Regulation of Rac and Cdc42 pathways by G(i) during lysophosphatidic acid-induced cell spreading.

The pertussis toxin-sensitive G protein, G(i), has been implicated in lysophosphatidic acid-induced cell mitogenesis and migration, but the mechanisms remain to be detailed. In the present study, we found that pertussis toxin blocks lysophosphatidic acid-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of constitutively active mutants of Rho family small GTP-binding proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that G(i)-induced cell spreading is mediated through the Rac and Cdc42 pathway. Transfection of constitutively active mutants of G alpha(i) and G alpha(11) and G beta gamma subunits enhanced spreading of pertussis toxin-treated cells. G beta(1) with G gamma(12), a major G gamma form in fibroblasts, was more effective for increasing cell spreading than G beta(1)gamma(2) or G beta(1) plus G gamma(12)S2A, a mutant in which Ser-2, a phosphorylation site for protein kinase C, is replaced with alanine. In addition, a protein kinase C inhibitor diminished G beta(1)gamma(12)-induced cell spreading, suggesting a role for phosphorylation of the protein. These findings indicate that both G alpha(i) and G beta gamma stimulate Rac and Cdc42 pathways with lysophosphatidic acid-induced cell spreading on fibronectin.

Most cell types respond to extracellular matrix proteins by adhering and then spreading out to acquire a flattened morphology. The cell spreading is also regulated by soluble factors from serum such as lysophosphatidic acid (LPA) 1 and plateletderived growth factor, which then induce cell growth and migration (1,2). The process is accomplished by complex dynamic rearrangements of the actin cytoskeleton, and the dynamics appear to be coordinated in space and time by intracellular signaling pathways involving Rho family small GTP-binding proteins (3,4), although the precise mechanisms are poorly understood.
Rho family members such as Rho, Rac, and Cdc42 are part of the Ras superfamily of small GTP-binding proteins that act as guanine nucleotide-regulated switches (3). Their activity is regulated primarily by two groups of proteins: guanine nucleotide exchange factors (GEF), which catalyze exchange of GDP for GTP; and GTPase-activating proteins, which stimulate hydrolysis of GTP to GDP. Upon binding to GTP, Rho family GTPbinding proteins interact with and activate various downstream effector proteins such as Rho kinases and p21-activated kinases (PAK). Rho stimulates the formation of actin stress fibers and focal adhesions, whereas Cdc42 activation triggers the extension of filopodia and Rac controls growth factor-stimulated membrane ruffling and formation of lamellipodia (3). It has been defined how soluble extracellular factors induce the assembly of focal adhesions and actin filaments in serumstarved adherent fibroblasts through activation of Rho. However, there are several reports that Rho family small GTPbinding proteins are also involved in the generation of intracellular morphological structures during cell spreading on an extracellular matrix (5)(6)(7). Activation of Rac and Cdc42 and consequent stimulation of PAK have been observed in NIH 3T3 cells in response to plating cells on fibronectin (5). With Rat1 cells, cell spreading was significantly reduced by expression of dominant negative mutants of Rac, Cdc42, and Rho (6). More recently, it was shown that plating Swiss 3T3 cells on fibronectin-coated dishes elicited a transient inhibition of Rho activation, suggesting the existence of an adhesion-dependent negative feedback loop (7).
LPA induces multiple cellular responses through a G protein-coupled receptor and activates Ras and Rho family GTPbinding proteins via different pathways (1). In fibroblasts, LPA stimulates cell growth and lowers cAMP levels in a pertussis toxin (PTX)-sensitive manner, suggesting coupling of the LPA receptor to G i family G proteins (8). LPA-induced activation of the Ras/mitogen-activated protein kinase cascade also occurs in a PTX-sensitive fashion, and G␤␥ subunits of G proteins transduce this effect (9,10). In contrast, in a variety of cells, stimulation of phospholipase C by LPA was shown to be PTXinsensitive, so that LPA receptor coupling to G q family G proteins may also be important (11). LPA induces the Rho-dependent formation of actin stress fibers and focal adhesions in quiescent Swiss 3T3 fibroblasts (12,13). G␣ 12 and G␣ 13 probably mediate this action, because microinjection of constitutively active mutants of G␣ 12 and G␣ 13 into fibroblasts triggers actin polymerization in a Rho-dependent manner (12,13).
It is well known that LPA induces cell migration, which is blocked by PTX, suggesting the involvement of G i . However, regulation of Rho family GTP-binding proteins by G i has not been clearly shown to date. In the present study, we found that PTX blocks LPA-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. We show here G i stimulation via the LPA receptor leads to Rac and Cdc42 activation during cell spreading.
Production of GST-CRIB-PAK1 cDNA was a generous gift from L. Lim (National University of Singapore). The Cdc42/Rac interactive binding domain (CRIB) of PAK1 (amino acids 67-150) (25) was amplified by polymerase chain reaction using PAK1 cDNA as a template, ligated into pCMV-Myc, and confirmed by DNA sequencing (20). The cDNA of Myc-CRIB was cloned into a pGEX-2T vector (Amersham Pharmacia Biotech). Expression and purification of GST fusion protein were performed in accordance with the manufacturer's instructions and published protocols (26). Escherichia coli BL21 cells transformed with the GST-Myc-CRIB construct were grown at 37°C, and expression of recombinant protein was induced by addition of 0.1 mM isopropyl-1thio-␤-D-galactopyranoside for 4 h. Cells were harvested, resuspended in buffer A (50 mM Tris-HCl, pH 8.2, 2 mM MgCl 2 , 0.2 mM Na 2 S 2 O 3 , 10% glycerol, 20% sucrose, 1 g/ml trypsin inhibitor, 0.2 mM phenylmethylsulfonyl fluoride), then sonicated on ice, and mixed gently for 30 min after addition of 10% Triton X-100 to a final concentration of 1%. Cell lysates were centrifuged at 4°C for 10 min at 12,000 ϫ g, and the supernatant was incubated with glutathione-coupled Sepharose 4B beads for 30 min at room temperature followed by further incubation at 4°C for 30 min. The beads were washed three times in lysis buffer A and once with phosphate-buffered saline. GST-Myc-CRIB fusion protein-bound beads were suspended in phosphate-buffered saline, and glycerol was added to a final concentration of 50% and stored at Ϫ20°C.
Transfection and the Cell Spreading Assay-NIH 3T3 cells were grown in Dulbecco's modified essential medium supplemented with 10% calf serum. For transient transfections, cells were plated at a density of 6 ϫ 10 5 cells/10-cm dish 24 h before transfection with LipofectAMINE Plus reagent according to the manufacturer's instructions (Life Technologies, Inc.). The final amount of the transfected DNA for a 10-cm dish was adjusted to 4 g containing 3.6 g of plasmid of interest or empty vector pCMV5 and 0.4 g of pEGFP-C3 (CLONTECH). Medium replacement with serum-free medium was performed after 24 h, and the cells were incubated for another 24 h, in the presence or absence of 20 ng/ml PTX for the last 16 h. They were then washed with phosphatebuffered saline, detached with trypsin-EDTA, and washed with Dulbecco's modified essential medium containing 0.3 mg/ml trypsin inhibitor and 1 mg/ml fatty acid-free bovine serum albumin. Cells were then resuspended in Dulbecco's modified essential medium, replated on fibronectin-coated glass coverslips or dishes, and incubated for 1 h, unless otherwise specified, in the absence or presence of 10 M LPA, 10 ng/ml platelet-derived growth factor, 10 ng/ml epidermal growth factor, and 5 M BIM. After incubation, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for immunocytochemistry or lysed with 1% SDS in 20 mM Tris-HCl, pH 8.0, 1 mM EDTA for immunoblot analyses. Untransfected cells were stained for F-actin with tetramethylrhodamine isothiocyanate-phalloidin. To quantitate cell spreading, images of cells were obtained using a laser scanning microscope (Fluo-view, Olympus), and the areas of fluorescence-positive or GFP-positive cells were measured using Fluoview image analysis software. For each experiment, the areas of 50 cells were quantitated. All results shown represent the mean Ϯ S.D. of at least three independent experiments. Data were analyzed by performing unpaired Student's t test. For immunoblotting, cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (27) or Tricine/SDS-polyacrylamide gel electrophoresis (28) for immunoblotting with various antibodies.
GTPase Pull-down Assay-This was performed essentially as described previously (26). After replating on fibronectin-coated dishes, cells were washed with phosphate-buffered saline, incubated for 5 min at 0°C in buffer B (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl 2 , 10% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, and 2 g/ml trypsin inhibitor) containing 1% Nonidet P-40, and centrifuged at 21,000 ϫ g for 5 min at 4°C. Aliquots were taken from the supernatant to compare protein amounts. For this purpose, incubation with bacterially produced GST-Myc-CRIB fusion proteins bound to glutathione-coupled Sepharose 4B beads at 4°C for 60 min was followed by washing three times in an excess of buffer B containing 0.5% Nonidet P-40. Proteins bound to the beads were eluted in Laemmli sample buffer (27) and then analyzed for bound Rac and Cdc42 by immunoblotting using monoclonal mouse antibodies against human Rac1 and Cdc42, respectively. Detection was with a chemiluminescence reagent (PerkinElmer Life Sciences) and densitometry analysis was performed using the LAS-1000 system (Fujifilm).

RESULTS
To investigate the role of G i signaling during cell spreading, serum-starved NIH 3T3 fibroblasts were incubated for 16 h in the absence or presence of PTX, replated on fibronectin-coated plastic dishes, then exposed to 10 M LPA for various times, and stained with rhodamine-phalloidin to label F-actin (Fig. 1). Cells incubated in the absence of PTX spread over a period of 60 min. This was markedly diminished with PTX treatment. To confirm that PTX abolishes only the function of G i , we examined the effects of PTX with induction by other stimulants known to enhance cell spreading or cell migration. Fibronectin alone (none), platelet-derived growth factor, and epidermal growth factor induced cell spreading, although the staining of F-actin in cell cortex was much weaker than that observed in LPA-stimulated cells, and this was not affected by PTX (Fig. 2). These results suggest that PTX specifically blocks G i activation via the LPA receptor. The apparently paradoxical result that spreading occurs equally on fibronectin in the presence or absence of LPA, but is inhibited by PTX only in the presence of LPA will be explained later (see "Discussion").
To examine which subtypes of Rho family GTP-binding proteins contribute to cell spreading induced by LPA, we transfected plasmids with various Rho family mutants into NIH 3T3 cells. Cells expressing dominant negative mutants of Rac (RacT17N) and Cdc42 (Cdc42T17N) were rounded, whereas dominant negative Rho (RhoT19N) was without effect (Fig. 3, A  and B). On the other hand, constitutively active mutants of Rac (RacG12V) and Cdc42 (Cdc42G12V) enhanced spreading of PTX-treated cells (Fig. 3, A and C). In contrast, the expression of the constitutively active form of Rho (RhoG14V) rather caused rounding of both PTX-treated and untreated cells (Fig.  3). The LPA receptor activates not only G i , but also other G proteins such as G q/11 and G 12/13 , which are PTX-insensitive, and G 12/13 is involved in Rho-dependent stress fiber formation (12). Therefore, it is possible that Rho is also activated during spreading. In fact, transfection of a dominant negative mutant of Rho restored cell spreading when the cells were treated by PTX (Fig. 3C). These observations suggest that the LPA receptor induces activation of Rac/Cdc42 and Rho through G i and probably G 12/13 , respectively. Rho-mediated cell rounding appears with inhibition of G i pathway by PTX.
To examine whether PTX indeed affected the activation of endogenous Rac and Cdc42 during cell spreading, we performed pull-down assays of Rac and Cdc42 using GST-Myc-CRIB-bound Sepharose beads. First, to confirm that GST-Myc-CRIB specifically binds to active forms of Rac and Cdc42 (29), GST-Myc-CRIB Sepharose beads were incubated with lysates from NIH 3T3 cells expressing FLAG epitope-tagged mutants of Rho family GTP-binding proteins. GST-Myc-CRIB beads retained constitutively active forms of Rac and Cdc42 but not dominant negative forms of Rac and Cdc42, indicating a high specificity for the GTP-bound state. Neither constitutively active nor dominant negative mutants of Rho were retained by GST-Myc-CRIB beads, consistent with published data (29) (Fig. 4A).
To determine whether Rac and Cdc42 were activated during LPA-stimulated cell spreading on fibronectin, cell lysates were incubated with GST-Myc-CRIB beads and the retained proteins were analyzed by immunoblotting with antibodies against Rac and Cdc42 (Fig. 4, B and C). In the control cells, Rac activity was increased 1.9-fold (Fig. 4B), and Cdc42 activity 1.7-fold (Fig. 4C). In contrast, Rac and Cdc42 activities did not significantly increase within 120 min in PTX-treated cells (Fig.  4, B and C). These results also indicate that Rac and Cdc42 activation is mediated by the G i pathway during LPA-induced cell spreading.
To examine further the involvement of Rac and Cdc42 activation in LPA-induced cell spreading, we transfected Myc-CRIB into NIH 3T3 cells and replated on fibronectin-coated coverslips in the presence of LPA. Expression of Myc-CRIB strongly inhibited cell spreading (control, 1592 Ϯ 156 m 2 ; Myc-CRIB, 461 Ϯ 43 m 2 ).
Next we determined which G protein subunit, G␣ or G␤␥, is responsible for cell spreading. First we transfected plasmids with several constitutively active mutants of ␣ subunits of G proteins and tested the restoration of spreading of PTX-treated cells in the presence of LPA (Fig. 5B). Constitutively active mutants of G␣ i2 (G␣ i2 Q205L) and unexpectedly G␣ 11 (G␣ 11 Q209L) restored cell spreading to the levels observed in Mock cells in the absence of PTX. Other active mutants, G␣ 12 and G␣ s (G␣ 12 Q229L and G␣ s Q227L), were not effective in PTX-treated cells (Fig. 5B). These active mutants, however, decreased cell spreading in the absence of PTX (Fig. 5A), suggesting that G␣ 12 and G␣ s cause cell rounding in these conditions. Then, we cotransfected G␤ 1 with various G␥ forms into NIH 3T3 cells. G␤␥ expression also restored spreading of PTXtreated cells (Fig. 6). To further test its involvement, we transfected ␤ARKct, which binds and sequesters free G␤␥, and showed this to reduce the LPA-induced cell spreading (Fig. 6).
We previously reported that the Ser-2 of the G␥ 12 subunit, a major G␥ in fibroblasts, is specifically phosphorylated by PKC and that this is blocked by PTX (23, 24), as shown in Fig. 6. We have also demonstrated that phosphorylation of G␥ 12 enhances migration of NIH 3T3 cells (16). Expression of G␤ 1 ␥ 12 was more effective at inducing cell spreading than that of G␤ 1 ␥ 2 or G␤ 1 with the mutant G␥ 12 S2A, in which a phosphorylation site is replaced with alanine (Fig. 6). To examine whether phosphorylation of G␥ 12 by PKC is involved in cell spreading, we tested the effect of the PKC inhibitor BIM (30) (Fig. 7). It significantly decreased cell spreading induced by G␤ 1 ␥ 12 as well as phosphorylation of G␥ 12 , while being essentially without effect on G␤ 1 ␥ 2 -induced cell spreading. It should be note that G␤ 1 ␥ 12induced cell spreading in the presence of BIM is comparable to G␤ 1 ␥ 2 -induced cell spreading. In PTX-untreated Mock cells, BIM suppressed LPA-induced cell spreading and phosphorylation of G␥ 12 . G q/11 is known to stimulate phospholipase C, which produces diacylglycerol, then activates PKC. Expression of G␣ 11 Q209L increased phosphorylation of G␥ 12 in PTXtreated cells (Fig. 7), and the treatment with BIM diminished cell spreading induced by G␣ 11 Q209L, but not by G␣ i2 Q205L (Fig. 7). Thus PKC-dependent phosphorylated G␥ 12 may partially contribute to G␣ 11 Q209L-induced cell spreading.
The dominant negative mutant of Rac inhibited cell spreading induced by G␣ i2 Q205L, G␣ 11 Q209L, and G␤␥. However, the dominant negative form of Cdc42 appeared to be less effective, especially with G␣ 11 Q209L-induced cell spreading. DISCUSSION It has been reported that PTX blocks cell growth and migration induced by LPA (1, 31). In the present study, we demon-strated that G i mediates LPA-induced cell spreading, one of important steps of growth and migration processes. LPA receptor is coupled not only with G i but also with G q and G 12 family G proteins. G␣ 12 and G␣ 13 , which activate Rho, induce the formation of actin stress fibers and focal adhesions (12,13). The present investigation indicated that G i activates Rac and Cdc42 during cell spreading stimulated by LPA. The evidence that the dominant negative mutant of Rho enhanced spreading of PTX-treated cells (Fig. 3C) suggests that this signal protein is also activated with LPA stimulation. Previous reports indicated that Rac down-regulates Rho activity in NIH 3T3 cells (32), or Rac may counteract Rho (33). In the latter case, PAK, which is activated by Rac and Cdc42, was found to block the phosphorylation of myosin light chain induced by Rho. Ren et al. (7) have described that plating Swiss 3T3 cells on fibronectin-coated dishes elicits a transient inhibition of Rho, followed by an activation phase, suggesting the existence of an adhesiondependent negative feedback loop. In addition, cell rounding observed by expression of G␣ 12 Q229L (Fig. 5A) suggests that G␣ 12 -mediated Rho activation caused cell rounding in these conditions. Taken together, the available information indicates FIG. 6. Effects of expression of ␤␥ subunits and ␤ARKct on cell spreading. NIH 3T3 cells were cotransfected with pEGFP-C3 and pCMV5 (Mock), G␤ 1 ␥ 2 , G␤ 1 ␥ 12 , G␤ 1 ␥ 12 S2A, or ␤ARKct, incubated in the absence or presence of 20 ng/ml PTX for 16 h, trypsinized, and replated on fibronectin-coated dishes or coverslips in the presence of 10 M LPA. After 60 min, GFP-positive cells were fixed and quantified for spreading. Lysates were immunoblotted with antibodies against G␤, G␥ 7 (␥), phospho-G␥ 12 (p-␥ 12 ), and Myc. Because the antibody against G␥ 7 reacts with G␥ 2 , G␥ 3 , G␥ 7 and G␥ 12 (22), it was used to detect G␥ 2  that activation of Rac and Cdc42 by G i promotes cell spreading and probably interferes with Rho-mediated cell rounding in LPA-stimulated cells (Fig. 9). On the other hand, it has been shown that Rac, Cdc42, and Rho were activated during cell spreading on fibronectin (5)(6)(7), and cells also tend to spread probably due to the down-regulation of Rho by Rac activation (Fig. 9). The degree of spreading may be determined by a balance between Rac/Cdc42 activation and Rho activation. The evidence that blockage of G i by PTX leads to cell rounding on fibronectin in the presence of LPA suggests that activation of Rho by G 12/13 and fibronectin is much larger than activation of Rac/Cdc42 by fibronectin (Fig. 9). This scheme explains the apparently paradoxical result (Fig. 2) that spreading occurs equally on fibronectin in the presence or absence of LPA, but is inhibited by PTX only in the presence of LPA.
It has been shown that G␣ i and G␤␥ released from G i by receptor stimulation regulate adenylyl cyclase and potassium channels (34). The present study demonstrated that they both contribute to LPA-induced cell spreading. The fact that expression of ␤ARKct partially inhibited cell spreading (Fig. 6) supported an involvement of G␣ i in addition to G␤␥. We have demonstrated previously that cotransfection of G␤ 1 with various G␥ forms into NIH 3T3 cells increases cell migration (16). Cell motility of G␥ 12 -expressing cells was much greater than those of G␥ 2 -, G␥ 5 -, and G␥ 7 -expressed cells. Because the effect of G␥ 12 was decreased by replacement of Ser2 by alanine, we concluded that protein phosphorylation enhances cell migration (16). The present study demonstrated G␤ 1 ␥ 12 to be more efficient at stimulating cell spreading than G␤ 1 ␥ 2 or G␤ 1 ␥ 12 S2A, consistent with results obtained for G␤␥-induced motility.
Since this was partially blocked by an PKC inhibitor, the existence of PKC-dependent and -independent pathways in G␣ 11induced cell spreading can be speculated. Phosphorylation of G␥ 12 in G␣ 11 Q209L-expressing cells points to an involvement in a PKC-dependent pathway. Although PTX-insensitive G q family G proteins would be expected to be activated by LPA in PTX-treated cells, cell spreading did not occur. The physiological significance of G␣ 11 -induced cell spreading remains uncertain.
A previous study demonstrated that increase of intracellular cAMP contents by forskolin or a cAMP phosphodiesterase inhibitor blocked LPA-induced cell spreading and migration of carcinoma cells (35). High levels of intracellular cAMP induced by forskolin also block chemotaxis of human embryonic kidney cells expressing the interleukin-8 receptor (36), and G␣ i and G␤␥ inhibit several types of adenylyl cyclase (34). It has, furthermore, been reported that LPA decreases cAMP levels in Swiss 3T3 cells in a PTX-sensitive manner (8). Therefore, suppression of cAMP levels by G i may be important in LPA-induced cell spreading. In fact, the expression of the constitutively active mutant G␣ s Q227L decreased cell spreading (Fig. 5A).
Small GTP-binding proteins are regulated by GEF and GTPase-activating proteins (3,37), and recent reports have indicated that G␣ 12 and G␣ 13 are able to bind directly to p115-RhoGEF (38,39) or PDZ-RhoGEF (40). G␣ 13 but not G␣ 12 stimulates the GDP-GTP exchange reaction of p115-RhoGEF. G␤␥ may also be associated with the NH 2 -terminal of Dbl, a FIG. 9. Schematic model for LPA-and integrin-dependent activation of Rho family GTP-binding proteins during cell spreading. LPA receptors stimulate both G i and G 12/13 , which lead to the activation of Rac and Cdc42 and Rho, respectively. Activated Rac and Cdc42 then induce cell spreading, whereas activated Rho induces cell rounding. Engagement of integrins with fibronectin leads to the activation of Rac, Cdc42, and Rho. In both cases, cells are spreading because Rac counteract Rho. A balance between Rac/Cdc42 activation and Rho activation may thus determine the degree of cell spreading. Blockage of G i pathway by PTX leads to cell rounding due to activation of Rho mediated by G 12/13 .
kind of RhoGEF (41), but its GEF activity toward Rho is not influenced by G␤␥ binding. With respect to GTPase-activating proteins, G␣ i specifically binds to that for Rap1, a member of the Ras family. Stimulation of the G i -coupled m2-muscarinic receptor translocates rap1GAPII from cytosol to the membrane and decreases the amount of GTP-bound Rap1 (42). Our finding that G␣ i and G␤␥ induce cell spreading in a Rac/Cdc42-dependent manner suggests the existence of GEF or GTPase-activating proteins, which directly interact with G␣ i or G␤␥ subunits.