A Rac1 Effector Site Controlling Mitogenesis through Superoxide Production*

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.

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.
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 p160 ROCK , a cytoplasmic component of the NADPH oxidase complex p67 PHOX , and the Rac-binding protein POR1 (reviewed in Ref. 11). Using chimeras made between Rac and the related GT-Pases 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)(13)(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 Racmediated 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 NaPO 4 ) 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 fluoresceinconjugated goat antibody to mouse immunoglobulin G and rhodaminelabeled 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 g/ml of each of the indicated constructs, and BrdUrd (10 M) 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 Ϫ20°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 M 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 g/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 g of the indicated expression vector using the CaPO 4 method. pCMV-GFP was used as a negative control. After 12 h of incubation with the DNA-CaPO 4 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 g of FLAG-tagged JNK1 and 10 g of expression vectors containing no insert, RacV12, or RacV12,⌬Ins using the CaPO 4 method. After a 12-h incubation with the DNA-CaPO 4 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 Cys 189 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-transferasec-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 Racinduced 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 G 1 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 superoxidegenerating 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 FIG. 1. Effects of RacV12 and RacV12,⌬Ins on membrane ruffling, JNK activation, and DNA synthesis. a, serum-starved COS1 cells were microinjected with expression plasmids encoding the indicated T7 epitope-tagged Rac mutants or with CMV-GFP as a vector control (Vect., 50 g/ml). 6 h after injection, cells were fixed and stained with rhodamine-labeled phalloidin to visualize actin filaments and membrane ruffles. b, COS1 cells were cotransfected with 10 g of FLAG-tagged JNK1 and 10 g of either RacV12 or RacV12,⌬Ins expression plasmids. JNK activity was measured by immunocomplex kinase assays with glutathione S-transferase-Jun as the substrate and visualized by autoradiography. Expression of JNK1 and Rac mutants was determined by Western blot analysis with polyclonal antibodies to JNK and monoclonal antibodies to T7. 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 H 2 O 2 (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 RacV12⌬Ins (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, FIG. 4. Production of superoxide by Rac mutants. a, COS1 cells were transfected with 10 g of the indicated construct, and NBT reduction was scored as in Fig. 2b. Values correspond to the means of three independent experiments, and the error bars represent the standard deviations. At least 100 cells were scored per condition in each experiment. b, schematic illustration of the effector pathways activated by different Rac mutants and their relative contribution to Rac-induced mitogenesis.
FIG. 2. Analysis of superoxide production using the NBT reduction assay. a, serum-starved COS1 cells were injected with expression vectors for pCGT-RacV12. Cells were then incubated for 10 h to allow the protein to express followed by a 1-h incubation in 0.5% NBT containing DMEM. In the lower panels, cells were incubated in 300 units/ml SOD (bovine erythrocyte source) 1 h previous to the addition of NBT. Top, cells expressing T7-tagged RacV12 as determined by indirect immunofluorescence using an anti-T7 monoclonal antibody. Middle, bright field micrographs of the corresponding fields showing the result of NBT reduction. Bottom, phase contrast micrographs of the corresponding fields. b, quantitation of superoxide production. COS1 cells transfected with the indicated constructs were incubated with NBT as above. Cells expressing the proteins as determined by immunofluorescence or autofluorescence in the case of GFP were scored for the presence of blue formazan precipitate. Values correspond to the means of three independent experiments, and the error bars represent the standard deviations. At least 100 cells were scored per condition in each experiment. 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 seruminduced 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 G 1 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 G 1 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 effectorbinding 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.