Regulation of RhoA Signaling by the cAMP-dependent Phosphorylation of RhoGDIα*

Background: cAMP-induced phosphorylation of RhoA has been considered to inhibit RhoA signaling, causing cell rounding. Results: Knockdown of RhoGDIα blocks cAMP-induced cell rounding, and RhoGDIα-WT expression but not RhoGDIα-S174A expression recovers. Conclusion: Phosphorylation of RhoGDIα likely inhibits RhoA by stabilizing a active RhoA-RhoGDIα complex. Significance: This may underlie Gs/cAMP-induced cross-talk with Gq/G13/RhoA signaling. RhoA plays a pivotal role in regulating cell shape and movement. Protein kinase A (PKA) inhibits RhoA signaling and thereby induces a characteristic morphological change, cell rounding. This has been considered to result from cAMP-induced phosphorylation of RhoA at Ser-188, which induces a stable RhoA-GTP-RhoGDIα complex and sequesters RhoA to the cytosol. However, few groups have shown RhoA phosphorylation in intact cells. Here we show that phosphorylation of RhoGDIα but not RhoA plays an essential role in the PKA-induced inhibition of RhoA signaling and in the morphological changes using cardiac fibroblasts. The knockdown of RhoGDIα by siRNA blocks cAMP-induced cell rounding, which is recovered by RhoGDIα-WT expression but not when a RhoGDIα-S174A mutant is expressed. PKA phosphorylates RhoGDIα at Ser-174 and the phosphorylation of RhoGDIα is likely to induce the formation of a active RhoA-RhoGDIα complex. Our present results thus reveal a principal molecular mechanism underlying Gs/cAMP-induced cross-talk with Gq/G13/RhoA signaling.

RhoA has been found to regulate cellular morphology, based on a number of observations. RhoA is a substrate of C3-toxin and Clostridium difficile toxin B by which it is inactivated through ADP-ribosylation at Asn-41 (12), and glycosylation at Thr-37 (13), respectively, resulting in morphological changes in which the cells become small and rounded (cell rounding). In 1996, Lang et al. (14) reported that PKA phosphorylates RhoA at Ser-188 in vitro and proposed that this plays a key role in the inactivation of this RhoGTPase. Their proposed mechanism is that RhoA-GTP is phosphorylated by PKA and extracted from the membrane in its GTP-bound form by RhoGDI␣, thus terminating RhoA-GTP signaling prematurely (14). They further postulated that this RhoA inactivation underlies the morphological changes of cytotoxic lymphocytes such as those induced by C3 toxin. It is noteworthy that even in its GTP-bound active form, phosphorylated RhoA may bind to RhoGDI␣. More recently many groups have reported similar findings for PKA or PKG in different types of cells, such as neuronal cells, renal fibroblasts, NIH3T3 cells, and so on, and speculated that a defective RhoA inactivation system might be related to diseases such as hypertension (15,16).
Some previous studies have demonstrated the phosphorylation of geranylgeranylated RhoA at Ser-188 in intact cells (17,18) and in vivo (19). Others have reported, however, that they could not detect the phosphorylation of RhoA in intact cells although many other studies have shown the in vitro phosphorylation of RhoA at Ser-188 by PKA or PKG (14,20,21). In addition, Bolz et al. (22) have reported that they failed to observe the PKG phospho-resistant effects of the RhoA-S188A mutant that were expected. By overexpression of the RhoA-S188A mutant in the resistant artery, they tried unsuccessfully to inhibit the effects of RhoA phosphorylation through the SNP-PKG pathway. These discrepancies might be due to the cells used, but another mechanism of inactivation of RhoA by PKA/PKG has also been postulated (23).
An alternative candidate for the PKA substrate may be RhoGDI␣. RhoGDI␣ plays important roles in RhoGTPase regulation systems as a chaperon (6,24,25) and is well known as a partner of phosphorylated RhoA. The PKA-induced phosphorylation of RhoGDI␣ at Ser-174 was reported previously by two groups. DerMardlrosslan et al. (26) suggested in their study that PKA phosphorylates RhoGDI␣ only at Ser-174 in vitro, whereas Pak1 phosphorylates RhoGDI␣ at both Ser-101 and Ser-174. Qiao et al. (27,28) demonstrated that PKA phosphorylates RhoGDI␣ at Ser-174 in vitro and when transfected into intact cells and speculated that phosphorylated RhoGDI␣ might inhibit RhoA. It must be noted, however, that these intact cell studies were conducted in the presence of endogenous RhoGDI␣, which should exist at a 3-fold higher level than RhoA, and the analyses failed to show any interaction between RhoA and phosphorylated RhoGDI␣.
In our current study, we observed that cardiac fibroblasts expressing a constitutively active and phosphorylation-resistant mutant RhoA, RhoA-G14V/S188A, undergo PKA-induced morphological changes, when RhoGDI␣ is co-expressed. This suggests that these morphological changes cannot be explained by the phosphorylation of RhoA alone. By performing a series of experiments involving: 1) a specific knockdown of endogenous RhoGDI␣ using siRNA; 2) re-expression of RhoGDI␣-WT or -S174A; 3) phosphorylation analysis in immunoprecipitated RhoGDI␣; and 4) coimmunoprecipitation studies between active RhoA and RhoGDI␣, we have elucidated in our present analyses that the cAMP-induced phosphorylation of RhoGDI␣ at Ser-174 likely plays a key role in the cAMP-induced sequestration of RhoA in the cytosol and in the morphological changes in cardiac fibroblasts.
Immunocytochemistry and Immunofluorescence Staining-Actin staining by Texas Red X-phalloidin (Invitrogen) was performed using standard procedures. Briefly, cells were placed onto the glass coverslips, washed twice with ice-cold PBS, and fixed with 4% fresh paraformaldehyde (Wako)/PBS for 10 min. After two further washes in PBS, the cells were permeabilized in 0.1% Triton X-100 for 5 min. After two further washes in PBS, the cells were blocked using 1% BSA/PBS at room temperature for 30 min and stained using Texas Red X/phalloidin (1:60) diluted in 1% BSA/PBS at room temperature for 30 min for actin.
cAMP Assay-cAMP accumulation in cardiac fibroblasts was assayed as described previously (32,33,36). Briefly, cardiac fibroblasts were seeded into 24-well plates at 0.2 ϫ 10 5 cells/ well. Twenty-four hours later, the medium was replaced with serum-free medium to which [ 3 H]adenine (2 Ci/ml, Amersham Biosciences) had been added. After a further 24 h, the cells were washed and incubated for 30 min with or without the indicated concentrations of agonists in the presence of 1 mM 3-isobutyl-1-methylxanthine. The reaction was terminated by removing the medium and lysing the cells in 5% trichloroacetic acid (500 l/well) containing ATP and cAMP (each at 1 mM). Membrane Localization Assay-Cardiac fibroblasts (6 ϫ 10 5 ) were seeded into 100-mm dishes or HEK293 cells (1.5 ϫ 10 6 ) were seeded into 60-mm dishes without or with siRNA and DNA transfection. Twenty-four hours later (cardiac fibroblasts) or 36 h later (HEK293 cells), when the cells were subconfluent, the medium was replaced with serum-free medium. At 48 h, the cells were harvested in ice-cold PBS after incubation with isoproterenol for 1 h and in the case of cholera toxin (CTX) 3 for 3 h. After centrifugation (400 ϫ g for 5 min at 4°C), the cells were lysed in hypotonic buffer (20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 2 mM MgCl 2 , 1 mM EGTA, proteinase inhibitors) for 10 min, and then sheared using Dounce homogenization (15 times). The nuclear fractions and unbroken cells were removed by centrifugation (800 ϫ g for 5 min at 4°C). The supernatants were then ultracentrifuged (160,000 ϫ g for 30 min at 4°C) and the membrane and soluble cytosol fractions were separated. The pellet was lysed as a membrane fraction in lysis buffer (40 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl 2 , 1 mM EGTA, 1% Triton X-100, and proteinase inhibitors) for 1 h at 4°C, and centrifuged (200 ϫ g for 5 min at 4°C). The supernatant was next mixed with the same volume of Laemmli sample buffer, and boiled for 5 min. Membrane protein extracts from the cardiac fibroblasts or HEK293 cells were then separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with monoclonal anti-RhoA antibody (1:500 dilution, Santa Cruz, 26C4) or anti-Myc antibody (1:2,000 dilution, MBL PL14).
The sequences of siRNA molecules targeting human RhoGDI␣ (Ser-698; Ambion) are, CCAGCATACGTACAG-GAAATT; and the homologous HA-B. taurus RhoGDI␣ sequences are, forward CCAGCACACGTACAGGAAAGG, and the added silent mutation to avoid siRNA interfering are: forward, CCAGCACACGTACCGCAAAGG. The underlined nucleotides are not complementary to the corresponding siRNA.
Transfection of siRNA alone or siRNA with DNA were performed using a standard protocol for Lipofectamine2000. First, rat cardiac fibroblasts were plated in 12-well dishes (0.2 ϫ 10 5 cells/well) and after 24 h, when confluence of the cells was about 50%, siRNA (each at 20 nM) and EGFP-C3 as a control or HA-RhoGDI␣-WT or -S174A in the pcDNA3.1 hygroϩ vector (0.4 g/well) were transfected to cells with Lipofectamine2000. Two days later, the cells were starved, and after an additional 24 h, the cells were collected using Laemmli sample buffer for Western blotting or fixed for immunocytochemistry. The bars indicate the ratio of the cells that responded to isoproterenol at 500 nM for 30 min. For each well, at least 100 cells were counted and the values shown represent mean Ϯ S.D. of triplicate determinations.
HEK293 cells stably expressing Myc-RhoA-G14V or Myc-RhoA-G14V/S188A were plated in 24-well dishes (1.5 ϫ 10 5 cells/24 well) or 60-mm dishes (1.5 ϫ 10 6 cells/well), and after 24 h, when the confluence of the cells was about 50 -60%, siRNA (5 nM) and HA-RhoGDI␣-WT or -S174A in the pcDNA3.1 hygroϩ vector (0.02 g/24-well or 0.2 g/60-mm dish) were transfected to the cells with Lipofectamine2000. 24 h later, the cells were re-seeded into collagen-coated glass or two 60-mm dishes, and after an additional 24 h, the cells were serum starved for 10 h, and collected using Laemmli sample buffer for Western blotting or fractionated for membrane analysis or fixed for immunocytochemistry.
Co-immunoprecipitation Assay-Both HA-RhoGDI␣ (WT or S174A) (0.48 g of DNA per 0.8 ϫ 10 6 cells/35-mm dish) and GFP-RhoA (G14V, G14V/S188A, or WT) (0.12 g of DNA per 0.8 ϫ 10 6 cells/35-mm dish) or constitutively active myc-RhoGTPases (RhoA-G14V, Rac1-G12V, or Cdc42-G12V) (0.12 g of DNA per 0.8 ϫ 10 6 cells/35-mm dish) were transfected into COS7 cells, and after 24 h, the medium was replaced with serum-free medium. After a further 48 h, the cells were stimulated with or without 200 M forskolin for 30 min. The reactions were terminated by washing with ice-cold PBS and the cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 40 mM NaCl, 10 mM EGTA, 10 mM sodium PP i , 100 mM NaF, 1 mM DTT, 1% Triton-X, and proteinase inhibitors) for 1 h at 4°C. The lysates were incubated with 1 g/sample of mouse monoclonal anti-GFP antibody (MBL, 1E4) or anti-Myc antibody (MBL, PL14) for 1 h at 4°C with rocking. This was followed by the addition of 20 l of Protein Gϩ solution (50% in resin bed) and tumbled for another 1 h at 4°C. After several further washes with immunoprecipitation buffer, the resins were vortexed and boiled after adding equal volume of 2ϫ Laemmli sample buffer. The proteins were separated by SDS-PAGE and Western blot analysis was performed using rabbit polyclonal anti-GFP (MBL, 1:4000) or anti-Myc (MBL, 1:2000) antibodies to analyze the immunoprecipitated GFP/Myc each or using anti-HA antibody (Roche Applied Science, 12CA5, 1:2000) to analyze co-immunoprecipitated HA-RhoGDI␣.
Statistical Method and Analysis-The statistical method used was the Dunnett's multiple comparison procedures. Averaged data of three independent experiments are shown, and error bars represent the S.D. These analyses were performed using Microsoft Excel software, *, p Ͻ 0.02.

Morphological Changes in the Cardiac Fibroblasts Are
Induced by cAMP Elevation and ROCK Inhibition-Cell rounding of cardiac fibroblasts, a characteristic morphological change, is induced by isoproterenol (Fig. 1A) at a 100 -500 nM dosage range with almost complete rounding induced at 1 M (data not shown). CTX (Fig. 1A) and forskolin (data not shown) also produce similar changes in cellular morphology, indicating that it is cAMP that primarily induces cell rounding. These changes are reversed in the absence of further stimulation for 24 h. In our current experiments, we inhibited the effector of RhoA, ROCK, using Y27632 (Fig. 1A, d), which induced a similar morphological change, as previously reported (39,40). This suggested that this morphological change is due to inhibition of the RhoA-ROCK signaling pathway. We also obtained a similar result in HEK293 cells (Fig. 1A, e and f).
We next confirmed that cAMP accumulation is induced by isoproterenol or CTX in cardiac fibroblasts (Fig. 1B). As previously indicated, we suspected that the morphological change accompanying cAMP accumulation in these may be the result of RhoA inhibition, and investigated whether RhoA is blocked by treatment with isoproterenol or CTX. It has been shown that activated RhoA is localized in the membrane fraction. As previously reported (14,21), RhoA in the membrane fraction of cardiac fibroblasts is decreased by treatments with cholera toxin (Fig. 1C) or isoproterenol (data not shown) and a similar result is observed in HEK293 cells (Fig. 1C). In parallel, phosphorylation of MYPT1 (Thr-696), which reflects Rho-associated kinase (ROCK activity), is decreased (Fig. 1C). These data suggest that cAMP inhibits RhoA activity.
A Phosphoresistant Mutant of RhoA Does Not Prevent Morphological Changes Induced by PKA Activation-Our results were consistent with previous reports that morphological changes in cardiac and other cell types are due to RhoA inactivation through PKA activation. We thus predicted that cells expressing RhoA-G14V/S188A would be resistant to CTX treatment, and that cells expressing RhoA-G14V would remain sensitive to this agent. Surprisingly, however, both RhoA-G14V and Rho-G14V/S188A were equally resistant to CTX treatment (Fig. 2). We speculated from this that the quantity of RhoA at the plasma membrane might exceed the capacity of endogenous RhoGDI␣ to sequester GTP-RhoA in the cytosol, and that remnant GTP-RhoA proteins might maintain cell morphology without being sequestered despite their PKA-dependent phosphorylation.
According to the reports by Michaelson et al. (41) the molar sum of RhoA, Rac1, and Cdc42 is approximately equal to the molar amount of RhoGDI␣, and the RhoGDI␣/RhoA molar ratio is between 3:1 and 8:1. Moreover, it has been reported that about 2-3% of the endogenous RhoA is located at the plasma membrane and that constitutively active RhoA, which is less than 10% of the total endogenous RhoA, is sufficient to induce changes in the actin cytoskeleton (42)(43)(44)(45). Based on these stoichiometric ratios of RhoA and RhoGDI␣, we speculated that the transient expression of RhoA-G14V increases RhoA localization at the plasma membrane, which exceeds the capacity of endogenous RhoGDI␣ to sequester it in complex form. To avoid any stoichiometric imbalance, we next transiently expressed a RhoA mutant with RhoGDI␣. Surprisingly, how- Cholera toxin (100 ng/ml) was added to the medium 3 h before adding isobutylmethylxanthine. cAMP accumulation was measured as described under "Experimental Procedures." Data are the mean Ϯ S.D. of three independent experiments. C, membrane localization of RhoA and phosphorylation of MYPT1 (Thr-696). The membrane localization of RhoA and phosphorylation of MYPT1 (Thr-696) in cardiac fibroblasts and HEK-293 was quantified using Western blotting. After a 24-h (cardiac fibroblasts) or 12-h (HEK293 cells) incubation in serum-free DMEM, the cells were incubated in serum-free medium without or with 1 g/ml of cholera toxin for 3 h and harvested for whole cell lysate for phosphorylated MYPT1 and total RhoA or fractionated for membrane RhoA. RhoA (in 5 g of membrane protein) and phosphorylation of MYPT1 (Thr-696) was detected by immunoblotting against a specific antibody as described under "Experimental Procedures." Each set of results is representative of at least two additional experiments. ever, cells expressing RhoA-G14V/S188A showed cAMP-induced cell rounding as prominently as cells expressing RhoA-G14V (Fig. 2). To resolve the question of why the PKAdependent morphological change occurs in cells expressing RhoA-G14V/S188A, we postulated that the PKA-induced phosphorylation of RhoGDI␣ at Ser-174 (26, 28) might inhibit activated RhoA and play an important role in the onset of morphological changes in cardiac fibroblasts.

siRNA of Rat RhoGDI␣ Inhibits the Endogenous Expression of RhoGDI␣ and Inhibits PKA-induced Morphological Change-
To test the hypothesis that phosphorylated RhoGDI␣ at Ser-174 sequesters RhoA at the plasma membrane and inactivates it, we first transiently expressed the RhoGDI␣-S174A mutant or RhoGDI␣-WT in cardiac fibroblasts and investigated the impact of this on cAMP-induced morphological changes. However, we observed no differences between the cells expressing RhoGDI␣-WT and those expressing RhoGDI␣-S174A. We thus speculated that endogenous phosphorylated RhoGDI␣ may cause cell rounding even in cells expressing RhoGDI␣-S174A. Our negative data contrast with the positive data reported from transient expression experiments in COS7 cells in another study (28).
To avoid any interference from endogenous RhoGDI␣, we next tried to deplete endogenous RhoGDI␣ by siRNA and then express RhoGDI␣-WT or its mutant exogenously. Two siRNAs targeting rat RhoGDI␣ (20 nM) were co-transfected with or without a HA-tagged B. taurus RhoGDI␣ expression construct into rat cardiac fibroblasts and successfully depleted the expression of endogenous rat RhoGDI␣ (Fig. 3A). In contrast, neither siRNA molecule disrupted the exogenous expression of HA-tagged B. taurus RhoGDI␣ (Fig. 3A) or other proteins such as MAPK (data not shown), confirming their specificity.
The effectiveness of the two RhoGDI␣ siRNAs was demonstrated in a rat RhoGDI␣ immunoblotting assay (Fig. 3A, upper lanes) and by immunofluorescent analysis of the expression of this protein (Fig. 3B, a and e). Most cells transfected with this siRNA mixture showed no morphological changes following CTX treatment (Fig. 3B, f and h). This observation, however, does not contradict the well established hypothesis that phosphorylated RhoA is sequestered by RhoGDI␣ in the cytosol.
Exogenously Transfected RhoGDI␣-WT, but Not Its S174A Mutant, Reverses the cAMP-induced Morphological Changes in Cells Depleted of Endogenous RhoGDI␣-We transfected rat cardiac fibroblasts with a wild type or S174A mutant HA-tagged B. taurus RhoGDI␣ together with the aforementioned siRNA mixture. We found that the expression of HA-tagged RhoGDI␣-WT or its S174A mutant was observed in about 10% of the cells, whereas transfection efficiency of the siRNAs was more than 90%. Therefore, almost all of the cells expressing HA-tagged RhoGDI␣ were co-transfected with siRNA (data not shown). The cells expressing HA-tagged RhoGDI␣-WT, but not those expressing RhoGDI␣-S174A, showed morphological changes following CTX treatment (Fig. 4A). The ratio of cells showing cAMP-induced morphological changes was quantitatively evaluated (Fig. 4B). The expression levels of these HA-RhoGDI␣ proteins were almost equal (Fig. 4B). These data suggest that the phosphorylation of RhoGDI␣-S174 by PKA is likely to play an important role in the PKA-induced morphological change of cardiac fibroblasts.
To confirm our hypothesis, we next made HEK293 cell lines stably expressing Myc-RhoA-G14V or Myc-RhoA-G14V/ S188A and investigated the correlation between cell rounding, active RhoA distribution, and MYPT1 phosphorylation. siRNA-induced knockdown of endogenous RhoGDI␣ and its rescue by HA-tagged B. taurus RhoGDI␣ operated successfully in HEK293 cells (Fig. 5A). We then transfected HEK293 cells stably expressing RhoA-G14V or RhoA-G14V/S188A with a wild type or S174A mutant HA-tagged B. taurus RhoGDI␣ together with the siRNA. We found that transfection efficiency of HA-tagged RhoGDI␣ or siRNA was more than 90% (data not shown). Without CTX treatment, neither of the two HEK293  2000 dilution). B, immunofluorescence staining. Cardiac fibroblasts were transfected with a combination of two siRNAs (#1 and #2, 20 nM each) targeting rat RhoGDI␣ and incubated with anti-RhoGDI␣ antibody (1:500 dilution) and Texas Red X-phalloidin (1:60 dilution) as described under "Experimental Procedures." Cholera toxin (100 ng/ml) was added to the medium 3 h before the assay (c and d and g and h). Each set of results is representative of at least two additional experiments.
stable cell lines showed cell rounding (Fig. 5B, a-d). After CTX treatment, the cells (in both cell lines) expressing HA-tagged RhoGDI␣-WT, but not those expressing RhoGDI␣-S174A, showed morphological changes (Fig. 5B, e-h). These paralleled with decreases in membrane Myc-RhoA proteins and MYPT1 phosphorylation (Fig. 5C). These data further support the notion that phosphorylation of RhoGDI␣-Ser-174 by PKA may play a key role in the PKA-induced morphological change and RhoA inhibition.
The Phosphorylation of RhoGDI␣ at Ser-174, but Not That of RhoA at Ser-188, Is Detectable in Intact Cells-We next examined whether endogenous RhoGDI␣ is phosphorylated at Ser-174 by PKA in intact cardiac fibroblasts. The phosphorylation of RhoGDI␣ at Ser-174 or RhoA at Ser-188 by CTX treatment was analyzed in whole cell lysates of cardiac fibroblasts using the respective anti-phosphoprotein antibodies, but none was Rat cardiac fibroblasts were transfected with a combination of two siRNAs (#1 and #2, 20 nM each) targeting rat RhoGDI␣ and with wild type or S174A mutant HAtagged B. taurus RhoGDI␣ (as described in Fig. 3A). The cells were immunostained with anti-HA antibody (1:2000 dilution) and Texas Red X-phalloidin (1:60 dilution). Cholera toxin was added to the medium 3 h before the assay. B, the ratio of cAMP-induced metamorphoses. Cardiac fibroblasts were transfected with the siRNA mix targeting rat RhoGDI␣ and with wild type or S174A mutant HA-tagged B. taurus RhoGDI␣ as described in A. After stimulation with 500 nM isoproterenol for 30 min, the cells were incubated with anti-HA antibody (1:2000 dilution) and Texas Red X-phalloidin (1:60 dilution). The ratio of the cAMP-induced metamorphosis of cardiac fibroblasts was quantified as described under "Experimental Procedures." No difference between the expression level of RhoGDI␣ WT and S174A was shown by Western blotting. Data are the mean Ϯ S.D. of three independent experiments. *, p Ͻ 0.02. Each set of results is representative of at least two additional experiments.  2000 dilution). B, immunofluorescence staining. HEK293 cells stably expressing Myc-RhoA-G14V or Myc-RhoA-G14V/S188A were transfected with siRNA targeting human RhoGDI␣ and with wild type or S174A mutant HAtagged B. taurus RhoGDI␣ (as described in A). The cells were immunostained with Texas Red X-phalloidin (1:60 dilution) as described under "Experimental Procedures." Cholera toxin (1 mg/ml) was added to the medium 3 h before the assay (e-h). C, membrane localization of RhoA and phosphorylation of MYPT1. HEK293 stably expressing Myc-RhoA-G14V or Myc-RhoA-G14V/ S188A transfected with siRNA targeting human RhoGDI␣ and with wild type or S174A mutant HA-tagged B. taurus RhoGDI␣ (as described in B) was harvested as a whole cell lysate for phospho-MYPT1(T696), total Myc-RhoA, and total HA-RhoGDI␣ or fractionated for membrane Myc-RhoA as described in the legend to Fig. 1C. Myc-RhoA (in 5 g of membrane protein) and phosphorylation of MYPT1 (Thr-696), total Myc-RhoA, and total HA-RhoGDI␣ were detected by immunoblotting against a specific antibody as described under "Experimental Procedures." Cholera toxin (1 g/ml) was added to the medium 3 h before the assay. Each set of results is representative of at least two additional experiments.
detected. Each phosphorylation event was then investigated in immunoprecipitated protein preparations. The phosphorylation of endogenous RhoGDI␣ at Ser-174 was detected by immunoprecipitation with a RhoGDI␣ antibody followed by immunodetection with an anti-phospho-RhoGDI␣ (Ser-174) antibody. The phosphorylation of RhoA on Ser-188 was not detected in the same way using the corresponding antibodies (Fig. 6A, left panel). In addition, similar results were obtained when 32 P phosphorylation of endogenous RhoGDI␣ and RhoA was investigated (Fig. 6A, left panel). These results were con- In cardiac fibroblasts, RhoGDI␣ or RhoA was immunoprecipitated (IP) using anti-RhoGDI␣ antibody or anti-RhoA antibody (1 g each), respectively. In COS7 cells, HA-RhoGDI␣ or Myc-RhoA were immunoprecipitated using anti-HA antibody or anti-Myc antibody (1 g each), respectively. Phosphorylated proteins were then detected using anti-phospho-RhoGDI␣-S174 antibody (1:1000 dilution) or anti-phospho-RhoA-S188 antibody (1:1000 dilution), respectively. For 32 P phosphorylation experiments, the cells were loaded with 100 Ci/ml of 32 P for 20 h before the experiments and 32 P-labeled phosphorylated proteins were detected by autoradiography. B, stoichiometric analysis of RhoGDI␣ phosphorylation. The phosphorylated and nonphosphorylated RhoGDI␣ in cardiac fibroblasts was separated by using Phos-tag gel as described under "Experimental Procedures." Phosphorylated RhoGDI␣ or RhoGDI␣ were detected using anti-phospho-RhoGDI␣-S174 antibody (1:1000 dilution, lane 1) or anti-RhoGDI␣ antibody (1:30,000 dilution, lanes 2 and 3), respectively. Arrow in lane 2 corresponds phosphorylated RhoGDI␣. C, cAMP regulates the coimmunoprecipitation of RhoGDI␣ with active RhoA. COS7 cells expressing GFP-RhoA (constitutively active G14V or G14V/S188A) and HA-RhoGDI␣ (wild type or S174A) were incubated with 200 M forskolin for 1 h and lysed. Then GFP-RhoA was immunoprecipitated using 1 g of anti-GFP antibody and the immunoprecipitated proteins were detected using anti-GFP antibody (1:2000 dilution) or anti-HA antibody (1:2000 dilution, for detecting co-immunoprecipitated HA-RhoGDI␣). The relative activity of coimmunoprecipitated HA-RhoGDI␣ was quantified as described under "Experimental Procedures." Data are the mean Ϯ S.D. of three independent experiments. *, p Ͻ 0.02. Each set of results is representative of at least two additional experiments. D, specificity for phosphorylated RhoGDI␣ interaction. COS7 cells expressing GFP-RhoA (G14V or WT (left panel)) or Myc-RhoA-G14V, Myc-Rac1-G12V, or Myc-Cdc42-G12V (right panel) together with HA-RhoGDI␣ (wild type) were incubated with 200 M forskolin for 1 h and lysed. Then GFP-RhoA or Myc-RhoGTPase was immunoprecipitated using anti-GFP antibody or anti-Myc antibody (1 g each), respectively, and co-immunoprecipitated HA-RhoGDI␣ was quantified as described in C. Each set of results is representative of at least two additional experiments. NOVEMBER 9, 2012 • VOLUME 287 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38711 firmed using exogenously tagged RhoGDI␣ and RhoA in COS7 cells (Fig. 6A, right panel).
The Phosphorylation of RhoGDI␣ at Ser-174 Increases Its Affinity for RhoA-To verify the effects of phosphorylation of RhoGDI␣ at Ser-174 on activation of the RhoA-RhoGDI␣ com-Based on these reports, we and others (22,23) have hypothesized that phosphorylation of RhoGDI␣ at Ser-174 may regulate RhoA activity and PKA-induced morphological changes. At least in rat cardiac fibroblasts and HEK293 cells, we have found in our present study that phosphorylation of RhoGDI␣ at Ser-174 by PKA is likely to increase the affinity of GTP-RhoA for RhoGDI␣, which may explain the cAMP-dependent sequestration of active RhoA away from the membrane to the cytosol and the consequent induction of cellular morphological changes.
A Novel Working Model of the cAMP-induced Regulation of the RhoA-RhoGDI␣ Cycle-Our findings in intact cells, combined with previous findings of others, suggest a biochemical basis for RhoA cycle regulation by G s /cAMP pathways (Fig. 7). In the resting state, geranylgeranylated and GDP-bound RhoA forms a complex with RhoGDI␣ via hydrophobic and chargecharge interactions (please note the discussed implications of this in the structure section) and accumulates in the cytosol. When extracellular stimuli facilitate the dissociation of RhoA from RhoGDI␣, RhoA anchors to the membrane. The G q /G 13coupled receptor (such as thrombin receptor, endothelin receptor, and angiotensin II type 1 receptor)-triggered activation of Rho-GEF promotes the RhoA activation step, i.e. GDP-GTP exchange, and the GTP-bound RhoA activates effector proteins such as ROCK or mDia. The RhoA inactivation step, i.e. GTP hydrolysis, is accelerated by GAP and the resulting GDP-RhoA is sequestered by RhoGDI␣ in the cytosol once more. The cAMP-induced phosphorylation of RhoGDI␣ at Ser-174 increases its affinity for the GTP-bound active form of RhoA, sequestering it in the cytosol and thereby inhibiting RhoA signaling. Morphologically, this cAMP-induced RhoA inactivation event produces cell rounding.
We have shown that at least more than 10% of RhoGDI␣ can be phosphorylated in cardiac fibroblasts (Fig. 6B). Previously, the RhoGDI␣/RhoA molar ratio was reported to be between 3:1 and 8:1 (41), and about 2-3% of RhoA seems to be located at the plasma membrane as active RhoA (42)(43)(44)(45). Therefore, phosphorylation of more than 10% of RhoGDI␣ would be enough to inhibit active RhoA, causing cell rounding.
Structure Aspects of the Association between RhoA and RhoGDI␣-The association between RhoA and RhoGDI␣ has been postulated to result at least on part from both hydrophobic interactions and charge-charge interactions between the C-terminal domains of the two proteins. The geranylgeranyl moiety bound to a cysteine within the C-terminal domain of RhoA, which is composed of a positively charged polybasic region (in the case of RhoA, RRGKKKSGC) can be inserted into a geranylgeranyl binding pocket of the C-terminal domain of RhoGDI␣, which has a negative charge. Ser-174 of RhoGDI␣ is located in a portion of the ␤ sheet of the geranylgeranyl binding pocket (49).
It is speculated that free RhoA (uncoupled from RhoGDI␣) is anchored at the plasma membrane. It is feasible that dual interactions (hydrophobic and charge-charge interactions) assist with the anchoring of free RhoA to the membrane (50). Because RhoGDI␣ binding masks the geranylgeranyl moiety and positive charges of the C-terminal domain of RhoA, a RhoA-RhoGDI␣ complex can remain stable in the cytosol.
At present, we do not know how the phosphorylation of RhoGDI␣ at Ser-174 impacts upon the RhoA-RhoGDI␣ complex. We may speculate that a conformational change in RhoGDI␣ induced by phosphorylation at Ser-174 may indirectly increase the affinity between RhoA and RhoGDI␣ and enable RhoA-GTP to bind to RhoGDI␣. An alternative but less likely possibility is that the added negative charge on RhoGDI␣ via phosphorylation may promote the charge-charge interactions. It has been reported that phosphorylation of the geranylgeranyl binding pocket of RhoGDI␣ by other kinases, such as at Ser-96 by PKCa (51), Ser-101 and Ser-174 by Pak1 (26), and Tyr-156 by SRC (52), promotes the dissociation of Rac despite the increasing negative charge at the geranylgeranyl binding pocket of RhoGDI␣.
Potential Dual Regulation of RhoA by RhoGDI␣-We have shown here that the cAMP-dependent phosphorylation of RhoGDI␣ at Ser-174 is likely to increase the affinity between RhoA-GTP and RhoGDI␣. If this mechanism underlies the inhibition of RhoA leading to morphological changes in cells, it may be possible that overexpression of RhoGDI␣ even without cAMP stimulation may mimic phosphorylation of RhoGDI␣. Indeed, we have observed that overexpression of RhoGDI␣ at high levels alone can cause cell rounding in HEK293 cells (data not shown). Furthermore, this phenomenon has been observed not only when wild type RhoGDI␣ is overexpressed but also FIGURE 7. Model of cAMP-induced regulation of RhoA-RhoGDI␣ cycle. At resting state, geranylgeranylated and GDP-bound RhoA forms a complex with RhoGDI␣ and accumulates in the cytosol. When extracellular stimuli facilitate the dissociation of RhoA from RhoGDI␣, RhoA anchors to the membrane. Receptor-triggered activation of Rho-GEF promotes an activation step, i.e. GDP-GTP exchange for RhoA and GTP-bound RhoA activates effector proteins (e.g. ROCK or mDia). The RhoA inactivation step, i.e. GTP hydrolysis, is accelerated by GAP and inactivated RhoA is sequestered by RhoGDI␣ to the cytosol once more. The cAMP-induced phosphorylation of RhoGDI␣ at Ser-174 increases its affinity for the GTP-bound active form of RhoA, and sequesters it in the cytosol. RhoA signaling is thereby inhibited. Morphologically, this cAMP-induced RhoA inactivation event produces cell rounding.
when RhoGDI␣-S174A is overexpressed. Miura et al. (53) have also reported that overexpression of RhoGDI␣ in Swiss 3T3 cells causes cell rounding accompanied by the disappearance of stress fibers.
In conclusion, the inhibition of RhoA signal can be induced by: 1) increases in RhoGDI␣ expression or 2) phosphorylation of RhoGDI␣. These dual regulatory events, and instances where they are defective, may therefore have physiological and pathophysiological implications.