From the
Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599,
¶ Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599
Received for publication, December 20, 2002
, and in revised form, March 20, 2003.
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ABSTRACT |
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INTRODUCTION |
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Administration of forskolin, an activator of adenylate cyclase, or dibutyryl cAMP to cells stimulates morphological changes that are strikingly similar to those observed upon introduction of the Rho-specific inhibitor C3-transferase (13). From this initial observation, cAMP- and cGMP-dependent kinase (PKA and PKG) were demonstrated to phosphorylate RhoA on Ser188 (13, 14), thereby joining RhoA with Rap1a and Rap1b as small GTPases regulated by carboxyl PKA phosphorylation (15, 16). Although PKA phosphorylation is linked to Rap activation (17, 18), evidence indicates that it negatively regulates RhoA function. RhoA phosphorylation promotes formation of RhoA·RhoGDI complexes (13, 19) and enhances the ability of RhoGDI to extract RhoA from membranes (13, 20). In support of enhanced RhoGDI binding, the ability of RhoA to cycle from membranes has been linked to cAMP and cGMP signaling within cells (21, 22). Functionally, constitutively active RhoA containing an S188A mutation is more effective in blocking actin dissolution promoted by dibutyryl cAMP (23) or 8-Bromo-cGMP (24), and constitutively active RhoA require a S188A mutation to promote stress fibers in cells co-transfected with constitutively active PKG (14).
On the other hand, the significance of RhoA phosphorylation to PKA/PKG regulation of its signaling is unclear considering recent reports. Thromboxane receptor stimulation promotes RhoA activation through a G13 and a PKA-sensitive pathway(s) (25). Manganello et al. (26) demonstrated recently that PKA phosphorylates G
13 subunit to promote
subunit uncoupling, thereby effectively shutting down receptor activation of RhoA. Moreover, PKG inhibited G
13 activation of serum response factor transcription by impeding activation of RhoA (27). Notably, PKG also inhibited serum response factor transcriptional activity promoted by constitutively active forms of Rho kinase, protein kinase N, or protein kinase C-related kinase 2, indicating that PKG antagonizes RhoA signaling downstream of effector regulation. Thus, an emerging picture is that PKA/PKG negatively regulates RhoA at multiple levels.
In this work, we expressed Ser188 phosphomimetic RhoA proteins, in the absence of constitutive RhoA activity and aberrant PKA/PKG signaling, to assess the contribution of PKA phosphorylation to RhoA function. We report here that addition of a charged group to Ser188 upon phosphorylation negatively regulates RhoA activity in vivo and indicates that this occurs through enhanced RhoGDI interaction rather than direct perturbation of GEF, GAP, or geranylgeranyl transferase activity.
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EXPERIMENTAL PROCEDURES |
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ConstructsRhoA mutations (S188A, S188D, S188E, C190A) and RhoG mutation (S187A) were created through PCR mutagenesis using the QuikChange mutagenesis kit (Stratagene). Mutations were confirmed by DNA sequencing, and cDNAs were subcloned into either pGEX4T-1 (Amersham Biosciences) or pCMV-Myc (Clontech) using EcoRI and XhoI restriction sites. pGEX4T-1-RhoGDI (human) was created by subcloning RhoGDI cDNA into pGEX4T-1 using BamHI and EcoRI restriction sites. pPro-HT-Dbl (DH/PH) was a gift of Dr. K. Rossman, University of North Carolina, Chapel Hill, NC.
Antibody ProductionAntibody production and purification was performed by Covance Research Products Inc. Rabbits were immunized with a phosphoserine peptide containing the proximal nine residues of RhoA (RRGKKKPSGC) thiol bonded to keyhole limpet hemocyanin. Antibodies were isolated by negative affinity purification with immobilized unphosphorylated RhoA peptide followed by positive affinity purification with immobilized phosphorylated RhoA peptide and low/high pH elutions.
Fusion ProteinsGST-Rho fusion proteins (GST, GST-RhoA, GSTRhoG, GST-Cdc42, GST-Rac1, GST-RhoGDI) were purified from BL21 Escherichia coli cells (Stratagene) using glutathione-Sepharose 4B (Amersham Biosciences). Proteins were eluted with free and reduced glutathione in Tris-buffered saline medium (50 mM Tris, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol) and stored in 30% glycerol. His6-Dbl DH/PH was purified from BL21 E. coli cells using nickel-nitrilotriacetic acid-Sepharose (Qiagen) and eluted with 20 units of tobacco etch virus protease (Invitrogen) according to the manufacturer's specifications. RhoGDI was produced by cleaving RhoGDI from GST-RhoGDI-Sepharose with 5 units of bovine thrombin (Sigma). RhoGDI was subsequently cleared with benzamidine-agarose (Sigma) to remove thrombin from incubation buffer. Recombinant geranylgeranyl transferase was purchased from Sigma. Phosphate-binding protein carrying an A197C mutation was purified and fluorescently labeled with N-[21-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide as described previously (28) from E. coli ANCC75 bacteria carrying plasmid pSN5182/7 (a kind gift of Dr. M. R. Webb, National Institute for Medical Research, London). His6-Larg DH/PH and His6-Vav2 DH/PH/CRD were kind gifts of Dr. M. Booden, University of North Carolina, Chapel Hill, NC. His6-DH/PH Dbs was a gift of Dr. K. Rossman, University of North Carolina, Chapel Hill, NC. Full-length p190RhoGAP was a generous gift of Dr. J. Settleman, Harvard Medical School.
Rho Protein PhosphorylationTo measure cPKA phosphorylation of Rho fusion proteins (see Fig. 1), 2.0 pmols of fusion proteins, 1.5 units of cPKA (Sigma), 330 pmols of ATP, and 3.3 pmols of [-32P]ATP (Amersham Biosciences) were incubated in phosphorylation buffer (50 mM Tris, 5 mM MgCl2,1mM dithiothreitol) at 30 °C for 20 min. Samples were removed, resolved by 15% SDS-PAGE, and then analyzed by autoradiography of dried gels. For large scale production of phosphorylated GST-RhoA, 120 µg (2.5 nmols) of GST-RhoA-Sepharose, 150 units of cPKA, and excess ATP (2 mM) were rotated in 1 ml of phosphorylation buffer for 90 min at 25 °C. Phosphorylation efficiency was estimated by scaling down reaction to 1% the amount of GST-RhoA and cPKA. Specifically, 1.2 µg (25 pmols) of GST-RhoA-Sepharose, 1.5 units of cPKA, 2.12 nmols of ATP, and 3.3 pmols of [
-32P]ATP (1:645 dilution) were incubated in 10 µl of phosphorylation buffer with rotation for 30 min at 25 °C. Reactions were washed extensively, and incorporation of 32P was calculated and corrected with values obtained from GSTSepharose reactions. On average, 0.0300.035 pmols of 32P was incorporated by RhoA into each reaction, indicating 8090% phosphorylation efficiency ((645 x 0.03 pmol)/25 pmol GST-RhoA). To measure phosphorylation of geranylgeranylated RhoA, 2.0 pmol of unmodified or prenylated RhoA (see below) were incubated with 1.5 units of cPKA, 330 pmols of ATP, and 3.3 pmols of [
-32P]ATP in phosphorylation buffer at 30 °C for 20 min. Samples were boiled to stop the reaction and then TX-114 was added to a concentration of 1%, and phase partitioning was performed as described previously for Rho proteins (29). The detergent phase was washed with three 20-fold volumes of phosphate-buffered saline to ensure removal of non-prenylated RhoA. Both aqueous and detergent phases were aliquoted and analyzed for RhoA phosphorylation by measuring release of radioactivity and visualizing by SDS-PAGE and autoradiography.
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In Vitro Guanine Nucleotide Exchange Factor AssaysFluorescence spectroscopic analysis of N-methylanthraniloyl (mant)-GTP (Biomol) incorporation into GDP-preloaded GST-Rho proteins was carried out using a FLUOstar fluorescence microplate reader at 25 °C similar to as described previously (30). 2 µM GST-RhoA or 1 µM prenylated GSTRhoA was prepared and allowed to equilibrate in exchange buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 50 µg/ml bovine serum albumin, 1% glycerol). 500 nM mant-GTP and varying amounts of DH/PH (Larg, Dbl, or Dbs) or DH/PH/CRD (Vav2) protein were added at the indicated time, and the relative mant fluorescence (excitation = 360 nm, emission = 460 nm) was monitored. Experiments were performed in duplicate for every condition.
GAP AssaysGAP assays were performed in a Spectramax Gemini XS fluorescence microplate reader (Molecular Devices) (excitation = 425 nm, emission = 465 nm) at 25 °C by incubating 1 µM GTP-loaded GST-RhoA together with 2.5 µM fluorophore-labeled phosphate-binding protein in the absence or presence of 250 or 750 pM purified p190RhoGAP in a buffer containing 20 mM Tris, pH 7.6, and 1 mM MgCl2 at a total volume of 100 µl. Upon hydrolysis of GTP, the resulting free Pi is bound rapidly (1.36 x 108 M-1 s-1 at 22 °C) and with high affinity (about 100 nM) by the phosphate-binding protein, resulting in a 13-fold increase in the fluorescence at 465 nm (28). Because of this, the observed change in fluorescence corresponds to the rate and amount of Pi released from the GTPase.
RhoGDI Competition Assays2 µM of the indicated GST-RhoA fusion protein was incubated for 10 min in exchange buffer containing 0.5, 1.0, or 2.0 µM RhoGDI to promote RhoA·RhoGDI complexes. Vav2 DH/PH/CRD and mant-GTP nucleotides were then added and allowed to equilibrate with mixing for 15 s, and the rate of mant-GTP incorporation was measured. Linear velocity of exchange was determined as described previously (31). Briefly, baseline and GEF-induced nucleotide exchange rates were calculated by dividing the change in emission at 460 nm by change in time. Values were averaged and standard deviations were calculated for each reaction. Velocity was considered linear as long as the regression value of the exchange slope was greater than 0.97. Data from these and all other experiments were considered significantly different if the p values, as determined by two-tailed t tests, were <0.02.
Geranylgeranyl Transferase AssaysGST or the indicated GSTRhoA-Sepharose (5 pmols), 100 pmols of 3H-geranylgeranyl pyrophosphate (PerkinElmer Life Sciences), and 0.36 units of geranylgeranyl transferase were incubated in Tris-buffered saline medium, pH 7.6, for 20 or 60 min. Samples were resolved by 15% SDS-PAGE, and gels were fixed, incubated in Amplify solution (Amersham Biosciences), and then exposed by autoradiography. Alternatively, reactions were collected at 20, 40, 60, and 80 min in duplicate, washed extensively, and placed in scintillation buffer, and the extent of 3H-geranygeranyl was quantified. To generate geranylgeranylated RhoA for kinetic assays, 50 µg (1 nmol) of GST-RhoA or phosphorylated GST-RhoA were incubated in solution with geranylgeranyl pyrophosphate (25 nmols) and in the absence or presence of 10 units of geranylgeranyl transferase overnight at 30 °C. Proteins were either GDP-loaded (GEF assays) or GTP-loaded (GAP assays) and eluted with small volumes of free glutathione in Trisbuffered saline containing 0.25% deoxycholate. Through dilutions, reactions contained either 0.025% (GEF) or 0.015% deoxycholate (GAP). Unmodified RhoA (no transferase) was utilized as an experimental control for the influence of prenylation on GTPase activity (see Fig. 5, C and D).
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Transfections and Production of Stable CellsNIH 3T3 fibroblast cells were transfected with the expression vectors indicated in each experiment according to the manufacturer's protocol using LipofectAMINE PLUS (Invitrogen). After introduction of the expression vectors for 3 h, the transfection medium was replaced with growth medium for 16 h. For creation of stable cell lines, NIH 3T3 cells were transfected with 1 µg of pPUR (Clontech) only or cotransfected with 0.05 µg of pPUR and 1 µg of pCMV-Myc RhoA construct and then selected in 10 µg/ml puromycin (Sigma). Clonal lines were established and screened for expression by Western blotting lysates with anti-c-Myc (clone 9E10; Sigma) monoclonal antibodies.
RhoA GTP Profile AssaysThe amount of activated, GTP-bound RhoA protein was measured using a technique similar to the method described by Ren et al. (32). Briefly, transfected or stable cells were lysed in 300 µl of 50 mM Tris, pH 7.4, 10 mM MgCl2, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, and protease inhibitors. 500750 µg of lysates were cleared at 16,000 x g for 5 min, and the supernatant was rotated for 30 min with 30 µg of GST-RBD (GST fusion protein containing the Rho-binding domain (RBD; amino acids 789) of Rhotekin) bound to glutathione-Sepharose beads. Samples were washed in 50 mM Tris, pH 7.4, 10 mM MgCl2, 150 mM NaCl, 1% Triton X-100, and protease inhibitors. GST-RBD pulldowns and lysates were then Western blotted with anti-c-Myc. To quantify GST-RBD pulldowns, Western blots of lysates and corresponding GST-RBD pulldowns from three unique experiments done in duplicate were scanned, and densitometry was performed using Metamorph imaging software.
Calculation of Cell SpreadingFor all experiments cells were replated in the absence of puromycin the day before experiments, trypsinized, washed twice in Dulbecco's modified Eagle's medium, and then suspended before plating for 30 min in Dulbecco's modified Eagle's medium and 0.5% fatty acid-free bovine serum albumin. Suspended cells were plated on fibronectin-coated coverslips for 20, 40, and 60 min. Coverslips were fixed and stained with Coomassie Blue (2% Brilliant Blue, 45% methanol, and 10% acetic acid) for 10 min and then washed with water and mounted. The relative areas of individual cells from Metamorph images were quantified with the use of NIH Image software. At least 50 cells taken from 10 arbitrary fields were counted from each coverslip, with two coverslips counted for every condition.
ImmunofluorescenceCells were plated and grown overnight on fibronectin-coated glass coverslips in the presence of serum. Cells were washed free of serum with serum-free medium and then incubated in serum-free media containing either Me2SO vehicle or 25 µM forskolin (Sigma) for 20 min. Cells were then fixed for 15 min in 3.7% formaldehyde in phosphate-buffered saline permeabilized for 5 min in 0.5% Triton X-100 in phosphate-buffered saline. Filamentous actin was labeled with Texas Red-conjugated phalloidin (Molecular Probes). Images were obtained on an Axiophot microscope (Zeiss) using a MicroMAX 5-MHz cooled CCD camera (Princeton Instrument) and Metamorph Image software (Universal Imaging Corp.).
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RESULTS |
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GEF Exchange against Phosphorylated RhoAGEFs bind GDP-bound Rho proteins and induce a transition state that promotes uptake of free GTP nucleotide, thereby stimulating Rho activation. It has been reported that cAMP inhibits GEF activation of RhoA in leukocytes (37), thereby raising the possibility that RhoA phosphorylation impairs GEF exchange. To address this hypothesis, GST-RhoA was phosphorylated in vitro with cPKA and compared with control protein for the ability of DH/PH domain-containing GEFs to promote nucleotide uptake. Using [-32P]ATP, GST-RhoA was estimated to be at least 8090% phosphorylated by cPKA. Basal incorporation of mant-GTP was identical for phosphorylated and control GST-RhoA proteins (Fig. 3A). Larg (Fig. 3A), Vav-2 (Table I), Dbl, and Dbs (not shown) all displayed similar exchange activity against control and phosphorylated GST-RhoA at multiple GEF concentrations. This finding also extended to phosphorylated and control geranylgeranylated RhoA proteins (Fig. 3B). Lastly, the phosphomimetics GST-RhoA(S188E) and GSTRhoA(S188D) were equivalent substrates as wild-type GSTRhoA or GST-RhoA(S188A) controls (Fig. 3C). These data demonstrate that phosphorylation of the RhoA tail does not directly interfere with GEF-induced exchange.
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RhoA Ser188 Phosphomimetics Display Increased Binding to RhoGDIcPKA-phosphorylation of RhoA has been reported to increase its affinity toward RhoGDI, therefore we examined the ability of RhoGDI to bind RhoA proteins using solution-phase binding competition of RhoGDI and GEF molecules. Briefly, GST-RhoA proteins were pre-incubated with no or varying amounts of full-length RhoGDI for 10 min to promote the formation of RhoA·RhoGDI complexes. Vav2 DH/PH/CRD and mant-GTP nucleotides were subsequently added and allowed to equilibrate with mixing for 15 s and then mant-GTP incorporation was measured. Vav2 exchange was linear over the initial 150200 s for phosphorylated, phosphoserine-mimetics, and control GST-RhoA proteins (see Fig. 4A and Tables I and II). As RhoGDI concentrations were increased, the velocity of nucleotide incorporation was both reduced and lengthened for all RhoA molecules (Tables I and II). Importantly, both phosphorylated and phosphomimetic GST-RhoA proteins exhibited a significant reduction in GEF exchange compared with control proteins at equal concentrations of RhoGDI (Fig. 4B). The bulkier S188E mutation resulted in slightly greater inhibition than the S188D mutation, suggesting tighter binding to RhoGDI. As prior experiments demonstrated that GEF exchange was equivalent for all RhoA proteins at multiple concentrations, these data provide additional evidence to previous reports that RhoA phosphorylation promotes or stabilizes RhoA·RhoGDI complex formation. Moreover, as these proteins are not post-translationally modified, these data indicate that adding a negative charge to the carboxyl terminus of RhoA enhances the protein-protein interactions of RhoA and RhoGDI.
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RhoA Phosphorylation Does Not Affect GAP ActivityRhoGAPs negatively regulate Rho activity by promoting the intrinsic GTP-hydrolyzing activity of Rho proteins. As cPKA phosphorylation is also hypothesized to negatively regulate RhoA, we analyzed whether p190RhoGAP activity against phosphorylated RhoA is perturbed in vitro. Basal and p190RhoGAP-induced GTP hydrolysis by phosphorylated, phosphoserine-mimetic, and control GST-RhoA proteins was essentially identical (Fig. 5, A and B). Moreover, p190RhoGAP activity against geranylgeranylated RhoA was also insensitive to PKA phosphorylation, although prenylated RhoA had a slightly lower basal and GAP-induced hydrolysis rate (Fig. 5, C and D). Together, these data indicate that phosphorylation of RhoA does not influence the ability of p190RhoGAP to bind and stimulate GTPase activity.
RhoA Phosphorylation Does Not Affect Geranylgeranyl Transferase ActivityIncubation of purified cellular membranes with cPKA promotes RhoA extraction from membranes by RhoGDI (13, 20). As non-prenylated RhoA is a cellular PKA target, we analyzed whether cPKA phosphorylation of RhoA impairs addition of a geranylgeranyl moiety as an additional level of regulation. Geranylgeranyl transferase possessed equivalent activity against phosphorylated and control protein GST-RhoA proteins (Fig. 6, A and B). Further, Ser188 mutants were also comparable substrates (Fig. 6C). Together, these data indicate that phosphorylation of the RhoA tail does not directly alter geranylgeranyl transferase modification of RhoA.
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Transient Expression of Ser188 RhoA MutantsNIH 3T3 cells transiently transfected with Myc epitope-tagged RhoA, RhoA(S188A), RhoA(S188D), or RhoA(S188E) displayed enhanced stress fibers compared with mock-transfected cells (data not shown), indicating that all transient proteins were functional. The extent of Myc-RhoA GTP loading was evaluated using GST-RBD pulldowns (32). Both phosphoserine-mimetic Myc-RhoA proteins, with Myc-RhoA(S188E) being the more significant, displayed reduced GTP loading in comparison with Myc-RhoA and Myc-RhoA(S188A) proteins (Fig. 7). Myc-RhoA(C190A) proteins were not appreciably GTP-loaded under these conditions and are included as a negative control for GTP loading. These data provide evidence that addition of a negative charge by phosphorylation of Ser188 is sufficient to negatively regulate RhoA activation.
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Stable Expression of Ser188 RhoA MutantsTo examine whether a functional differences exists between Ser188 mutant RhoA proteins, stable clones of NIH 3T3 cells expressing Myc-RhoA, Myc-RhoA(S188A), or Myc-RhoA(S188E) were isolated. Two independent clones were chosen for each based solely on expression level and characterized further. Western blots revealed that Myc-RhoA migrates at higher apparent molecular weight than the endogenous RhoA proteins; therefore it was evident that all clones express Myc-RhoA protein at a significantly lower level than endogenous RhoA (Fig. 8A). GST-RBD pulldowns established that, contrary to transiently expressed proteins; there is little or no difference in the GTP-loading profile of stably expressed mutant Myc-RhoA proteins, with the exception of the non-prenylated mutant, Myc-RhoA(C190A) (Fig. 8B).
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Activity of Stably Expressed RhoA ProteinAs RhoA is hypothesized to antagonize cell spreading (34), NIH 3T3 clones were allowed to spread on fibronectin and collected at the indicated time, and rates of cell spreading were quantified (Fig. 9A). Both Myc-RhoA(S188E) clones spread at a similar rate as a pool of mock-transfected clones, whereas all the Myc-RhoA and Myc-RhoA(S188A) clones spread significantly slower. Additionally, a pool of Myc-RhoA(S188E)-expressing cells (five clones) that express a higher amount of Myc-RhoA (Fig. 9B, inset) still spread significantly faster than a corresponding pool of Myc-RhoA(S188A)-expressing cells (Fig. 9B).
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RhoA signaling has been found to antagonize the morphological affects of cAMP (23); therefore the effects of forskolin on stress fiber organization were examined for the stable clones. Mock-expressing cells revealed dissolution of stress fibers following forskolin activation of adenylate cyclase (Fig. 10, panel above line). A minority of cells expressing wild-type Myc-RhoA were protected from filamentous actin disassembly, whereas cells expressing Myc-RhoA(S188A) were completely protected against the morphological affects of cAMP (Fig. 10, panels below line). On the other hand, both Myc-RhoA(S188E)-expressing clones uniformly displayed stress fiber dissolution (Fig. 10, panels below line). Together, these data provide evidence that addition of a negative charge to Ser188 by phosphorylation is sufficient to attenuate RhoA activity.
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DISCUSSION |
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Unlike Rap1a and Rap1b, which are easily isolated as phosphoproteins from cells (15, 16), a clear demonstration of intracellular RhoA phosphorylation is lacking. Lang et al. (13) were able to immunoprecipitate only small amounts of 32P-labeled RhoA from intact cells, whereas Essler et al. (38) reported recently that a significant charge shift of RhoA was not observed following cAMP stimulation of HUVEC cells. To address this problem, we created antibodies that specifically recognize the phosphorylated form of RhoA. When expressed in cells, the non-prenylated RhoA (C190A) was a cellular target of PKA following forskolin stimulation and during cell spreading, an event associated with low RhoA (32, 34) and high PKA activity (35, 36). Although limited by antibody constraints, these results provide the first conclusive evidence, at least in the case of non-prenylated RhoA, that Ser188 is phosphorylated in response to cellular PKA activation in vivo.
Treatment of cells with cAMP or cGMP agonists is associated with translocation of RhoA from membranes (13, 14). Nonprenylated RhoA is efficiently phosphorylated within the cell (Fig. 2), suggesting that it may be a target of PKA regulation. We therefore analyzed whether Ser188 phosphorylation controls RhoA cycling by impeding prenylation. In vitro assays demonstrated that geranylgeranyl transferase activity was not sensitive to the presence of a phosphate on Ser188. As events of Rho protein prenylation are poorly understood, it remains possible that phosphorylation of this residue affects post-translation modification of RhoA through mechanisms not reflected by in vitro assays. However, regulating RhoA cycling at the step of prenylation appears to be an inefficient and long term approach for a kinase that depends on localized and transient activity (reviewed in Ref. 39).
We found that transiently expressed phosphomimetic Myc-RhoA proteins were poorly loaded with GTP nucleotides compared with control proteins. We also demonstrated that Myc-RhoA(S188E) protein signaling is significantly attenuated compared with control proteins and that this difference was not reflected in the GTP-loading profiles of stably transfected cells. Both of these results are consistent with enhanced sequestration of Myc-RhoA(S188E) by RhoGDI. In the case of transiently expressed protein, sequestration by RhoGDI would reduce the accessibility of GEFs, as is observed in vitro (Tables I and II). In the case of stably expressed proteins, Myc-RhoA(S188E) that has become GTP-loaded over time could still be aberrantly sequestered by RhoGDI. This would be reflected by low RhoA activity in the cell (see Figs. 9 and 10) but not by RBD pulldowns because of a limitation of the assay. The lysis conditions of RBD pulldown assays requires disruption of both RhoA(GTP)·membrane and RhoA(GTP)·effector complexes to form RhoA(GTP)·GST-RBD complexes that are driven by the vast molar excess of GST fusion protein. Not unexpectedly, we found that the lysis conditions completely disrupt RhoA·RhoGDI complexes,2 and have been unable to find lysis conditions that liberate RhoA(GTP) for RBD pulldown assays and preserve RhoA·RhoGDI binding. It is probable, therefore, that a portion of the stably expressed and GTP-loaded Myc-RhoA(S188E) protein is released from inactive RhoA(GTP)·RhoGDI pools, in addition to active RhoA-(GTP)·effector complexes. Thus, GST-RBD pulldowns, while an effective tool for measuring RhoGAP-mediated inhibition (34) or GEF activation of RhoA (31), will not reflect RhoGDI inhibition by sequestration.
In addition to enhanced binding to RhoGDI, Myc-RhoA(S188E) proteins may have lower binding affinity to an effector, such as Rho kinase (23). However, we found that constitutively active GST-RhoA(63L, S188E) was as efficient as GST-RhoA(63L, S188A) proteins in pulling down Rho kinase from NIH cell lysates.3 Further, stably expressed phosphomimetic RhoA proteins bound the RBD domain of the Rho effector Rhotekin (Fig. 8B). Therefore, we propose that attenuation of phosphomimetic RhoA activation and activity is because of cytosolic sequestration of RhoA driven by enhanced RhoGDI interactions that are observed in vitro.
The data presented here provide information regarding RhoA phosphorylation in context of regulatory protein interactions and demonstrate that the addition of a negative charge to Ser188 is sufficient to diminish both RhoA activation and activity within the context of a cell. As PKA also uncouples GEF activation from receptor stimulation (26, 37) and antagonizes microfilament integrity by directly phosphorylating and inhibiting myosin light chain kinase (40), it is becoming clear that PKA inhibits RhoA signaling pathways at multiple levels. It will be important in future work to establish where and when RhoA phosphorylation occurs within the cell and to address whether PKA phosphorylation works in parallel with RhoGAP-mediated inhibition of RhoA during events of cell protrusion and migration.
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FOOTNOTES |
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To whom correspondence should be addressed: Lineberger Comprehensive Cancer Center, CB 7295, University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-1904; Fax: 919-966-1856; E-mail: hawkeye{at}med.unc.edu.
1 The abbreviations used are: GEF, guanine nucleotide exchange factor; cPKA, catalytic domain of protein kinase A; GAP, GTPase activating protein; DH, Dbl homology; PH, pleckstrin homology; CRD, cysteine-rich domain; GST, glutathione S-transferase; mant, N-methylanthraniloyl; RBD, Rho-binding domain of Rhotekin; RhoGDI, Rho guanine-dissociation inhibitor; ggRhoA, geranylgeranylated RhoA; PKA, cAMP-dependent kinase; PKG, cGMP-dependent kinase.
2 S. M. Ellerbroek and K. Burridge, unpublished results.
3 S. M. Ellerbroek and K. Burridge, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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