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(Circulation Research. 2003;93:848.)
© 2003 American Heart Association, Inc.

Cellular Biology

G Protein ß 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

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 tvy{at}uic.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 Gß subunits, transducin. We have determined that Gß subunits but not G subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that Gß subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, Gß 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 Gß 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.

Key Words: Rho GTPases • guanine nucleotide exchange factor • actin cytoskeleton • serum response element • reactive oxygen species

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Guanine 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 Gß subunits have been implicated in activation of individual RhoGEFs. Thus, it was shown that G13 could activate several GEFs: p115RhoGEF, LARG, and PDZ-RhoGEF.^5–7 Similarly, Gß 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 p47^phox, p67^phox, gp91^phox, and gp22^phox is activated by Rac to produce ROS in phagocytic leukocytes and other nonphagocytic cells.¹³

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 Gß 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
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

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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thrombin and LPA Stimulate p114RhoGEF via Gß 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.

View larger version (45K): [in this window] [in a new window]   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 G13 were used as a probes to detect expression of p114RhoGEF or G13. 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.

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 p114RhoGEF-dependent activation of SRE-mediated transcription of a luciferase reporter gene.¹⁸ NIH3T3 cells were transiently transfected with p114RhoGEF and SRE-driven luciferase reporter. To correct for variations in transfection efficiency, an expression vector coding for ß-galactosidase was cotransfected with the above constructs, and the expressed ß-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.

View larger version (13K): [in this window] [in a new window]   Figure 2. Agonist-induced activation of SRE via p114RhoGEF is mediated by Gß12 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-ßgal. 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 µmol/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, Gß subunits are functionally coupled to p114RhoGEF to induce SRE activation. 50 ng of each construct (G13Q226L, G12Q229L, GqR145C, Gi2Q205L, Gß1, G2, and p114RhoGEF) was transfected for SRE measurement. Presented data are the mean±SEM from three independent experiments. C, SRE activation induced by G13Q226L is potentiated by p115RhoGEF. NIH3T3 cells were transfected with 50 ng pSRE.L, 50 ng pCMV-ßgal, 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.

Because G protein-coupled receptors activated by LPA could activate multiple heterotrimeric G proteins, including G12, G13, Gq, and Gi, 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 constitutively active GTPase-deficient mutants of G13, G12, Gq, and Gi2. Without p114RhoGEF transfection, activated mutants of G13, G12, and Gq induced SRE activation (Figure 2B), which was consistent with previously reported data.¹⁹ Gi2 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), G13-induced SRE activation was strongly potentiated in the cells expressing p115RhoGEF, a guanine nucleotide exchange factor known to be activated by G13.⁵ This result demonstrates that G subunit could activate a suitable GEF.

Because receptor stimulation of heterotrimeric G proteins results in a release of Gß subunits that are capable of stimulation of downstream signaling pathways, we have also analyzed the effect of Gß1 and G2 subunits, the best characterized Gß combination^20,21 on p114RhoGEF-induced SRE activation. Coexpression of plasmids encoding Gß1 and G2 isoforms strongly enhanced the p114RhoGEF-dependent activation of SRE (Figure 2B). However, neither Gß1 nor G2 alone affected p114RhoGEF-dependent activation of SRE (data not shown). Further, the functional Gß subunits scavenger, transducin,²² inhibited both LPA-dependent and Gß12-dependent potentiation of SRE activity induced by p114RhoGEF (Figure 2A). This result indicated that both exogenous and endogenous Gß subunits could activate p114RhoGEF.

Gß Subunits Stimulate p114RhoGEF via Interaction With Its DH/PH Domain
Because DH/PH domains are the hallmarks of RhoGEF proteins¹ and the Gß subunits have been shown to interact with PH domains of other signaling molecules,²³ we hypothesized that Gß12 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 domain and proline cluster (aa 648 to 952) (Figure 3A). NIH3T3 cells were transfected with p114RhoGEF constructs together with Flag-tagged Gß1 and G2 subunits. p114RhoGEF polypeptides were immunoprecipitated from cell lysates using Myc antibody and presence of Gß subunit was determined using Flag antibody. Data showed that Gß1 subunit interacted with full-length p114RhoGEF, DH-PH domain, and C-terminal domain (Figure 3B). Apparently, lower Gß1 binding to full-length p114RhoGEF was due to the lower expression of the full-length p114RhoGEF. We concluded that Gß1 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 Gß1-binding domain of p114RhoGEF is required for Gß12-mediated signaling. Data showed that SRE activation by DH/PH domain of the p114RhoGEF was further enhanced by Gß12 subunits, whereas C-terminal domains of p114RhoGEF did not affect SRE activity (Figure 3C).

View larger version (27K): [in this window] [in a new window]   Figure 3. Gß12 subunits interacted with p114RhoGEF. A, Schematic representation of the domain structure of p114RhoGEF and its deletion mutants. B, Coimmunoprecipitation of p114RhoGEF with Gß12 subunits. NIH3T3 cells grown in 10-cm2 dishes were transiently transfected with vector alone or various Myc-tagged constructs of p114RhoGEF together with Flag-tagged Gß1 subunit and G2 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, Gß12 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.

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.

View larger version (26K): [in this window] [in a new window]   Figure 4. p114RhoGEF activates RhoA and Rac1 but not Cdc42. NIH3T3 cells were transfected with 5 µg 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-ßgal, 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.

To further support data of p114RhoGEF-dependent activation of RhoA and Rac1 proteins, we have determined the involvement of specific Rho GTPases in p114RhoGEF-dependent SRE activation. Because RhoA, Rac1, and Cdc42 are known to activate SRE,¹⁸ we have used dominant-negative mutants of these GTPases to inhibit p114RhoGEF-induced 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 rhodamine-phalloidin 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-containing cells was calculated from at least 400 Myc-positive cells counted. In 40% of the cells expressing full-length 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.

View larger version (31K): [in this window] [in a new window]   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 serum-starved 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.

p114RhoGEF Induces Reactive Oxygen Species (ROS) Formation
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 phosphate-buffered 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.

View larger version (21K): [in this window] [in a new window]   Figure 6. p114RhoGEF induces reactive oxygen species formation. A, TNF--induced generation of ROS. NIH3T3 cells were loaded with 10 µmol/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 µmol/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 µmol/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.

Finally, the generation of ROS was observed in the cells expressing p114RhoGEF (Figure 6B). Importantly, 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 p67^phox subunit from the cytoplasm to the membrane,¹³ we have determined if p114RhoGEF could induce translocation of p67^phox from the cytoplasm to the membrane. We have constructed the GFP-tagged p67^phox subunit and transiently expressed it in NIH3T3 fibroblasts. In unstimulated cells, GFP-p67^phox subunit was localized throughout the cytoplasm (Figure 7A), verifying that the localization of GFP-p67^phox subunit was similar to the localization of endogenous p67^phox subunit. Stimulation of the cells with 1 µmol/L LPA for 10 minutes resulted in translocation of the GFP-p67^phox subunit to the plasma membrane (Figure 7A). We then analyzed distribution of p67^phox subunit in the cells transfected with domains of p114RhoGEF. NIH3T3 cells were cotransfected with GFP-p67^phox subunit and Myc-tagged constructs of p114RhoGEF. Localization of p67^phox subunit in a Myc-positive cells showed that both full-length p114RhoGEF and its DH/PH domain induced translocation of the p67^phox subunit to the plasma membrane (Figure 7B). Importantly, C-terminal domain did not affect the intracellular localization of p67^phox subunit.

View larger version (21K): [in this window] [in a new window]   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 µmol/L LPA for 10 minutes. B, NIH3T3 (80 000) cells were seed on the gelatin-precoated coverslips in 12-well plates and were transiently cotransfected with either 5 µg 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 µg pcDNA3, pcDNA3-p67phox, 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 µg 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.

We next determined the association of p67^phox with membrane fraction using Western blotting analysis of total cell 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 p67^phox (Figure 7C). Importantly, in a positive control experiment, RacV12 also induced translocation of p67^phox subunit to the plasma membrane fraction.

As binding of Rac to p67^phox 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 p67^phox 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 p67^phox in complex with Rac1-binding domain was analyzed using Rac1 or p67^phox 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, p67^phox 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 p67^phox subunit. Taken together, these data demonstrated that p114RhoGEF could induce ROS formation likely via activation of NADPH oxidase.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have demonstrated for the first time that Gß 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.

View larger version (19K): [in this window] [in a new window]   Figure 8. Proposed model for the ligand-dependent activation of p114RhoGEF. Stimulation of G protein-coupled receptors resulted in release of Gß 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.

Gß subunits have been implicated in the regulation of Rho GTPase-driven cellular events. Thus, it was reported that Gß subunits could induce stress fiber formation and focal adhesion assembly in a Rho-dependent manner.²⁵ One of the possible mechanisms for such Gß-dependent activation of Rho GTPases is activation of guanine nucleotide exchange factors. Two GEFs, Ras-GRF1 and P-Pex1, that could be activated by Gß subunits have been recently identified.^8,9 Interestingly, both proteins are Rac-specific guanine nucleotide exchange factors. Because Gß subunits could be released on stimulation of all G protein-coupled receptors, recognition that Gß 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.

Gß subunits are known to interact with PH domains of several signaling proteins. Thus, Gß subunits interact with PH domains of ß-adrenergic receptor kinase, ßARK (see review²³), Bruton tyrosine kinase,³ and phospholipase ßC2²⁶ and phospholipase ßC3.²⁷ It is believed that the interaction of Gß with the PH domains results in the activation of these enzymes. Because p114RhoGEF contains the PH domain, it is possible that Gß subunits activate p114RhoGEF via interaction with its PH domain. It is our future challenge to address the molecular mechanism of Gß-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 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 host-defense 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: p40^phox, p47^phox, p67^phox, p22^phox, and gp91^phox. In the basal state, p40^phox, p47^phox, and p67^phox exist in the cytosol as a complex, whereas p22^phox and gp91^phox 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 p67^phox to the membrane, and promote interaction between activated Rac1 and p67^phox.

In conclusion, the results presented here suggested that ligand stimulation of the novel guanine nucleotide exchange factor p114RhoGEF might activate RhoA- and Rac1-mediated 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.

	Footnotes

 
Original received April 14, 2003; revision received September 15, 2003; accepted September 16, 2003.

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