G Protein βγ Subunits Stimulate p114RhoGEF, a Guanine Nucleotide Exchange Factor for RhoA and Rac1
Regulation of Cell Shape and Reactive Oxygen Species Production
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 Gβγ subunits, transducin. We have determined that Gβγ subunits but not Gα subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that Gβγ subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, Gβγ 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 Gβγ 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.
- Rho GTPases
- guanine nucleotide exchange factor
- actin cytoskeleton
- serum response element
- reactive oxygen species
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 Gα and Gβγ subunits have been implicated in activation of individual RhoGEFs. Thus, it was shown that Gα13 could activate several GEFs: p115RhoGEF, LARG, and PDZ-RhoGEF.5–7⇓⇓ Similarly, Gβγ 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
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 Gβγ 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
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
Thrombin and LPA Stimulate p114RhoGEF via Gβγ 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 [α-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 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 β-galactosidase was cotransfected with the above constructs, and the expressed β-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 Gα12, Gα13, Gαq, and Gαi, 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 Gα13, Gα12, Gαq, and Gαi2. Without p114RhoGEF transfection, activated mutants of Gα13, Gα12, and Gαq induced SRE activation (Figure 2B), which was consistent with previously reported data.19 Gαi2 did not affect SRE activity. Cotransfection of p114RhoGEF with indicated Gα subunits did not result in additional potentiation of SRE activity (Figure 2B), suggesting that involvement of Gα subunits in the regulation of p114RhoGEF is unlikely. In a positive control experiment (Figure 2C), Gα13-induced SRE activation was strongly potentiated in the cells expressing p115RhoGEF, a guanine nucleotide exchange factor known to be activated by Gα13.5 This result demonstrates that Gα subunit could activate a suitable GEF.
Because receptor stimulation of heterotrimeric G proteins results in a release of Gβγ subunits that are capable of stimulation of downstream signaling pathways, we have also analyzed the effect of Gβ1 and Gγ2 subunits, the best characterized Gβγ combination20,21⇓ on p114RhoGEF-induced SRE activation. Coexpression of plasmids encoding Gβ1 and Gγ2 isoforms strongly enhanced the p114RhoGEF-dependent activation of SRE (Figure 2B). However, neither Gβ1 nor Gγ2 alone affected p114RhoGEF-dependent activation of SRE (data not shown). Further, the functional Gβγ subunits scavenger, transducin,22 inhibited both LPA-dependent and Gβ1γ2-dependent potentiation of SRE activity induced by p114RhoGEF (Figure 2A). This result indicated that both exogenous and endogenous Gβγ subunits could activate p114RhoGEF.
Gβγ Subunits Stimulate p114RhoGEF via Interaction With Its DH/PH Domain
Because DH/PH domains are the hallmarks of RhoGEF proteins1 and the Gβγ subunits have been shown to interact with PH domains of other signaling molecules,23 we hypothesized that Gβ1γ2 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 Gβ1 and Gγ2 subunits. p114RhoGEF polypeptides were immunoprecipitated from cell lysates using Myc antibody and presence of Gβ subunit was determined using Flag antibody. Data showed that Gβ1 subunit interacted with full-length p114RhoGEF, DH-PH domain, and C-terminal domain (Figure 3B). Apparently, lower Gβ1 binding to full-length p114RhoGEF was due to the lower expression of the full-length p114RhoGEF. We concluded that Gβ1 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 Gβ1-binding domain of p114RhoGEF is required for Gβ1γ2-mediated signaling. Data showed that SRE activation by DH/PH domain of the p114RhoGEF was further enhanced by Gβ1γ2 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-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.
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-α, a recognized ROS inducer24 for 15 minutes to allow maximum oxidant accumulation during this period. Control cells showed little fluorescence. In contrast, TNF-α 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.
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 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 μmol/L LPA for 10 minutes resulted in translocation of the GFP-p67phox 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 Myc-positive 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 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 p67phox 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.
In the present study, we have demonstrated for the first time that Gβγ 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.
Gβγ subunits have been implicated in the regulation of Rho GTPase-driven cellular events. Thus, it was reported that Gβγ subunits could induce stress fiber formation and focal adhesion assembly in a Rho-dependent manner.25 One of the possible mechanisms for such Gβγ-dependent activation of Rho GTPases is activation of guanine nucleotide exchange factors. Two GEFs, Ras-GRF1 and P-Pex1, that could be activated by Gβγ subunits have been recently identified.8,9⇓ Interestingly, both proteins are Rac-specific guanine nucleotide exchange factors. Because Gβγ subunits could be released on stimulation of all G protein-coupled receptors, recognition that Gβγ 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.
Gβγ subunits are known to interact with PH domains of several signaling proteins. Thus, Gβγ subunits interact with PH domains of β-adrenergic receptor kinase, βARK (see review23), Bruton tyrosine kinase,3 and phospholipase βC226 and phospholipase βC3.27 It is believed that the interaction of Gβγ with the PH domains results in the activation of these enzymes. Because p114RhoGEF contains the PH domain, it is possible that Gβγ subunits activate p114RhoGEF via interaction with its PH domain. It is our future challenge to address the molecular mechanism of Gβγ-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: 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 Rac1-mediated signaling pathways and results in physiologically significant cellular events.
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.
Original received April 14, 2003; revision received September 15, 2003; accepted September 16, 2003.
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