rac described thus far in fibroblasts, induction of membrane ruffling and JNK activation.
|
Rac proteins have been shown to play a critical role in mitogenesis and
oncogenesis. In Swiss 3T3 cells, RacV12 stimulates the transition from
G1 to the S phase of the cell cycle (2). In addition,
RacV12 promotes focus formation in NIH3T3 cells when coexpressed with a
constitutively active form of Raf1 kinase (1). To determine whether the
insert region of Rac is required for its mitogenic activity, quiescent
REF-52 cells were microinjected with expression plasmids encoding the
Rac mutants, and cell cycle progression was measured by BrdUrd
incorporation. In REF-52 cells, microinjection of RacV12 alone was not
sufficient for the induction of DNA synthesis. However, coinjection of
RacV12 with the membrane-targeted form of Raf1, Raf-CAAX, resulted in a
synergistic stimulation of DNA synthesis. In contrast, RacV12,Ins
failed to stimulate DNA synthesis when coinjected with Raf-CAAX (Fig.
1c). These results suggest a role for the Rac insert region
in mediating effector interactions that are essential for Rac-induced
cell proliferation. This conclusion is further supported by our
findings that a Rac mutant containing a single amino acid substitution
in the insert region (K130N) was also defective in stimulating DNA
synthesis, despite retaining its ability to activate JNK cascade and
induce membrane ruffling (data not shown).
Because the Rac insert region has been implicated in the activation of NADPH oxidase in phagocytic cells (3, 15), we investigated the relationship between Rac-mediated superoxide production and mitogenesis in nonphagocytic cells. COS1 cells were transfected with Rac mutants expression plasmids, and superoxide production was examined using the NBT reduction assay (21). This assay involves the incubation of cells with NBT, which when reduced forms an insoluble purple precipitate, and has been used to demonstrate a superoxide-generating NADPH oxidase system in fibroblasts (22). Cells expressing RacV12 were positive for NBT staining, as detected by bright field microscopy (Fig. 2a). The RacV12-dependent NBT reduction was inhibited by the incubation of transfected cell with SOD, indicating that NBT staining is because of Rac-induced superoxide production (Fig. 2a). This finding is consistent with earlier reports implicating Rac in the regulation of intracellular reactive oxygen species production in nonphagocytic cells (17, 23).
|
It should be noted that the NBT reduction assay was not sufficiently
sensitive to detect Rac-mediated superoxide production in REF-52 cells.
Another method for the detection of increase in intracellular reactive
oxygen species involves the loading of cells with the fluorophore
DCFDA, which fluoresces upon interaction with
H2O2 (17). Using this method, we were able to
detect an increase in DCFDA fluorescence in cells injected with RacV12. In contrast, no increase in DCFDA fluorescence was detected in cells
injected with RacV12Ins (not shown). Thus the results obtained in
REF-52 cells and COS1 cells with respect to the relative abilities of
Rac mutants to stimulate the production of reactive oxygen species are
qualitatively similar. However, because the DCFDA loading method does
not permit a quantitative analysis in which protein expression and
production of reactive oxygen species can be correlated on a per cell
basis, we have used the NBT reduction assay in the subsequent
experiments. As illustrated in Fig. 2b, virtually all cells
expressing RacV12 were positive for NBT staining. The frequency of
NBT-stained cells was reduced to nearly background levels in cells
expressing RacV12,
Ins (Fig. 2b), indicating that this
mutant failed to induce superoxide production. Likewise, Raf-CAAX was
also deficient in superoxide production. These observations together
with the finding that RacV12,
Ins lacks mitogenic activity suggest a
role for superoxide production in mediating the effects of Rac on cell
proliferation.
To further test this idea, we examined the effects of various agents that interfere with superoxide generation or accumulation on RacV12-induced mitogenesis. Treatment of RacV12 expressing cells with the specific flavoprotein inhibitor diphenylene iodonium (DPI) and SOD had no appreciable effect on Rac-induced membrane ruffling or JNK activation (Fig. 3, a and b). The anti-oxidant N-acetyl cysteine (NAC) had no effect on Rac-induced membrane ruffling but moderately inhibited Rac-induced JNK activation. In addition, these inhibitors had no effect on the levels of expression of Rac or JNK as determined by Western blot analysis (not shown). In contrast, NAC, DPI, and SOD blocked the stimulation of DNA synthesis induced by coinjection of RacV12 and Raf-CAAX (Fig. 3c). All inhibitors were used at the minimal concentration, which results in the complete inhibition of Rac-induced superoxide production, as determined by DCFDA fluorescence. Furthermore, at these concentrations, DPI and SOD had no effect on serum-induced DNA synthesis (Fig. 3c). In contrast, NAC induced a 70% inhibition of DNA synthesis, indicating that this agent exerts a more general inhibitory effect on cell growth. We conclude that Rac-mediated superoxide production is specifically required for its growth promoting effects. Significantly, DPI exerted an inhibitory effect on DNA synthesis if added at intervals up to 10 h after injection. Beyond this interval DPI addition had no effect on the ability of RacV12 and Raf-CAAX to stimulate DNA synthesis. In REF-52 cells, the G1 phase of the cell cycle is approximately 18 h long (not shown). Thus it appears that production of reactive oxygen species is required up to mid G1 phase.
|
Using effector-binding loop mutants of Rac, it has been recently
demonstrated that the activation of PAK and JNK are not required for
Rac-induced mitogenesis (8-10). On the other hand, a correlation was
observed between Rac-induced actin polymerization and cell cycle
progression (8, 9). Our finding that RacV12,Ins retained the ability
to induce membrane ruffling but was no longer able to stimulate DNA
synthesis indicates that Rac-induced actin polymerization is not
sufficient for its growth promoting activity. This result is consistent
with a recent report suggesting that membrane ruffling is not
sufficient for the full transforming activity of Rac (10). Utilizing
the NBT reduction assay, the capacity of the Rac effector-binding loop
mutants RacV12,L37 and RacV12,H40 to stimulate superoxide production
was tested. The RacV12,L37 mutant activates JNK but is defective in
inducing actin polymerization, whereas the RacV12,C40 induces actin
polymerization but is defective in JNK activation (Fig.
4b and Ref. 8). Both mutants
were as effective as RacV12 in inducing superoxide production (Fig.
4a). However, as shown previously, only the RacV12,H40 is
functional in promoting cell proliferation and transformation (8).
Therefore, although necessary, superoxide production is not sufficient
for the mitogenic activity of Rac. Together, these observations suggest
that both Rac-induced actin polymerization and superoxide production
are required for Rac-controlled cell proliferation (Fig.
4b). It remains to be determined whether additional
Rac-mediated signals contribute to its mitogenic activity.
|
Superoxide generation has been frequently implicated in the control of normal cell growth and the promotion of malignant transformation (24-27). Our findings indicate that Rac-induced superoxide production is a critical mediator of mitogenic signaling. The molecular identification of the cellular targets of Rac-mediated superoxide generation should provide insights into the mechanisms linking reactive oxygen species and growth control.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Linda VanAelst and Patrick Hearing for providing plasmids and Arie Abo and David Lambeth for helpful discussion.
![]() |
FOOTNOTES |
---|
* This work was supported by American Heart Association Grant 9650340N and National Institutes of Health Grant CA55360.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 516-632-9738;
Fax: 516-632-8891; E-mail: barsagi{at}asterix.bio.sunysb.edu.
1 The abbreviations used are: PAK, p21-activated kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BrdUrd, bromodeoxyuridine; NBT, nitro blue tetrazolium; DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; JNK, Jun kinase; CMV, cytomegalovirus; GFP, green fluorescence protein; SOD, superoxide dismutase; DPI, diphenylene iodonium; NAC, N-acetyl cysteine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|