The Function of the p190 Rho GTPase-activating Protein Is Controlled by Its N-terminal GTP Binding Domain*

p190 is a GTPase-activating protein (GAP) for the Rho family of GTPases. The GAP domain of p190 is at the C terminus of the protein. At its N terminus, p190 contains a GTP binding domain of unknown significance. We have introduced a mutation (Ser 36 3 Asn) into this domain of p190 that decreased its ability to bind guanine nucleotide when expressed as a hemagglutinin (HA)-tagged protein in COS cells. In vitro, both the wild type and S36N mutant HA-p190 proteins showed similar GAP activities toward RhoA, but when expressed in NIH 3T3 fibroblasts only wild type p190 appeared able to function as a RhoGAP. Wild type HA-p190 induced a phenotype of rounded cells with long, beaded extensions similar to that seen when Rho function is disrupted by ADP-ribosylation. HA-p190(S36N), although expressed at a similar level to the wild type protein, had no dis-cernible effect on the cells. The beaded extension phenotype induced by wild type HA-p190 required GAP function. A GAP-defective mutant, p190(R1283A), had no effect on cell morphology. Moreover, the beaded extension phenotype could be suppressed by co-expression of a gain-of-function Rho mutant, RhoA(G14V), or Rac mutant, Rac1(G12V). Activation of the Jun kinase (JNK) via muscarinic receptors was

Actions of the Ras-like GTPases are tightly controlled and are dictated by the state of nucleotide binding, which alters the conformation of discrete "switch regions" in the proteins (3). The change in conformation allows the GTPases to interact with their appropriate targets (4). Regulation of the Ras-like GTPases is accomplished by GEFs (guanine nucleotide exchange factors), which catalyze the exchange of GDP for GTP on the GTPases, and GAPs (GTPase-activating proteins), the function of which is to catalyze the hydrolysis of bound GTP to GDP (5).
GAPs have been identified that act specifically either on Ras or Rho GTPases. RasGAPs include p120 (6) and neurofibromin (7), whereas p190, Bcr, and RhoGAP act on the Rho subfamily of GTPases (8). Many of these GAPs possess, in addition to the sequences responsible for enhancing GTP hydrolysis, domains that are found in diverse signal transduction components. For example, the p190 RhoGAP contains a C-terminal catalytic domain and at its extreme N terminus a region with significant sequence similarity to the guanine nucleotide binding domain of the Ras superfamily of GTPases (9). This region is most similar to the yeast Rab GTPase, Ypt1, but it contains unusual amino acid residue substitutions within key motifs. Through this N-terminal region, p190 has been shown to bind GTP, and mutations within the GTP binding domain of p190 exhibit altered nucleotide binding affinities (10). However, the significance of this GTP binding domain is unknown.
In addition to their ability to enhance hydrolysis of GTP, some GAPs may be downstream effectors of the GTPases with which they interact. The p120 RasGAP is believed to be involved in signal transduction events (11). It recognizes tyrosine-phosphorylated p190 RhoGAP (12,13), and in cells transformed by tyrosine kinases, most of the p120 RasGAP is found complexed to p190 (14). The formation of a p120-p190 complex may provide a means for the Ras and Rho GTPase pathways to coordinate their signals. The p190 RhoGAP is predominantly cytoplasmic, although changes in localization have been reported to occur when cells are stimulated with certain growth factors. In murine fibroblasts that overexpress c-Src, for example, stimulation with epidermal growth factor induces a rapid and transient condensation of p190 RhoGAP and p120 RasGAP into arc-like structures radiating away from the nucleus (15). Cheng et al. (16) have shown that in chronic myelogenous leukemic cells the association of p190 with p120 parallels an increase in actin polymerization and cell adhesion induced by phorbol esters. However, these treatments have not been shown directly to alter the GAP activity of p190.
A new p190 RhoGAP isoform has been discovered, called p190-B. This protein is expressed in a number of cell types and is clustered at adherence sites where the cell membrane at-taches to fibronectin (17). This interaction could establish a transmembrane link between integrins and the Rho family of GTPases, but there is no evidence to date that integrins modulate p190 GAP activity.
The existence of an unusual GTP binding domain (GBD) in p190 suggests the interesting possibility that it functions to control the RhoGAP domain. To investigate this hypothesis, we created a mutant p190 protein in which Ser 36 was changed to Asn. This mutation is homologous to the Ras(S17N) mutation that generates a protein with decreased affinity for nucleotide binding (18). By using this mutant protein we showed that binding of GTP is required for p190 RhoGAP function within the cell, even though in vitro RhoGAP activity is not affected. We speculate that regulatory factors tightly control the GTP/ GDP ratio of p190 which in turn regulates its GAP activity and hence modulates the Rho GTPases.

EXPERIMENTAL PROCEDURES
Production of Epitope-tagged p190 and GFP-fused p190 -A HindIII-EcoRI fragment was isolated from p190 cDNA clone 391 (9) provided by R. Weinberg and J. Settleman (Massachusetts Institute of Technology). This fragment, which lacks the 5Ј 670 base pairs of the p190 open reading frame, was subcloned into pBS. The first 670-base pair fragment was amplified by polymerase chain reaction (PCR), to include a 5Ј BamHI site. The PCR product was cut with BamHI and HindIII and subcloned into the pBS-p190 plasmid. The reconstructed p190 open reading frame was excised from pBS using BamHI and EcoRI and subcloned into pKH3, which attaches, in frame, a triple HA1 tag to the N terminus (19). To make GFP-p190, the BamHI-EcoRI p190 fragment was inserted into a vector (K7-GFP) that contains a BamHI-EcoRI cloning site in the same frame as pKH3, 3Ј to the GFP open reading frame (20).
Creation of p190 Mutants-The mutation Ser 17 3 Asn in Ras creates a mutant GTPase that cannot stably bind guanine nucleotides. The equivalent residue in p190 is Ser 36 . We mutated this residue to Asn to produce p190(S36N) by overlap polymerase chain reaction (PCR) (21) using pKH3-p190 as a template, a mismatched 5Ј primer and a 3Ј primer about 800 base pairs downstream. The PCR product, containing the mutation, was digested with BamHI and HindIII and then inserted in place of the wild type p190 fragment in pKH3. To create a GAPdefective p190, a point mutation was introduced so as to alter Arg 1283 3 Ala. This residue is highly conserved in GAP domains and is required for their catalytic activity (22). An R1283K mutation in p190 was recently shown to destroy the p190 RhoGAP activity (23). To create p190(R1283A), a fragment of p190 extending from the XhoI site to the EcoRI site was subcloned into the pBluescript II KS phagemid (Stratagene) in which the KpnI site was previously destroyed. Using pKH3-p190 as a template, a mismatched 5Ј primer containing the p190 NsiI site at about position 3800 and sequence encoding for the Arg 1283 to Ala mutation was used for amplification together with a 3Ј primer about 200 base pairs downstream that contained a KpnI site. The PCR product was digested with KpnI and NsiI and then inserted in place of the wild type fragment in the Bluescript-p190(XhoI-EcoRI) construct. The p190 fragment containing R1283A was cut out of Bluescript from the XhoI to EcoRI site and inserted in place of the wild type p190 fragment in pKH3. The regions of p190 in which the mutations were introduced were sequenced to verify the mutations were present. The p190(1-266) and p190(1-266)S36N truncation mutants were created by PCR which added a stop codon and an EcoRI site after position 266 in the nucleotide sequence. These fragments were then inserted into pKH3.
Cell Culture and Transfection-NIH 3T3 and COS cells were cultured and transfected by the CaPO 4 method as described previously (19). Amounts of plasmid transfected are described in the figure legends.
Immunofluorescence-For immunofluorescence, NIH 3T3 cells on Lab-Tek glass slides were transfected, allowed to grow 38 -40 h, and then fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 min. Fixed cells were then permeabilized with Ϫ20°C methanol for 2 min and blocked in 2% dried milk in Tris-buffered saline plus 0.1% Tween 20 (TBS-T) for at least 1 h. Cells were then incubated with either a 1:400 dilution of the anti-HA1 antibody 12CA5 or a 5 g/ml concentration of anti-p190 antibody (Transduction Laboratories) in 2% bovine serum albumin/TBS-T for 1 h at 37°C. After washing with TBS-T, detection of antibody was accomplished by incubating the cells with a 1:1000 dilution of secondary antibody (Cy3-conjugated goat anti-mouse) in 2% bovine serum albumin/TBS-T for 1 h. Cells were washed with TBS-T and mounted with Vectashield (Vector). Fluorescence was detected using a Nikon Diaphot inverted microscope with either an oil or water immersion 60ϫ objective and recorded using a Photometrics 200 CCD camera. Images were processed using Adobe Photoshop. For detection of expressed GFP-fusion proteins, cells were fixed as above and directly mounted with Vectashield, prior to examination by epifluorescence microscopy.
Time Lapse Microscopy-NIH 3T3 cells were grown on 35-mm plates and transfected with 5 g of pRK7GFP-p190 plasmid. The GFP-p190 was imaged by fluorescence microscopy using a 20ϫ objective lens. Time lapse began approximately 36 h post-transfection and continued over a period of 7 h at 37°C. Images were captured using a Hamamatsu camera and processed using OpenLab and Adobe Photoshop software.
Immunoprecipitation and Immunoblotting-Cells were lysed by adding a buffer containing 25 mM HEPES (pH 7.4), 300 mM NaCl, 5 mM MgCl 2 , 1% Triton X-100, 0.1 mM EDTA, 1 mM ATP, 20 mM ␤-glycerophosphate, 10 g of leupeptin/ml, 0.1 mM Na 3 VO 4 , and 1 mM phenylmethylsulfonyl fluoride and rocking on ice for 20 min. Cells were then scraped into centrifuge tubes and pulled through a 26-gauge needle 5 times. The lysates were clarified by centrifugation at 14,000 ϫ g. Soluble lysates were incubated with 3 l of 12CA5 antibody for 1 h at 4°C at which time 30 l (50/50 slurry) of protein A-Sepharose beads were added for an additional 1-h incubation at 4°C. The recovered antibody-antigen complex was washed 5 times in wash buffer (25 mM MOPS (pH 7.1), 150 mM NaCl, 1 mM dithiothreitol (DTT), 1% Nonidet P-40, 1% sodium deoxycholate and 1 mM phenylmethylsulfonyl fluoride) and washed 2 times in wash buffer (25 mM HEPES (pH 7.4), 300 mM NaCl, 10 mM MgCl 2 , 1 mM ATP, 100 M GTP, and 1 mM NaPO 4 ). Detection of proteins was by immunoblotting as described previously (20) with the following changes: blots were incubated with either a 1:6000 dilution of the 12CA5 antibody in TBS or a 1:500 dilution of the anti-p190 antibody.
Thin Layer Chromatography (TLC)-COS cells in 10-cm plates were incubated in 3 ml of phosphate-free Dulbecco's modified Eagle's medium containing 0.5 mCi of [ 32 P]orthophosphate/plate at 37°C for 4 h. Cells were then lysed, and proteins were immunoprecipitated as above with the following change: to the RIPA wash buffer was added 10 mM MgCl 2 to keep the nucleotide bound to the immunoprecipitated proteins. Once the proteins were washed, bound nucleotides were eluted from the proteins by heating the recovered antibody-antigen complex to 68°C in the following buffer: 20 mM HEPES (pH 7.4), 100 mM NaCl, 5 mM EDTA, 5 mM GTP, and 5 mM GDP. An aliquot of the eluted nucleotides was then spotted onto a polyethyleneimine-cellulose plate, resolved by TLC in 0.75 M KH 2 PO 4 (pH 3.4) (10), and visualized by autoradiography. GTP/GDP ratio was calculated after background (empty vector) subtraction by assuming that all the phosphates were at equilibrium with the 32 P label and using the formula %GTP ϭ GTP/ 1.5(GDP) ϩ GTP. GTP and GDP values were determined by Phosphor-Imager analysis.
RhoGAP Assay-5 g of RhoA protein, which was produced in Escherichia coli as GST-RhoA and cleaved with thrombin to remove the GST, was loaded with 20 Ci of [␥-32 P]GTP (3000 Ci/mmol); the labeled protein was then incubated with immunoprecipitated wild type or mutant p190 proteins for the specified time points. RhoGAP activity was detected as the loss of [␥-32 P]GTP bound to RhoA. In control experiments (data not shown), RhoA loaded with [␣-32 P]GTP showed no significant reduction in 32 P counts when in the presence of either wild type or mutant p190 proteins. The amount of 32 P bound to protein was determined through a filter binding assay (24).
JNK Assay-NIH 3T3 cells, which stably express the human m1 type (hm1) muscarinic receptor (19), were transfected with plasmids as indicated in Fig. 8. Transfected cells were stimulated with either 100 l of distilled water (control) for 15 min, 100 M carbachol for 15 min, or 100 g of anisomycin/10-cm plate of cells for 20 min. The cells were lysed in the following buffer: 25 mM HEPES (pH 7.4), 300 mM NaCl, 1.5 mM MgCl 2 , 0.5 mM DTT, 20 mM ␤-glycerophosphate, 1 mM Na 3 VO 4 , 0.1% Triton X-100, 20 g of aprotinin/ml, 10 g of leupeptin/ml, 1 mM phenylmethylsulfonyl fluoride, and 1 M okadaic acid. The lysates were clarified by centrifugation at 14,000 ϫ g. Aliquots of detergent-soluble protein extract were removed from each sample tube and were fractionated by SDS-gel electrophoresis on 12% polyacrylamide gels. The proteins were then transferred onto nitrocellulose membrane and immunoblotted as above. HA-tagged proteins were immunoprecipitated from the remaining detergent-soluble extracts and washed 3 times in Buffer A (2 mM Na 3 VO 4 and 1% Nonidet P-40 in phosphate-buffered saline), washed 1 time in Buffer B (100 mM MOPS (pH 7.5), 0.5 M LiCl), and washed 1 time in Kinase Buffer (12.5 mM MOPS (pH 7.5), 12.5 mM ␤-glycerophosphate, 7.5 mM MgCl 2 , 0.5 mM EGTA, plus 0.5 mM NaF and 0.5 mM Na 3 VO 4 ). Immunoprecipitated proteins were resuspended in 30 l of Kinase Buffer, to each reaction tube was added 2 g of purified GST-cJun-(1-79) and 2 Ci of [␥-32 P]ATP. The reaction tubes were incubated at 30°C for 20 min at which time the reactions were halted by the addition of 10 l of 4ϫ Laemmli sample buffer. Immunocomplex kinase reactions were fractionated by SDS-gel electrophoresis on 12% polyacrylamide gels and autoradiographed.

Creation and Characterization of Mutant p190 Proteins-
Recombinant p190 protein expressed in SF9 insect cells is bound primarily to GTP (10). To investigate the function of nucleotide binding to p190, it was necessary to create a mutation that eliminated binding of GTP. We therefore created a mutation within the p190 guanine nucleotide binding domain (GBD) that is homologous to the Ser 17 3 Asn mutation in Ras (Fig. 1A). This Ras mutant has a low affinity for both GTP and GDP and associates tightly with Ras-specific exchange factors. When expressed in cells, Ras(S17N) acts as a dominant-negative mutant that prevents the activation of endogenous Ras (25). We mutated the Ser 36 of p190 to Asn to produce p190(S36N) (Fig. 1A). Neither this mutant nor wild type p190 bound nucleotide when expressed in bacteria. Therefore, to establish whether p190(S36N) could stably bind GTP, COS cells were transiently transfected with wild type or mutant plasmids and were labeled with [ 32 P]orthophosphate. The HAtagged p190 proteins were immunoprecipitated from cell lysates with a monoclonal antibody (12CA5) directed against the HA epitope. Bound nucleotide was eluted from the immunoprecipitated proteins and detected by thin layer chromatography. As shown in Fig. 1B, HA-p190 expressed in COS cells was specifically associated with both GDP and GTP. After correction for nonspecific binding, the fractional association with GTP of p190 appears to be about 12%. In accordance with prediction, the p190(S36N) mutant did not specifically bind any guanine nucleotide, although it was expressed at a level similar to that of the wild type protein. The p190 protein, when expressed in SF9 insect cells, can also bind GTP (10). Nonetheless, the remote possibility remained that the wild type p190 was associated through its C-terminal GAP domain with a GTPase that was complexed with 32 P-labeled guanine nucleotides. To eliminate this possibility, we expressed the isolated, HA-tagged N-terminal GBDs (residues 1-266) of wild type and S36N p190 in COS cells. As can be seen from Fig. 1B, the wild type GBD was bound to both GTP and GDP in a ratio similar to that of the full-length HA-p190. The isolated GBD containing the S36N mutation did not detectably bind nucleotide. These results strongly support the conclusion that, when expressed in mammalian cells, the N-terminal GBD of p190 binds to both GDP and GTP and that the S36N mutant, like the analogous S17N mutant of Ras, possesses a greatly reduced affinity for guanine nucleotides.
Overexpression of Wild Type but Not Mutant p190 Proteins Induces Beaded Extensions in NIH 3T3 Fibroblasts-To determine the effect of the S36N mutation on p190 function, both wild type and S36N p190 were expressed by transient transfection as HA-tagged proteins in NIH 3T3 fibroblasts. The cells were fixed and stained for HA. Overexpression of wild type HA-p190 produced a distinct phenotype in which the cells rounded up and extended very long, beaded, dendritic-like processes, as observed by indirect immunofluorescence (Fig. 2). This phenotype is similar to that seen when either C3 botulinum toxin or RhoGDI is microinjected into Swiss 3T3 cells (26,27). A similar phenotype was produced when HA-p190 was expressed in COS and BHK cells (data not shown). Fig. 2 shows representative cells that had been transfected with p190, p190(S36N), or with a GAP-defective mutant, p190(R1283A) (23), and stained with an anti-HA antibody. Remarkably, only those cells transfected with wild type p190 exhibited the beaded extension phenotype. Cells transfected with either the S36N or R1283A mutant looked normal, even though expression levels of the mutants and wild type p190 were similar, as determined from the fluorescence intensities.
The Phenotypic Changes Induced by p190 Overexpression Result from Cell Body Retraction Accompanied by Formation of Long, Beaded Extensions-To investigate how the p190 overexpression phenotype was produced, NIH 3T3 cells were transfected with a vector that encodes a GFP-p190 fusion protein and monitored by time lapse imaging, beginning 36 h post- transfection. Fig. 3 shows typical transfected cells undergoing retraction of the cell body accompanied by the extension of long, dendritic processes. Cells were monitored for 7 h during which time they continued to extend processes and move around the surface of the plate. These data indicate first that GFP-p190 induces the same phenotype as does the ectopic expression of HA-p190 and second that the phenotype is produced not simply by retraction but also by the active elongation of dendrite-like processes.
The Phenotype Produced by p190 Is Dose-dependent-To confirm that the lack of effect of p190(S36N) on cell morphology was not a result of expression differences, we transfected both COS and NIH 3T3 cells with a range of plasmid doses. In these experiments 5, 10, or 25 g of pRK7, pKH3-p190, or pKH3-p190(S36N) were transiently transfected into cells along with a plasmid containing the gene for green fluorescent protein (GFP) that was used as a marker for transfected cells. Cells were allowed to grow for 2 days, at which time green fluorescent cells were counted and scored for morphology. A fraction of the cells was harvested for immunoblot detection of p190 proteins. Fig. 4 shows a positive correlation between amount of p190 plasmid introduced into the cells, expression of p190, and incidence of the beaded extension phenotype. However, even when 5 times more p190(S36N) plasmid than p190 plasmid was transfected into the cells, there was no evidence of significant morphological changes induced by HA-p190(S36N). These results confirm that p190(S36N) is nonfunctional when expressed in either NIH 3T3 or COS cells.
p190 RhoGAP Activity Is Essential for Induction of Morphological Changes-The similarity between the beaded extension phenotype induced by the overexpression of p190 in NIH 3T3 cells, and that induced by botulinum C3 toxin or RhoGDI (26,27), argues that this phenotype is a consequence of the p190 RhoGAP activity, which depletes the cell of Rho:GTP (and/or Rac:GTP). To test this hypothesis, we used three independent approaches. First, we ascertained that the GBD domain was not sufficient for induction of the beaded extension phenotype. NIH 3T3 cells were transfected with plasmids expressing the isolated HA-tagged GBD domain, residues 1-266, of wild type p190. Transfected cells were fixed and stained for HA. Immunofluorescence showed that cells expressing HA-GBD appeared normal. Therefore, the GBD of p190 is not responsible for the induction of the beaded extension phenotype.
In the second approach, we created a GAP-defective p190. The GAPs of many small GTPases contain a conserved Arg residue that is essential for catalyzing nucleotide hydrolysis (22,28). This residue is located at position 1283 in p190 (Fig.  1A). While our study was in progress, the mutation of Arg 1283 to Lys in p190 was found to destroy its RhoGAP activity (23). We created in p190 a point mutation in this Arg residue, to produce HA-p190(R1283A). To test the GAP activity of the mutant, it was expressed in COS cells, immunoprecipitated with anti-HA antibody, and incubated with recombinant RhoA complexed to [␥-32 P]GTP. As shown in Fig. 5, Rho-directed GAP activity of p190 was easily detectable, but the R1283A mutant did not catalyze GTP hydrolysis on RhoA, even though it was present in the immunoprecipitates at much higher levels than the wild type and S36N mutant p190 proteins. The 1283 mutant was then transfected into NIH 3T3 cells, which were fixed and stained with anti-HA antibody. As can be seen from Fig. 2, although p190(R1283A) was expressed at a level comparable with that of wild type p190, it did not induce the formation of beaded extensions. This result supports the hypothesis that the GAP activity of p190 is required for the induction of morphological changes in NIH 3T3 cells.
As a third, independent approach, we asked whether the co-expression of Rho or Rac could reverse the phenotype induced by p190. We reasoned that a gain-of-function Rho mutant, which is constitutively GTP-bound and is resistant to GAP activity (29, 30), would likely counteract the effects of FIG. 3. Morphological changes associated with p190 overexpression result from cell body retraction accompanied by extension of long, beaded processes. NIH 3T3 cells were grown on 35-mm plates and transfected with 5 g of pRK7GFP-p190 plasmid. GFP-p190 images were captured by fluorescence microscopy with a 20ϫ objective lens at the indicated times using a Hamamatsu camera and Open Lab software. Selected images were processed using Adobe Photoshop 4.0, and pixel intensities were inverted to make the thin dendrite-like processes more clearly visible.

FIG. 4. Dose dependence of the beaded extension phenotype produced by overexpression of p190 protein.
Increasing amounts of plasmid DNA, as indicated, were transfected into COS cells. Plasmids used were as follows: pRK7sGFP ϩ pRK7, pRK7sGFP ϩ pKH3-p190, and pRK7sGFP ϩ pKH3-p190(S36N), where GFP was used as a marker for transfected cells. pRK7 is the parent vector of pKH3 and lacks the triple HA1 epitope tag sequence. For each transfection 20 random fields containing at least 200 cells were scored by eye for cell morphology. After scoring, the same cell cultures were lysed, and extracts were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-p190 antibody. Data are mean percent Ϯ S.D. Results are representative of three independent experiments. p190 most effectively, but increased levels of wild type Rho might partially reverse the effects also. NIH 3T3 cells were therefore co-transfected with HA-p190 and HA-RhoA, the gainof-function mutant HA-RhoA(G14V) and, as a negative control, with the dominant-interfering mutant HA-RhoA(S19N).
As can be seen from Fig. 6A and Table I, RhoA(G14V) completely inhibited the induction of beaded extensions by p190. Wild type RhoA caused a partial inhibition, reducing the incidence of beaded extensions from about 25 to 8% of the transfected cells. The RhoA(S19N) mutant produced cells of abnormal phenotype that were difficult to score consistently. The values given are, however, minimum estimates, and the S19N mutant did not block expression of the beaded extension phenotype. Importantly, the co-expression of the RhoA constructs did not perturb the expression of p190 (Fig. 6B). Therefore, inhibition of the beaded extension phenotype was specific to RhoA:GTP function and was not a result of the inhibition of p190 expression. RhoA(S19N) is believed to act by sequestering upstream Rho exchange factors, thereby reducing production of endogenous Rho:GTP. One might therefore predict that  (vector), pKH3-p190, pKH3-p190S36N, or pKH3-p190(R1283A) were lysed, and the indicated immunoprecipitated proteins were used in a GAP assay as described under "Experimental Procedures." Samples were filter-bound at the given time points. Data are mean Ϯ S.E. from triplicate samples. Relative levels of the exogenously expressed proteins in the immunoprecipitates were determined by anti-p190 immunoblot. Note that the GAP-defective p190(R1283A) was expressed at much higher levels than the wild type p190. RhoA(S19N) alone could induce the formation of beaded extensions. However, this effect was not observed, although the majority of cells expressing RhoA(S19N) were abnormally shaped. The discrepancy points to the possibility that p190 acts in vivo as a GAP not only on Rho but also on Rac and/or Cdc42, as has also been shown by others (31)(32), and that the beaded extension phenotype arises as a consequence of the inhibition of function of multiple GTPases. Alternatively, we cannot rule out the possibility that RhoA(S19N) was not able to adequately limit exchange activity on endogenous RhoA under the cellular conditions tested, and this resulted in an inability to inhibit all the endogenous RhoA in the cell.
To test this hypothesis we asked whether a gain-of-function Rac mutant (G12V) could counteract p190. Co-expression studies using Rac1(G12V), wild type Rac1, and the dominant interfering mutant, Rac1(S17N), produced results very similar to those produced with RhoA (data not shown). Therefore, p190 GAP may act on both Rho and Rac within the intact cell, particularly under conditions where p190 is overexpressed (33).
The GTP Binding Domain (GBD) of p190 Regulates GAP Activity in Vivo but Not in Vitro-Although the GBD is located at the extreme N terminus of p190, and the GAP domain is at the C terminus, one could imagine an allosteric interaction that could allow the GBD to regulate GAP activity directly. To test this possibility, we expressed HA-tagged p190 and the S36N mutant in COS cells, and then immunoprecipitated the proteins from cell lysates and assayed them for GAP activity using recombinant RhoA loaded with [␥-32 P]GTP. As shown in Fig. 5, both the wild type and the S36N mutant p190s displayed similar in vitro RhoGAP activities. Immunoblotting of the same lysates revealed similar levels of expression of these two proteins (Fig. 5). Therefore, despite the inability of p190(S36N) to induce the beaded extension phenotype, the mutant is not defective in GAP activity in vitro. We interpret this result to mean that the GBD of p190 modulates p190 GAP activity indirectly, perhaps by interaction with a regulatory factor.
To investigate this hypothesis further, we asked whether the co-expression of the isolated GBD could interfere with p190 function. Cells were therefore transfected with HA-p190 plus wild type HA-p190(1-266) or with HA-p190(1-266)S36N. Both of these isolated GBD fragments expressed efficiently, at molar levels about 10-fold better than that of the full-length HA-p190 ( Fig. 1). The unmutated GBD had no effect on the incidence of the beaded extension phenotype induced by p190 (Table II). However, expression of the S36N GBD significantly lowered the percent of cells displaying this phenotype, without reducing the level of expression of p190 (Fig. 7). Our interpretation of these data, which is consistent with the results described previously, is that the GBD of p190 must be in the GTP-bound state to allow GAP function within the cell. The S36N mutation in the GBD, analogous to the S17N mutation of Ras, reduces its affinity for guanine nucleotides but enhances its affinity for a guanine nucleotide exchange factor (GEF). Overexpression of the isolated S36N GBD would sequester this exchange factor, thereby reducing the fraction of full-length p190 in the functional, GTP-bound state.
p190 Inhibits Carbachol-mediated JNK Activation-We wished to test the model described above in an assay that was independent of cell morphology. Gain-of-function mutants of the Rac/Rho family of GTPases can stimulate activity of the Jun N-terminal protein kinase (JNK) (2). Activation of JNK by G-protein-coupled receptors, such as the muscarinic receptors, is also mediated by Rac/Rho GTPases (34). We therefore predicted that the overexpression of HA-p190 would block this activation, by suppressing the formation of Rac/Rho:GTP. A similar block to JNK activation has been demonstrated previously, using the isolated catalytic domain of p190 (31).
To test this prediction, cells expressing the human muscarinic receptor, subtype 1 (hmr1), were transfected with HAtagged JNK and wild type or S36N mutant p190 and were stimulated with the muscarinic agonist carbachol. JNK was immunoprecipitated from cell lysates and assayed using GSTc-Jun as a substrate. As shown in Fig. 8, carbachol stimulated JNK by about 3.5-fold. Co-expression of wild type p190 reduced this activation. Importantly, the S36N mutant, although expressed at a level similar to that of wild type p190, did not inhibit JNK activation. Rather, the S36N mutant reproducibly elevated the basal JNK activity by about 2-fold and enhanced the effect of carbachol. The most likely explanation of these data is that the S36N p190 is acting as a dominant interfering mutant to inhibit the function of endogenous p190GAP, thereby elevating basal Rac/Rho:GTP levels. By analogy with the S17N Ras mutant, we propose that S36N p190 sequesters  along with the full-length p190 protein in NIH 3T3 cells The data represent combined results from independent experiments, the number (n), of which is noted in parentheses. For each experiment, 100 -400 cells were scored for phenotype. For each condition 4 g of each plasmid was transfected into cells on 2-well Labtek slides. A vector encoding GFP was included in all transfections as a marker for transfected cells.

Plasmids transfected
Cells with beaded extensions a Represents a significant difference from transfection with pKH3-p190 alone. p value is Ͻ 0.01 (unpaired t test). Values shown are Ϯ 1 S.E. (%). a nucleotide exchange factor for the p190 GBD. In the absence of a functional exchange factor, the GTP/GDP ratio of the p190 GBD falls, which (by an unknown mechanism) then disables the p190 GAP domain. DISCUSSION This study presents the first evidence of a role for guanine nucleotide binding to the N-terminal GBD of the p190 Rho/ RacGAP. The isolated C-terminal GAP domain has been shown previously to stimulate the GTPase activity of Rho, Rac, and Cdc42 in vitro and to cause both a loss of stress fibers and cell shape changes consistent with an inhibition of Rho function (8). No linkage between the N-terminal GBD and the C-terminal GAP domains has previously been reported, and in vitro studies have shown that point mutants in the GBD that are defective in GTP binding possess a similar GAP activity to wild type p190 (10). We now demonstrate that, in transfected NIH 3T3 cells, the nucleotide bound state of the p190 Rho/RacGAP GBD influences cellular morphology and JNK activity, effects that are likely to be mediated by the GAP activity of p190.
NIH 3T3 cells transfected with wild type, HA-tagged, p190 Rho/RacGAP exhibited a pronounced rounding of the cell body and beaded, dendritic-like extensions similar to those seen when Rho function is impaired by C3 botulinum toxin or by injection of RhoGDI (26,27). Remarkably, the expression at similar levels of a mutant p190 defective in GTP binding (S36N) did not induce this phenotype. Wild type p190, but not the S36N mutant, also inhibited the activation of JNK by muscarinic receptors. Importantly, and in agreement with previous studies (10), the GAP activities of the immunoprecipitated wild type and S36N mutant p190s toward RhoA were similar, confirming that, in vitro, the GAP domain and GTP binding domain can function independently of one another. We demonstrated that the beaded extension phenotype induced by expression of wild type p190 is mediated by the Rho/Rac GAP activities of the protein in three ways. First, a point mutant in the C-terminal GAP domain, which abolishes detectable GAP activity toward recombinant RhoA, was incapable of inducing the beaded extension phenotype. Second, the overexpression of the isolated N-terminal GBD was also unable to induce this phenotype. Third, the phenotype induced by wild type p190 was completely suppressed by co-expression of a gain-of-function Rho mutant, RhoA(G14V), which is insensitive to RhoG-APs (29,30). These data taken together strongly support the hypothesis that the GAP activity of p190 is required for induc-tion of the biochemical and phenotypic changes occurring in cells that overexpress p190 and that within the intact cell, GTP-binding by the N-terminal GBD is essential for the accomplishment of this Rho family-directed GAP activity.
It is important to note that overexpression of a dominantnegative Rho mutant, RhoA(S19N), did not produce a phenotype identical to the overexpression of p190 in NIH 3T3 cells. There are at least two explanations for this result. First, the dominant-negative RhoA(S19N) is presumed to act by sequestering Rho-specific exchange factors (GEFs) and therefore prevents the activation of endogenous Rho GTPases. The specificities of the GEFs sequestered by RhoA(S19N) may differ in regard to which GTPase they inactivate. For example, the guanine nucleotide exchange factors Dbl and Ost are able to catalyze exchange on both RhoA and Cdc42 but not Rac (35,36). The p190 GAP also possesses differing affinities for the various members of the Rho family in vivo and in vitro. Although p190 has a 9-fold greater affinity for Rho than Rac or Cdc42 (8), the catalytic efficiency of p190 for Rac is not substantially different from that of other known Rho family GAPs (33). Moreover, although microinjection experiments of the p190 catalytic domain have suggested p190 is specific for Rho (8), more recent data demonstrate that p190 also inhibits both Rac and Cdc42 function (31,32). Therefore, the set of Rac/Rho GTPases inactivated by these two reagents, RhoA(S19N) and p190, may also differ and result in the production of distinct cellular phenotypes. Alternatively, protein expression of RhoA(S19N) might not have been sufficient to adequately sequester RhoA exchange factors. A second possibility is that p190 possesses functions additional to its GAP activity that can produce phenotypic changes in cells that overexpress the protein. However, our present data are not consistent with this latter hypothesis.
We have shown that wild type HA-tagged p190 behaves as a functional Rho/RacGAP when expressed in intact cells, but that the S36N mutant p190, which fails to bind GTP, appears to be GAP-defective in vivo. The data therefore argue that the Nterminal GTP binding domain can regulate the GAP activity toward RhoA either by altering the accessibility of Rho:GTP to the GAP domain of p190 due to mislocalization of p190, or via interactions with factors that regulate GAP activity. We have considered each of these hypotheses. To test whether the loss of GTP binding to p190 causes the protein to mislocalize within the cell, such that it is inaccessible to Rho and Rac, we used confocal laser microscopy to visualize cells co-expressing HAtagged and GFP fusion p190 proteins. However, we were unable to detect any obvious difference in distribution of the two proteins (data not shown).
Another possibility is that p190 wild type and S36N associate differentially with cellular factors. One obvious candidate is the p120 RasGAP which has been shown to be capable of high affinity association with tyrosine-phosphorylated p190 (12)(13)(14)37). We have not been able to detect any regulatory effect of p120 binding on p190 RhoA GAP activity. For example, the co-expression of p120GAP in COS cells with p190 did not significantly alter the specific activity of p190 RhoGAP under conditions in which both proteins were co-precipitated (data not shown). Furthermore, under the conditions used for our in vitro GAP assays, neither wild type nor S36N were associated with detectable amounts of endogenous p120 RasGAP (data not shown). Both the wild type and S36N p190 were tyrosinephosphorylated to the same level. We conclude that neither the state of p190 tyrosine phosphorylation nor association with p120 GAP are responsible for the effect of ectopic p190 expression or the inability of the mutant p190(S36N) to induce that effect. FIG. 8. Expression of HA-p190 in NIH 3T3 cells decreases JNK activation in response to carbachol. Cells were transfected with either 2 g of pKH3-JNK ϩ 20 g of pKH3, 2 g of pKH3-JNK ϩ 20 g of pKH3-p190, or 2 g of pKH3-JNK ϩ 20 g of pKH3-p190S36N. Cells were treated with agonist (carbachol (C) or anisomycin (A)) or sterile water (control(Ϫ)). JNK activity was assayed using GST-cJun as a substrate, as described under "Experimental Procedures." For the autoradiograph, the gel was exposed to film for 16 h. 100 g of anisomycin/ 10-cm plate was used as a positive control for JNK activation. Values for JNK activity are means Ϯ range (n ϭ 2).
In the GDP-bound state, p190 may associate with an inhibitory factor. However, this model predicts that the expression of the S36N mutant GBD would sequester the inhibitory factor and thereby enhance the beaded extension phenotype induced by co-transfecting wild type p190. This was not the case. Rather, the S36N GBD partially suppressed the induction of the beaded extension phenotype which we discuss in further detail below. We cannot, however, discount this model entirely because the factor may be abundant and was not fully sequestered by the mutant GBD.
An alternative model is that p190:GTP associates with an effector or target protein that is a co-activator of the GAP domain. Precedent exists for this model in the Ran GTPase system. For instance, although Ran GAP is a highly efficient catalyst of GTP hydrolysis in vitro, RanBP1 can stimulate its activity severalfold and appears to be required in vivo (38,39). In our model the mutant p190(S36N) would not bind the coactivator and so could not function as an efficient GAP within the intact cell. One might argue that co-expression of the isolated GBD would sequester the co-activator and thereby suppress the effect of the wild type p190. However, we find that neither the full-length p190 nor the isolated GBD are exclusively GTP-bound when expressed in COS cells, and it may not be technically feasible to express a high enough concentration of GTP-bound GBD within the cells to sequester a significant fraction of co-activator.
Both models suggest that the p190 GAP activity is controlled by the guanine nucleotide-bound state of its GBD. Guanine nucleotide binding would then be regulated by other factors. A p190 GBD-GAP activity has already been described in lysates from rat fibroblasts (10), but it remains uncharacterized. The reverse process would be catalyzed by a p190 GBD exchange factor, or GEF. Our data provide evidence in favor of the existence of such a p190-GEF. We showed that the expression of p190(S36N) enhanced basal JNK activity and enhanced the JNK activity stimulated by carbachol, which acts through a Rac/Cdc42-mediated pathway (34). These effects are consistent with a sequestration of a p190-GEF by the S36N mutant, which reduces the endogenous level of GTP-bound p190, shutting down the p190 GAP activity and thereby increasing the level of Rac:GTP, which in turn activates JNK. Second, we showed that the overexpression of the isolated S36N(GBD) mutant partially suppresses the beaded extension phenotype induced by p190. Again, the sequestration of a p190-GEF would reduce the fraction of p190 exhibiting Rho/Rac GAP activity and would thereby inhibit the expression of the phenotype.
Maruta and co-workers (40) have reported that the S36N GBD mutant is capable of transforming NIH 3T3 cells, whereas the overexpression of the wild type GBD can inhibit Ras-dependent transformation. Although we have not observed similar phenotypic effects, these results are nonetheless consistent with our model, in which the S36N mutant would sequester an exchange factor for the p190 GBD leading to a potentiation of Rho/Rac activities, which might in turn alter cell growth characteristics. Overexpression of the wild type GBD at very high levels may compete for a GAP for p190, leading to an attenuation of Rho/Rac signaling which might then suppress transformation by Ras (41).
Together, these data provide evidence of a novel mechanism for the control of Rho family GTPases through a GAP that is regulated via its own GTPase domain. They further imply that an unknown signal transduction pathway impinges on p190 GAP. The major challenge will be to identify the regulatory factors and targets for the p190 GBD and to determine the signals to which they respond.