Endothelin 1 Stimulates β 1 Pix-Dependent Activation of Cdc42 Through the G sα Pathway

Abstract

β₁Pix (PAK-interacting exchange factor) is a recently identified guanine nucleotide exchange factor (GEF) for the Rho family small G protein Cdc42/Rac. On stimulation with extracellular signals, GEFs induce the exchange of guanosine diphosphate to guanosine triphosphate, resulting in the activation of the small guanosine 5C-triphosphatases. This activation enables the signal to propagate to downstream effectors. Herein, we show that G_sα stimulation by cholera toxin increased Cdc42 activation by endothelin-1 (ET-1), whereas pertussis toxin had no effect. H-89, a protein kinase A (PKA) inhibitor, strongly inhibited Cdc42 activation by ET-1. Moreover, the overexpression of β₁Pix enhanced ET-1–induced Cdc42 activation. The essential role of β₁Pix in ET-1–induced Cdc42 activation was evidenced by the blocking of Cdc42 activation in cells expressing β₁Pix mutant lacking the ability to bind PAK (β₁Pix SH3m[W43K]) or mutant lacking GEF activity (β₁PixΔDH). The overexpression of mutant lacking the pleckstrin homology domain β₁PixΔPH, which is unable to bind phospholipids, had no effect on Cdc42 activation. These results demonstrate that β₁Pix, along with PKA, plays a crucial role in the regulation of Cdc42 activation by ET-1.

Introduction

The Rho family small guanosine 5C-triphosphatases (GTPases) have emerged as key regulators that mediate extracellular signaling pathways, leading to the formation of polarized actin-containing structures such as stress fibers, membrane ruffles, lamellipodia, and filopodia. Besides changes in cytoskeletal architecture, these GTPases mediate diverse biologic events, including stimulation of DNA synthesis, cellular transformation, and signaling to the nucleus (1, 2).

Rho GTPases cycle between inactive guanosine diphosphate (GDP)–bound and active guanosine triphosphate (GTP)–bound forms. Interconversion between these two forms is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins, and guanine nucleotide dissociation inhibitors. GEFs of the Dbl family stimulate activation of Rho GTPases by catalyzing GDP/GTP exchange of these G proteins (3–5). All members of this family contain the Dbl homology (DH) domain, which is responsible for catalytic activity. GEF proteins are activated in various ways, including phosphorylation by protein kinases (4–6). GEFs also contain a pleckstrin homology (PH) domain that is responsible for the interaction with phospholipids. PAK-interacting exchange factor (Pix) family proteins consist of two isoforms, αPix and βPix, and recently a new splice variant of βPix, designated as β₂Pix, has been identified (7). The human Pix family bind tightly through an N-terminal Src homology 3 (SH3) domain to a conserved proline-rich PAK sequence located at the C terminus and are colocalized with PAK to form activated Cdc42- and Rac1-driven focal complexes (8). Recently, Pix has been shown to form a trimolecular complex with PAK1 and p95PKL (also known as G protein–coupled receptor [GPCR] kinase-interacting target) (9). Furthermore, tyrosine-phosphorylated p95PKL can bind paxillin (10, 11) and therefore provides the link between Pix/PAK and focal complexes through this interaction. The presence of several domains allows Pix to interact with a variety of signaling proteins and suggests that Pix might have an important role in mediating the effects of extracellular signals (12–14).

In the present study, we demonstrated that the stimulation of a subunit of G_s protein by cholera toxin enhanced Cdc42 activation by endothelin-1 (ET-1). The overexpression of β₁Pix in mesangial cells enhanced Cdc42 activation by ET-1. We also showed that this activation is blocked by protein kinase A (PKA) inhibitor H-89.

Materials and Methods

Cell Culture and Transfection.

All materials for cell culturing were purchased from Invitrogen (Carlsbad, CA). Previously characterized simian virus 40–transformed human mesangial cells (HMCs) (15) were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) in a 37°C humidified incubator with 5% CO₂. Transient transfection of cells with mammalian expression vectors was performed using Lipofectamine 2000 (Life Technologies, Gaithersburg, MD) according to the manufacturer’s instructions.

Pulldown Assays of Rho Family GTPases.

Cells were transfected with empty vector, Myc-tagged β₁Pix, or its mutants for 24 hrs. After stimulation with ET-1 for 5 mins, cells were lysed in lysis/wash buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl₂, 1 mM EDTA, 1% glycerol, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). To measure the active GTP-bound form of endogenous Cdc42 in the cell lysates, we performed pulldown assay (Cytoskeleton, Denver, CO) using recombinant glutathione S-transferase (GST)–tagged PAK1-PBD·PaK-binding domain (PDK). Aliquots (500 μg) of the supernatants mixed with glutathione agarose with 10 μg of GST·PAK1·PBD were precipitated by centrifugation. Complexes were boiled in a Laemmli sample buffer and then separated on 15% SDS polyacrylamide gels. The separated proteins were immunoblotted using specific anti-Cdc42 antibody.

Reverse Transcription Polymerase Chain Reaction (PCR) Analysis.

Total RNA isolated from rat mesangial cells was reverse transcribed using Superscript reverse transcriptase (Invitrogen), oligo (dT) primers (Invitrogen), and deoxynucleotide triphosphate as specified by the manufacturer. β₁Pix was amplified by means of PCR using TITANIUM Taq polymerase (Clontech Laboratories, Inc., Palo Alto, CA) in the presence of deoxynucleotide triphosphate, the forward primer 5′-GGAATTCCAT-GACTGATAACGCCAACAGCCAA-3′, and the reverse primer 5′-GCTCTAGAGCTAGATTGGTCTCATCC-CAAGCAGG-3′. The PCR products were subjected to electrophoresis in a 1% acrylamide gel, and the results were visualized using a bioimaging analyzer. The β₁Pix cDNA was cut with EcoRI and XbaI and inserted into the EcoRI-XbaI site of pcDNA3.1/Myc-His vector. β₁Pix mutants β₁Pix SH3m(W43K), β₁PixΔDH, and β₁PixΔPH were made using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) (16).

GDP/GTP Exchange Assays.

The exchange assays were performed as previously described (17). For GTPγS binding, 2 μg of the recombinant GTPases were initially incubated for 5 mins in 60 μl of loading buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM EDTA, 0.2 mM dithiothreitol, 100 μM adenosine monophosphate–purine nucleoside phosphorylase [AMP-PNP], and 10 μM GDP) at room temperature. MgCl₂ was then added to a final concentration of 5 mM, and the incubation continued for an additional 15 mins. Finally, aliquots (20 μl) of GDP-loaded GTPases were mixed with 100 μg of lysates from cells overexpressing c-Myc-β₁Pix or c-Myc-β₁Pix(L238R, L239S) diluted in reaction buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM MgCl₂, 100 μM AMP-PNP, 0.5 mg/ml bovine serum albumin, and 5 μM [³⁵S]GTPγS) to initiate the exchange reaction (final volume, 100 μl) at room temperature. Aliquots (15 μl) of samples were taken at various time points from the reaction mixture and added to 10 ml of ice-cold phosphate-buffered saline. Bound and free nucleotides were separated by filtration through BA85 nitrocellulose filters. For the GDP dissociation assay, 10 μM radiolabeled [³H]GDP was used in the loading buffer instead of GDP, and 1 mM GTP was used in the reaction buffer instead of [³⁵S]GTPγS.

Results

We first sought to determine whether β₁Pix regulates Cdc42 activation by ET-1. Pix family proteins are GEFs for the small GTPase proteins Cdc42/Rac (8) and have been shown to signal via these proteins. Therefore, we studied the effect of β₁Pix and its inactive mutants on ET-1–induced Cdc42 activation in HMCs. In our experiments, Cdc42 activation was measured after ET-1 treatment of HMC-overexpressing wild-type β₁Pix or its mutants β₁Pix SH3m(W43K), β₁PixΔDH, or β₁PixΔPH (8). ET-1 induced Cdc42 activation in cells expressing empty vector, and this activation was enhanced by β₁Pix overexpression. By contrast, β₁PixΔDH, which lacks GEF activity, and SH3 domain–mutated β₁Pix SH3m(W43K), which lacks the ability to bind to PAK, significantly decreased ET-1–induced Cdc42 activation (Fig. 1A). The expression of β₁PixΔPH had no effect on Cdc42 activity. This result indicates that PAK (or another SH3 domain) and GEF activity of β₁Pix are essential for the regulation of Cdc42 activation by ET-1, whereas the PH domain is not.

To demonstrate that Cdc42 is specifically activated by ET-1, we treated the cells with toxin B, which specifically inhibits Cdc42. Figure 1B shows that toxin B completely blocked Cdc42 activation by ET-1.

We wanted to confirm that β₁Pix is working as a GEF for Cdc42. In vitro assays measured the Cdc42 GEF activity of β₁Pix (Fig. 2 ). β₁Pix enhanced the incorporation of GTPγS into purified Cdc42, whereas β₁Pix had no effect of GTPγS incorporation into the dominant negative form of Cdc42 (Fig. 2A ). In reciprocal experiments, [³H]GDP dissociation from purified Cdc42 was enhanced by immunoprecipitated c-Myc–tagged β₁Pix but not by the β₁Pix(L238R, L239S) mutant, which has GEF activity (Fig. 2B ). The GDP release assay confirmed that β₁Pix is a GDP/GTP exchange factor for Cdc42.

Specific small GTPases are activated by the phosphorylation of GEF (7, 8). Results of a previous study showed that PKA phosphorylates β₁Pix on Ser516 and Thr526 (16). Accordingly, we examined whether this kinase regulates ET-1–induced Cdc42 activation. For this purpose, we used H-89, a PKA inhibitor. Cells were starved for 24 hrs and stimulated with ET-1 for 15 mins. Preincubation with H-89 abolished ET-1–induced Cdc42 activation (Fig. 3). This result strongly indicates that PKA acts upstream to regulate Cdc42 activation. This finding is in agreement with previous data showing that the overexpression of a nonphosphorylatable form of β₁Pix, β₁Pix(S516A, T526A), prevents ET-1–induced Cdc42 activation (16). Collectively, the results herein strongly support the theory that β₁Pix phosphorylation by PKA occurs upstream of Cdc42 activation. This phosphorylation results in β₁Pix activation and its trans-location to focal adhesion complexes where it activates Cdc42. Indeed, H-89 treatment of the cells overexpressing β₁Pix or the expression of β₁Pix(S516A, T526A) blocked β₁Pix translocation to focal adhesion complexes (16).

It appears that activation of Cdc42 can be mediated by different GPCRs. ET-1 receptors are coupled to different G proteins depending on the cell type. In an attempt to determine which G protein is involved in ET-1–induced Cdc42 activation, we used different toxins. Cholera toxin, which adenosine diphosphate-ribosylates and permanently activates the α subunit of G_s protein, enhanced Cdc42 activation by ET-1 (Fig. 4, lane 3). Inhibition of G_i/o proteins by pertussis toxin had no effect on ET-1–induced Cdc42 activation (Fig. 4 , lane 4), indicating that G_sα protein is transducing the signal from ET-1 receptors downstream to Cdc42. This result corroborates our findings that PKA acts upstream of Cdc42 activation by ET-1. It is well established that the G_sα signaling pathway induces the activation of PKA through the production of cyclic adenosine monophosphate (cAMP) by adenylate cyclase.

Discussion

There is accumulating evidence that GPCR agonists activate small GTPases that, in turn, modulate a variety of biologic responses, including cell differentiation and growth (12). It has been shown that the cAMP analogue 8-Br-cAMP can stimulate Cdc42 (17), and in this study we tested the hypothesis that G_sα may mediate ET-1–induced Cdc42 activation. Treatment of HMCs by cholera toxin, which permanently activates G_sα proteins, enhanced ET-1–induced Cdc42 activation, whereas pertussis toxin, which inhibits G_i/o proteins, had no effect. This result suggests that G_sα mediates the activation of Cdc42 by ET-1. However, we cannot rule out the existence of other mechanisms that may activate adenylate cyclase that do not directly involve the G_sα subunit.

We showed that ET-1 stimulates Cdc42 by a PKA-dependent mechanism, as ET-1–induced Cdc42 activation was inhibited in the presence of the selective PKA inhibitor H-89. It has been proposed that PKA can directly modulate activity of some small GTPases. Activation of Rap1 and inhibition of RhoA were ascribed to their phosphorylation by PKA (18, 19). In line with these studies, we show herein that β₁Pix overexpression enhanced ET-1–induced Cdc42 activation, whereas deletion of the DH domain or the mutated SH3 domain strongly inhibited Cdc42 activation by ET-1. Moreover, it has been previously shown that β₁Pix can be phosphorylated by PKA in vitro on Ser516 and Thr526 (16). The overexpression of β₁Pix(S516A, T526A) inhibited Cdc42 activation and β₁Pix translocation to focal adhesion complexes (16).

The discovery of GEFs, GTPase-activating proteins, and GDP dissociation inhibitors has improved our understanding of how small G proteins are regulated. It is possible that subunits of activated heterotrimeric proteins coupled to GPCR can directly bind to GEFs, as recently demonstrated for G_α12/G_α13 and PDZ-RhoGEF (20–22). In addition, GPCR can regulate small G proteins through other pathways triggered by heterotrimeric G proteins, including tyrosine kinases, protein kinase C, and cAMP (23, 24).

Figure 1.
Role of β₁Pix in ET-1–induced Cdc42 activation. (A) HMCs were cotransfected with empty vector alone, Myc-tagged β₁Pix(WT), or the β₁Pix mutants β₁Pix SH3m(W43K), β₁PixΔDH, or β₁PixΔPH. After 24 hrs of transfection, cells were stimulated with ET-1 (100 nM) for 5 mins, and active Cdc42 was measured as described in the “Materials and Methods” section. (B) Cells were treated with toxin B (10 ng/ml) for 3 hrs before stimulation with ET-1 (100 nM) for 5 mins. The data are representative of three independent experiments.

Figure 2.
β₁Pix is a specific guanine nucleotide exchange factor for Cdc42. (A) Time course for the binding of [³⁵S]GTPγS to purified Cdc42(wt) or dominant negative Cdc42. Purified Cdc42 (solid circles) or its dominant negative form (open circles) were preloaded with GDP and then added to reaction incubations containing [³⁵S]GTPγS together with aliquots from HMC cell lysates overexpressing c-Myc–tagged β₁Pix. (B) Time course of the dissociation of [³H]GDP from purified Cdc42. Purified Cdc42 was preloaded with [³H]GDP and then added to reaction incubations containing 1 mM GTP together with aliquots from HMC cell lysates overexpressing β₁Pix (solid circles) or the β₁Pix(L238R, L239S) mutant (open circles). The data are expressed as means ± SE of three independent experiments.

Figure 3.
Effect of PKA inhibitor on ET-1–induced Cdc42 activation. HMCs were treated with H-89 (10 μM) for 45 mins and then stimulated with ET-1 (100 nM) for 15 mins. Activated Cdc42 was measured as described in the “Materials and Methods” section. The data are representative of three independent experiments.

Figure 4.
Effect of cholera and pertussis toxins on Cdc42 activation by ET-1. (A) HMCs were treated with cholera toxin (200 ng/ml) or pertussis toxin (100 ng/ml) for 4 hrs before ET-1 stimulation. Active Cdc42 was measured using pulldown assay. (B) Quantitative analysis of the results obtained in (A) by densitometry.

Footnotes

This study was supported by a grant from the American Heart Association grant (A.C.) and grant HL22563 from the National Institutes of Health (A.S.)

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