Loop 6 of RhoA Confers Specificity for Effector Binding, Stress Fiber Formation, and Cellular Transformation*

Hui ZongDagger §, Narayan Raman, Leigh A. Mickelson-YoungDagger §, Simon J. AtkinsonDagger , and Lawrence A. QuilliamDagger §parallel

From the Dagger  Department of Biochemistry and Molecular Biology and the § Walther Oncology Center,  Division of Nephrology, Indiana University School of Medicine, Indianapolis, Indiana 46202

    ABSTRACT
Top
Abstract
Introduction
References

Rho family GTPases regulate multiple cellular processes, including cytoskeletal organization, gene expression, and transformation. These effects are achieved through the interaction of GTP-bound proteins with various downstream targets. A series of RhoA/Rac1 and Rho/Ras chimeras was generated to map the domain(s) of RhoA involved in its association with two classes of effector kinase, represented by PRK2 and ROCK-I. Although the switch 1 domain was required for effector binding, the N terminus of Rho (residues 1-75) was interchangeable with that of Rac. This suggested that the region of Rho that confers effector binding specificity lay further C-terminal. Subsequent studies indicated that the "insert domain"(residues 123-137), a region unique to Rho family GTPases, is not the specificity determinant. However, a determinant for effector binding was identified between Rho residues 75-92. Rac to Rho point mutations (V85D or A88D) within loop 6 of Rac promoted its association with PRK2 and ROCK, whereas the reciprocal Rho(D87V/D90A) double mutant significantly reduced effector binding capacity. In vivo studies showed that microinjection of Rac(Q6IL/V85D/A88D) but not Rac(Q6IL) induced stress fiber formation in LLC-PK epithelial cells, suggesting that loop 6 residues conferred the ability of Rac to activate ROCK. On the other hand, the reciprocal Rho (Q6IL/D87V/D90A) mutant was defective in its ability to transform NIH 3T3 cells. These data suggest that although Rho effectors can utilize a Rho or Rac switch 1 domain to sense the GTP-bound state of Rho, unique residues within loop 6 are essential for determining both effector binding specificity and cellular function.

    INTRODUCTION
Top
Abstract
Introduction
References

The Ras superfamily of GTPases is involved in the regulation of multiple cellular processes, including signal transduction to induce growth and differentiation (Ras), membrane trafficking (Rab), nuclear transport (Ran), and cytoskeletal regulation (Rho) (1-3). The best characterized members of the Rho family of GTPases are RhoA, Rac 1, and CDC42Hs, which regulate stress fiber formation, lamellipodia formation and membrane ruffling, and filopodia formation, respectively (2). Rac and CDC42 also regulate transcription via activation of the JNK and p38 mitogen-activated protein kinase cascades, whereas all three Rho family members can activate the serum response factor (4, 5) and promote cell cycle progression (2, 6).

Ras superfamily proteins act as molecular switches, alternating between inactive GDP-bound and active GTP-bound states. GTP binding induces a conformational change in two domains of Ras, referred to as switch 1 and switch 2 (7). In the case of Ha-Ras, effector protein binding and activation occurs primarily through association with the switch 1 domain located in loop 2 and beta -strand 2 (residues 32-40) (3). Indeed, point mutations within this region of Ras can differentially disrupt its interaction with various downstream targets (8-11). Mutations in the Rac switch 1 domain similarly perturb specific effector protein interactions (12-14). However, additional regions of Rac have been implicated in effector binding and/or activation. These include an alpha -helix located between residues 123-137 that is unique to Rho family members (henceforth referred to as the "insert domain") and a region located between residues 143-175 (15, 16).

Studies by Hall and colleagues (16, 17) indicated that in contrast to Ras, the effector-binding domain of Rho extends beyond switch 1. For example, a Rac73Rho chimera, lacking Rho N-terminal sequences could still induce stress fiber formation (16). Therefore, we set out to determine which regions of Rho are involved in its interaction with the downstream effectors PRK2 and p160 ROCK (18). PRK2 is a serine/threonine protein kinase isolated by us and others (5, 19-22) that shares a Rho binding domain, HR1,1 with PRK1/PKN, PRK3, Rhotekin, and Rhophilin (see Ref. 18 and Fig. 2). The exact function of these proteins is currently unknown. Meanwhile, the Rho kinases (ROKalpha /ROCK-II/Rho-kinase and ROKbeta /ROCK-I/p160 ROCK) that mediate Rho-induced stress fiber formation possess a distinct Rho-binding structure (23), referred to herein as RBD. Although HR1 and RBD do not share primary sequence homology, they are both highly charged and are predicted to contain significant alpha -helical content. It was therefore of interest to determine whether or not the same domains of Rho recognized both classes of effector-binding motif.

Using Rho/Rac chimeras, we demonstrate here that multiple domains of Rho are involved in effector binding. Although switch 1 domain residues common to both Rho and Rac are required for binding Rho-effectors, unique residues in loop 6 of Rho confer specific interaction with Rho targets. In vivo data demonstrated that loop 6 of Rho not only distinguishes between Rho and Rac effectors but also is required for their physiological activation. Deletion of the Rho insert domain (residues 123-137) severely disrupted Rho-induced transformation but did not significantly disrupt PRK2 or ROCK-I binding to Rho in vitro. The loss of transformation resulting from insert domain deletion may have been due to requirement of the insert helix for activation of an additional Rho target. However, we cannot rule out the possibility that its deletion resulted in disruption of the adjacent loop 6 structure.

    EXPERIMENTAL PROCEDURES

Materials-- [alpha -32P]GTP (3,000 Ci/mol) was purchased from Amersham Pharmacia Biotech. Vent DNA polymerase and all restriction enzymes were purchased from New England Biolabs. PCR cloning vector pCR Blunt was from Invitrogen. M2-FLAG and hemaglutinin antibodies were from Sigma and Berkeley Antibody Co., respectively.

Construction of Plasmids-- All the Rho mutants and Rho/Rac chimera constructs used in this study are summarized in Fig. 1. Rho and Rac mutants are offset by two codons due to the longer N terminus of RhoA. The Rac(Val12)143Rho, Rac73Rho143Rac, and Rho(G14V/T37A) constructs have been described previously (16) and were provided by A. Hall (University College, London, United Kingdom). Rho(F39A), Rho(D87V/D90A), Rac(V85D/A88D), Rac(V85D), and Rac(A88D) mutants were generated by site-directed mutagenesis using PCR. Rho122Rac139Rho (RhoDelta Rac) was created by PCR using long oligonucleotides that replaced the Rho insert domain (codons 123 to 138) with that of Rac. RhoDelta Ras mutations were also created using this strategy, replacing codons 122-141 of Rho with codons 121-127 of Ha-Ras. The Rho75Rac and Rac73Rho chimeras were made by swapping the N terminus of Rho/Rac, using the PvuII site at codon 59 (residues 59-73 are identical between Rho and Rac). Site-directed mutagenesis was used to generate wild type Rho75Rac92Rho and Rac73Rho90Rac using Rho75Rac92Rho(Leu63) and Rac73Rho90Rac(Leu61) (16) as PCR templates. Rho92Rac and Rac90Rho were constructed by swapping the N-terminal 59 amino acids of Rho75Rac92Rho and Rac73Rho90Rac, taking advantage of the codon 59 PvuII common to the chimeras. All mutants were subcloned into pGEX-2T (Amersham Pharmacia Biotech) to generate GST fusion protein or into pZIP (24) or pCGN for expression in mammalian cells. Codons 905-1046 of p160 ROCK were PCR-amplified from a human small intestine Marathon-ready cDNA library (). Codons 1-284 (HR1), 36-113 (HR1a), 133-208 (HR1b), and 210-284 (HR1c) were amplified from PRK2 by PCR. All constructs were subcloned into pGEX-2T and pMal-c (New England Biolabs) to generate GST and maltose-binding protein (MBP) fusion proteins, respectively. pCMV2-FLAG-PRK2 has been described previously (5). All cDNAs created for this study by PCR were confirmed by dideoxynucleotide sequencing.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1.   Summary of Rho/Rac chimeras used in this study. Closed bars represent Rho sequences; open bars represent Rac or Ras sequences. Nonchimeric mutants are indicated with hatched boxes. Rho and Rac mutants are offset by two codons due to the longer N terminus of RhoA.

Expression of GST Fusion Proteins-- GST-fused Rho GTPases were expressed in the BL21(DE3)lysE strain of Escherichia coli. Following a 3-h induction with 0.2 mM isopropyl-1-thio-beta -D-galactopyranoside at 37 °C, cells were pelleted and resuspended in lysis buffer (50 mM Tris, pH 7.6, 100 mM NaCl, 5 mM MgCl2, 100 µM GDP, 0.5% Nonidet P-40, 1 mM dithiothreitol, 1.9 mg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). After sonication to lyse the cells, the supernatant was tumbled with glutathione-agarose beads (Sigma) at 4 °C overnight. Beads were then washed three times with lysis buffer and stored at 4 °C. GST-fused HR1 and RBD were purified similarly except for using 1 mM EDTA instead of MgCl2 and GDP in the lysis buffer. MBP fusion proteins were also purified in a similar manner except for using amylose beads (New England Biolabs) and the following lysis buffer: 20 mM Tris, pH 7.4, 200 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol, 1.9 mg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Rac proteins for microinjection studies were cleaved from GST using 10 units of thrombin in 50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 2.5 mM CaCl2, 1 mM dithiothreitol at 4 °C overnight. After removing thrombin with p-aminobenzamidine beads (Sigma), the supernatants were dialyzed against 15 mM Tris, pH 7.6, 150 mM NaCl, 5 mM MgCl2, 0.1 mM dithiothreitol at 4 °C for 12 h and concentrated using an Amicon Centricon filter, as described (25). The active Rac protein concentration was estimated by [32P]GTP loading (26).

Rho Overlay Assay-- Bead-bound Rho proteins were loaded with [alpha -32P]GTP by incubation for 20 min at 30 °C in 50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM EDTA, 5 µM GTP, 10 µCi of [alpha -32P]GTP, and 1 mM dithiothreitol. MgCl2 was added to 10 mM to stop the reaction, and free nucleotide was removed by washing beads three times with the loading buffer containing 10 mM MgCl2 in place of GTP/EDTA. Proteins were then eluted by incubating beads with 100 µl of 20 mM glutathione, 100 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM MgCl2, 10% glycerol, and 1 mM dithiothreitol on ice for 20 min. HR1a and RBD proteins were run on SDS-polyacrylamide gel electrophoresis gels, transferred to polyvinylidene difluoride membrane, and blocked in 1% bovine serum albumin, 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, and 0.05% Tween-20 for 2 h. The membrane was then incubated for 15 min at 4 °C with 25 pmol of [alpha -32P]GTP-bound Rho (determined by scintillation counting) in blocking buffer supplemented with 10 mM MgCl2 and 100 µM GDP. Following extensive washing in binding buffer, the membrane was autoradiographed at -80 °C to visualized the binding of Rho to its effectors.

Rho Precipitation Assay-- The GTP binding capacity of each Rho mutant was determined as above, using [alpha -32P]GTP. 2.5 µg of active GST-fused GTPases was then preloaded with unlabeled GTPgamma S or GDP and eluted off glutathione-agarose beads as described above. Subsequently, the proteins were incubated with 5 µg of MBP-fused HR1 or 1 µg of MBP-fused RBD immobilized on amylose beads in 50 mM Tris, pH 7.6, 50 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol, and 50 µM GTP at 4 °C for 1 h. Beads were washed three times in the above buffer (except for 0.1% Triton X-100), and bound proteins were separated by SDS-polyacrylamide gel electrophoresis. The binding of GST-Rho/Rac to HR1 and RBD was then determined by Western blotting using "in-house" anti-GST polyclonal antibody and ECL (Amersham Pharmacia Biotech) detection.

PRK2 Precipitation Assay-- COS cells were transfected with pCMV2-FLAG-Prk2 using LipofectAMINE (Life Technologies, Inc.). After 2 days, cells were washed three times with cold PBS and lysed in 20 mM Tris, pH 7.4, 50 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 1.9 mg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. 2 µg of active bead-bound GTPases was preloaded with GTPgamma S as described above. Equal amounts of cell lysate supplemented with 10 mM MgCl2 and 50 µM GTP were incubated with each GTPase at 4 °C for 2 h. Beads were washed three times with 20 mM Tris, pH 7.4, 50 mM NaCl, 10 mM MgCl2, 0.1% Nonidet P-40, 10% glycerol, and 1 mM dithiothreitol. The binding of PRK2 to immobilized GTPases was determined by Western blotting with M2 anti-FLAG antibody.

Exchange Assay-- WT RhoA was preloaded with [alpha -32P]GTP as described above. 25 pmol of active Rho was then incubated with 400 µg of GST (as control) or different amounts of GST-HR1a (0.5-12.5 nmol/16-400 µg) on ice for 1 h to allow ligand binding. Subsequently, 500 µl of GTP exchange buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and 50 µM GTP) was added to each sample, and exchange reactions were incubated at 37 °C. Aliquots were removed at the indicated time points, and reactions were terminated by addition of 500 µl of ice-cold GTP exchange buffer. Protein-bound nucleotide was then captured on BA85 nitrocellulose filters (Schleicher & Schuell), which were washed three times with 1 ml of cold GTP exchange buffer prior to scintillation counting.

Microinjection and Fluorescence Microscopy-- LLC-PK cells were maintained in 1:1 Dulbecco's modified Eagle's medium/Ham's F-12 medium supplemented with 10% fetal calf serum. For microinjection experiments, cells were plated on coverslips and grown to >90% confluence. Cells were then co-microinjected with the indicated Rac proteins (60 µg/ml), botulinum exoenzyme C3 (10 µg/ml), and fluoroscein-conjugated dextran as described.2 C3 exoenzyme was also prepared as described.2 After 45 min, cells were fixed and stained with rhodamine-conjugated phalloidin, and F-actin was visualized using a Bio-Rad MRC 1024 confocal microscope.

NIH 3T3 Cell Transfection-- For transformation assays, NIH 3T3 cells were transfected with 2 µg of empty pZIP vector or vector encoding Rho(Q63L), Rho(Q63L/D87V/D90A) or RhoDelta Ras(Leu63) along with 1 µg of additional pZIP vector or pZIP-Raf(Y340D), as indicated, using the calcium phosphate precipitation procedure (27). Cells were maintained in regular growth medium, and transforming foci were fixed and stained with crystal violet after 12-16 days of culture. To create stable cell lines, NIH 3T3 cells were transfected with 0.5 µg of pCGN plasmids containing the above mutants and selected on growth medium supplemented with 200 µg/ml hygromycin B. At least 100 colonies were pooled and immunoblotted for HA-tagged Rho expression approximately 3 weeks posttransfection.

    RESULTS

The HR1a Domain of PRK2 Is Sufficient for Its Interaction with Rho-- PRK2 and the related kinase PKN/PRK1 possess three tandem copies of a 60-70-amino acid sequence at their N terminus that has been referred to as homology region 1 (HR1) (Ref. 19 and Fig. 2a). Single HR1 units are also present in other Rho-binding proteins (Rhophilin and Rhotekin) and appear to represent a common Rho binding motif (18). Another Rho-binding motif, RBD (Fig. 2b), that is present in ROCK does not share significant primary sequence homology with HR1 (23). Both domains, however, are highly charged and predicted to contain significant alpha -helical content. We therefore established in vitro binding assays to determine whether these two distinct structures interacted with common domains of Rho. Fig. 2c shows that interaction of RhoA-GTP with the HR1 region of PRK2 occurs primarily via the N-terminal-most repeat, HR1a. Weaker interaction was detectable with the HR1b but not with the HR1c domain. This is in agreement with Flynn et al. (28), who similarly found that Rho predominantly bound to the HR1a domain of PRK1/PKN. Binding of Rho to both HR1a and HR1b was determined to be GTP-dependent. RhoA-GTP also bound to the RBD of p160 ROCK (Fig. 2d). Although ROCK-RBD has been reported to interact with Rac (13, 14), under our assay conditions (Fig. 2d) and those described by Leung et al. (29), there was no interaction of RBD with WT Rac1-GTP. We therefore generated a series of Rho/Rac chimeras to map which domains of Rho are required for its specific interaction with PRK2 and ROCK-1.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2.   Rho-GTP specifically interacts with the HR1a domain of PRK2 and the RBD of ROCK. a, diagram indicating the presence of HR1a, HR1b, and HR1c putative Rho-binding domains, at the N terminus of PRK2. HR2, catalytic domain and a Nck-binding domain (5, 19) are also shown. b, diagram showing the RBD of ROCK-I, located between the coiled-coil and pleckstrin homology (PH) domain (18). The catalytic domain and cysteine-rich region are also shown. c, the indicated MBP-HR1 fusion proteins were immobilized on amylose beads and incubated with GST-Rho that had been loaded with GTP or GDP. Co-precipitation of Rho by MBP fusion proteins was then determined by Western blotting with anti-GST antibody. NS indicates a nonspecific band present in all but the HR1a lanes. Results are representative of at least two experiments. d, both HR1a and RBD bound to Rho-GTPgamma S but not Rac-GTPgamma S in the Rho precipitation assay.

The Switch 1 Domain of Rho Influences Effector Binding-- Introduction of residues from Rho into the switch 1 domain of Rac has previously been shown to disrupt various Rac functions (12). However, we found that a Rac73Rho chimera (containing residues 1-73 of Rac1) still bound to HR1 and RBD, suggesting that either a Rac or a Rho switch 1 domain could support Rho-effector interaction (see Fig. 3). In contrast, introduction of a Rho T37A mutation into Rho disrupted its ability to bind the PRK2-HR1 domain. A similar observation was previously reported for PKN (30). However, this mutation may disrupt the interaction of Thr37 with Mg2+ and the gamma -phosphate of GTP that is necessary for the switch 1 domain to adopt its active conformation (7, 26, 31). Therefore, we also created a mutation in Phe39 that, like Thr37, is conserved between Rac and Rho. The F39A mutation totally abolished the ability of Rho to bind to both HR1 and ROCK-RBD, suggesting that the switch 1 domain does indeed contribute to effector binding but does not determine the specificity of Rho-effector interaction. The GTP dependence of Rho-effector binding (Fig. 2) was also supportive of the involvement of switch 1. However, because Rac does not normally interact with PRK2 or ROCK (5, 29), then the C-terminal two-thirds of Rho must determine effector binding specificity.


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 3.   Mutation of conserved residues within the Rho switch 1 domain but not its replacement by Rac sequences can disrupt binding to HR1a and RBD. a, using the Rho overlay assay (see under "Experimental Procedures"), GST-RBD and GST-HR1a were found to bind to [alpha -32P]GTP-labeled RhoA but not Rac1. Mutation of Rho switch 1 residues 37 and 39 or mutations that exchanged the C-terminal two-thirds of Rho with Rac disrupted Rho-effector binding. b, to confirm these findings, interaction of mutants was assessed using the Rho precipitation assay. The same GST-Rho/Rac mutants described in a were incubated with amylose-immobilized MBP-HR1, and binding of Rho proteins was determined by Western blotting. Weak binding of effectors with Rho(T37A) and Rho75Rac were more evident in this assay due to the sharper resolution of Rho protein bands. NS indicates a nonspecific band appearing in all lanes. Data are representative of at least three independent experiments.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 4.   Rho/Rac chimeras localize the Rho-effector binding determinant to residues 76-122. To address binding of Rho effectors to the insert domain, this region (residues 123-138) was swapped with the equivalent residues of Rac (RhoDelta Rac) or Ras loop 8 (RhoDelta Ras). Although the RhoDelta Ras mutant did not bind as efficiently, both chimeras associated with HR1a and RBD in the overlay assay. A Rac73Rho143Rho containing the central portion of Rho bound to HR1a and RBD, but a Rac143Rho chimera containing the C terminus of Rho did not. Together, these data narrow the Rho-specific binding determinant to residues 76-122. The numbers below each lane show (phosphorimager) quantitation of the Rho-effector binding data and indicate the ability of each chimera to bind GTP (a measure of protein viability). Data are representative of at least three experiments.

The Insert Domain Is Not Required for Rho Effector Interaction-- The highly charged insert domain has been postulated to be an effector binding determinant and has been reported to be required for Rac-induced activation of the neutrophil NADPH oxidase (15, 32, 33). Because the HR1 and RBD sequences are also highly charged and could possibly associate with the insert domain via ionic interaction, we exchanged the entire insert domain of Rho with that of Rac. As seen in Fig. 4, the Rho123Rac138Rho chimera (RhoDelta Rac) still bound efficiently to HR1a and RBD, suggesting that the insert domain did not dictate Rho effector binding specificity.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5.   A sequence encompassing residues 76-92 is responsible for specific interaction of Rho versus Rac with HR1 and RBD. a, as shown in Fig. 3, Rho75Rac had reduced effector binding ability, whereas Rac73Rho still bound to HR1 and RBD in the Rho precipitation assay. A Rho92Rac chimera also bound strongly to effectors, but the complementary Rac90Rho chimera had no detectable interaction with HR1 or RBD, narrowing the Rho-effector binding determinant to residues 76-92. This was confirmed using a Rac73Rho90Rac chimera. Data are representative of 3-5 experiments. b, although Rac did not associate with HR1, a previously described F78S mutant (34) was found to weakly bind to it in a GTP-dependent manner. Data are representative of two experiments. c, in the Rho precipitation assay, GTPgamma S-bound Rac(Q61L) and Rac(F78S) associated with MBP-RBD.

Although introduction of the Rac insert domain did not lead to loss of Rho-effector binding, this sequence possesses a very similar conformation and charge distribution to that of Rho, having highly charged residues on the outer surface of the helix and long-chain hydrophobic residues on the inner side facing loop 6 (34, 35). So, just as residues 1-73 of Rac could replace residues 1-75 of Rho, it was possible that the insert domains may also be interchangeable. To determine whether the insert domain is involved in Rho-effector binding, we removed this region, replacing it with the corresponding sequence from loop 8 of Ras. This swap was equivalent to that previously made in CDC42 by McCallum et al. (36), which also removes prolines 138 and 141 to minimize disruption of the global structure. This RhoDelta Ras mutant still interacted with PRK2 and ROCK-RBD (Fig. 4), indicating that the insert domain is not essential for PRK or ROCK family binding. Although RhoDelta Ras did not bind to effectors quite as effectively as WT Rho (~60% binding), it was not as efficiently expressed in E. coli and bound fewer mol of GTP/mg of total protein (Fig. 4), suggesting that the decreased binding may just reflect recombinant protein instability.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 6.   Rho residues 87 and 90 in loop 6 confer specific interaction with HR1 and RBD. a, alignment of RhoA residues 76-92 with the equivalent sequence of Rac. Identical residues are indicated by asterisks, and unique residues are in boldface. The most divergent residues, 87 and 90, are shown in larger type. The bar indicates the positions of loop 5, beta -strand 4, loop 6, and alpha -helix 3 in the Rho-GTPgamma S three-dimensional structure (44). b, using the Rho precipitation assay, double mutation of Rho aspartate residues 87 and 90 to those present in Rac resulted in a dramatic decrease in binding to both Rho effectors. Introducing these aspartate residues into positions 85 and 88 of Rac, together or alone, resulted in a gain of RBD and HR1 binding. There was no binding to Rac or the MBP control. Data are representative of at least three experiments.

Rac/Rho Chimeras Define a Novel Effector-binding Domain in RhoA-- To identify regions of Rho outside of the classic switch domains that confer effector binding specificity, we used an additional series of Rho/Rac chimeras. Although a Rac143Rho chimera was ineffective, Rac73Rho143Rac still bound to both HR1 and RBD (Fig. 4). This suggested that a region between residues 75 and 143 of Rho is sufficient for effector binding. This notion is supported by the observation that Rac73Rho143Rac induced stress fiber formation in Swiss 3T3 cells as effectively as Rho (16). The significant decrease in binding of HR1 and RBD to Rac73Rho143Rac, as with RhoDelta Ras, was very likely due to the poor GTP binding capacity of Rac73Rho143Rac (Fig. 4). Because the RhoDelta Rac and RhoDelta Ras chimeras that still bound Rho targets encompassed residues 123-138, we concluded that the Rho-effector-binding domain lay between residues 75 and 123. This was confirmed using a Rho122Rac chimera, which bound to both effectors.3 Additionally, because a Rho92Rac but not a Rac90Rho chimera could efficiently interact with HR1a and RBD (Fig. 5a), the Rho-effector-binding sequence was further narrowed down to Rho residues 76-92.

Rac Residue 78 Weakly Influences Rho-Effector Binding-- Although we previously found no interaction between full-length PRK2 and Rac1-GTP in precipitation experiments (5), it has been reported that Rac can weakly bind to this kinase (20). However, this binding discrepancy may have been due to the use of a Rac F78S mutant (34) in the latter study. Because residue 78 is located within the residue 76-92 region defined above, we compared the ability of Rac(WT) and Rac(F78S) to bind to Rho effectors. As shown in Fig. 5, b and c, the Rac(F78S) mutant bound, albeit weakly, to PRK2-HR1 and ROCK-RBD under conditions where Rac(WT) did not. Although this may help resolve differences in the literature regarding Rho-effector binding specificity (5, 12, 13, 20, 29, 37), the weak signal suggested that other residues within the 76-92 region must contribute to PRK2 and ROCK binding.

Loop 6 Confers Rho Protein Effector Binding Specificity-- A region spanning residues 74-90 has previously been shown to be required for Rac binding to the Rac GAP, BCR (16). Therefore we next looked at the ability of a Rac73Rho90Rac chimera to bind to HR1a and RBD. Introducing residues 76-92 of Rho into Rac conferred HR1 and RBD binding capability on Rac (Fig. 5a). The reciprocal Rho75Rac92Rho chimera was unstable, making it difficult to assess its effector binding properties.

Comparison of Rho residues 76-92 with the equivalent sequence of Rac (residues 74-90) indicated several differences (Fig. 6a). Most notably, the negatively charged Asp residues 87 and 90 of RhoA loop 6 were hydrophobic Val and Ala residues in Rac. Because loop 6 is a surface exposed region of Rho, it represents a potential second effector-binding site. Therefore, we swapped these loop 6 residues between Rac and Rho. Introduction of a V85D/A88D double mutation into Rac (RacDD) conferred strong HR1 and RBD binding activity (Fig. 6b). Furthermore, the introduction of the reciprocal (D87V/D90A) mutation into loop 6 of Rho (RhoVA) resulted in a partial loss of binding to RBD and almost complete loss of binding to HR1 (Fig. 6b). This data suggested that loop 6 is at least one of the determinants of Rho-effector binding specificity. To establish whether a single Asp residue was responsible for effector binding, the residue 85 and 88 mutations were introduced individually into Rac. Both of these mutants were capable of conferring HR1 and RBD binding, although less effectively than RacDD. Interestingly, the RhoVA mutant bound to RBD more effectively than to HR1 (Fig. 6b). A similar binding difference was also observed with Rho75Rac (Fig. 5a), suggesting that RBD was less dependent on loop 6 for Rho binding.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   Loop 6 of Rho is also required for association with full-length PRK2. Full-length, FLAG-tagged PRK2 was expressed in COS cells and precipitated from cell lysates using the indicated GTPgamma S-bound GTPases immobilized on glutathione-agarose beads. PRK2 was detected using anti-FLAG antibody. The far right lane (Lysate) indicates the position of PRK2 Western blotted from the cell lysate. As observed for the isolated HR1 and HR1a domains, full-length PRK2 bound to Rac chimeras containing Rho loop 6 residues. Binding did not require the Rho insert domain. Data are representative of at least two experiments.

To confirm that the binding pattern observed with isolated Rho binding domains reflected interactions of the full-length proteins, we expressed FLAG-tagged PRK2 in COS cells as described previously (5) and used GST-Rho/Rac proteins to precipitate it from cell lysates. Consistent with the isolated HR1 data, PRK2 bound to all Rho/Rac chimera containing the Rho loop 6 region (Fig. 7).

HR1a Inhibits Rho Guanine Nucleotide Exchange-- If Rho effectors interact with loop 6 of Rho, they will occupy the groove created between the switch 1 and insert domains (Fig. 8), occluding the GTP-binding pocket. Consequently, effector binding would reduce the GTP exchange rate of Rho. To test this hypothesis, we measured the rate of [alpha -32P]GTP release from Rho in the presence and absence of excess GST-HR1a. As shown in Fig. 9, the presence of HR1a inhibited the rate of GTP exchange in a dose-dependent manner. Despite having a higher affinity for Rho-GTP than HR1, preliminary data indicated that the ROCK-RBD could not inhibit GTP release under similar conditions.3


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 8.   Three-dimensional representation of Rho A showing proximity of Rho-effector interaction domains.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 9.   The HR1a domain of PRK2 inhibits GTP release from RhoA. To examine the effect of HR1a-Rho interaction on nucleotide exchange, RhoA was loaded with [alpha -32P]GTP and incubated with GST (400 µg) or various concentrations of GST-HR1a at 37 °C (up to 400 µg). Nucleotide exchange was monitored at the indicated times using a nitrocellulose filter binding assay. Increasing the GST-HR1a concentration resulted in a dose-dependent inhibition of GTP release. Representative of two experiments performed in duplicate.

Loop 6 of Rho Is Required for Stress Fiber Formation and Cellular Transformation-- Although the physiological role of PRK2 is not yet certain, it is well established that ROCK mediates Rho-induced stress fiber formation (2, 23, 38). Therefore, to determine whether the interaction of Rho loop 6 with RBD resulted in ROCK activation, serum-starved LLC-PK epithelial cells were microinjected with activated Rac(Q61L) or Rac(Q61L)DD, and stress fiber formation was determined. Cells were co-injected with botulinum exoenzyme C3 to prevent any secondary activation of endogenous Rho by Rac. Microinjection of Rac(Q61L)DD resulted in the appearance of numerous actin stress fibers (Fig. 10). In contrast, Rac(Q61L) was only capable of inducing membrane ruffles. This suggested that the addition of two Rho loop 6 residues into Rac enabled it to function as Rho to activate ROCK, leading to stress fiber formation in vivo. Due to the low yield of GST-Rho(Q63L)VA in E. coli, it was not possible to determine whether mutation of loop 6 of Rho would prevent its activation of ROCK. However, if loop 6 of Rho is required for effector activation, then the DD right-arrow VA mutation of Rho(Q63L) should result in a loss of transforming potential. Therefore, we examined the ability of Rho(Q63L) and Rho(Q63L)VA to cooperate with the partially activated Raf(Y340D) mutant to induce transforming foci in NIH 3T3 cells. Whereas Rho(Q63L) synergized with Raf(Y340D) to generate foci, Rho(Q63L)VA was considerably less effective (Fig. 11a). Interestingly, the RhoDelta Ras(Q63L) mutant, which lacks the insert domain, was also unable to cooperate with Raf(Y340D) to induce transformation, even though it retained the ability to bind to both PRK2 and ROCK-I. It was not possible to determine the expression of the RhoDelta Ras in NIH 3T3 cells because the only available Rho antibody is directed to the (deleted) insert domain. Therefore, to confirm the expression of Rho mutants in NIH 3T3 cells, HA epitope-tagged constructs were generated in the vector pCGN, and stable cell lines were established. Western blotting of cell lysates with anti-HA antibody indicated that the Rho mutants were expressed at similar levels (Fig. 11b).


View larger version (157K):
[in this window]
[in a new window]
 
Fig. 10.   Rho loop 6 residues confer Rac with the ability to induce stress fiber formation in porcine epithelial cells. LLC-PK kidney epithelial cells were microinjected with buffer containing activated Rac(Q61L) or Rac(Q61L)DD as indicated. Cells were co-injected with C3 ADP-ribosyltransferase to prevent endogenous Rho activation and fluorescein-conjugated dextran to detect injected cells. After 45 min, cells were fixed and stained with rhodamine-conjugated phalloidin to detect F-actin, and injected cells were visualized by confocal microscopy. Upper panels show rhodamine-phalloidin staining; lower panels identify fluorescein-dextran-injected cells. Introduction of Rac(Q61L) induced the formation of peripheral membrane ruffles (middle column), whereas Rac(Q61L)DD, containing the double Asp mutation, induced extensive stress fiber formation (right column). Scale bar in lower left panel represents 20 µm.


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 11.   Mutation of Rho loop 6 or deletion of the insert domain disrupts transformation of NIH 3T3 cells. a, NIH 3T3 cells were transfected with empty pZIP vector or the indicated Rho mutants in the absence (upper panels) or presence (lower panels) of Raf(Y340D). Transforming foci were visualized by staining with crystal violet after 12-16 days in culture. Representative data are shown from five independent experiments performed in quadruplicate. b, NIH 3T3 cells were transfected with the Rho/Rac mutants in the vector pCGN, which provides an N-terminal HA tag. Following stable selection on hygromycin B, Rho protein expression levels were determined on a pooled population of cells using anti-HA antibody.

Rac Residue 61 and Rho Residue 63 also Influence Rho-Effector Binding-- At the outset of these studies we used Rac(Q61L) and Rho(Q63L) activating mutations equivalent to those found in activated Ras (39) because these proteins bound to Rho effectors more efficiently than WT Rac and Rho. However, Rac(Q61L)-GTP with no additional mutations was found to bind to both Rho effectors (Figs. 5 and 12). It was also found that Rac(Q61L, V85D)-GDP and Rac(Q61L, A88D)-GDP bound to HR1 very efficiently in contrast to their wild type counterparts (Fig. 12). Similarly, a Rho(Q63L) mutant compensated for the loss of binding of RhoVA to HR1. However, G12V mutations, equivalent to those found in oncogenic Ras, bound to effectors less efficiently than WT mutants/chimeras.3 This suggested that Rac- and Rho-effector interactions are best assessed using a WT protein background. Although Rac(Q61L) interacted with the ROCK-RBD in vitro (Fig. 5c), it did not induce stress fiber formation after microinjection into the LLC-PK cells (Fig. 10). This suggested that additional sequences, within loop 6, were required for activation of ROCK in vivo. However, we have not ruled out the possibility that Rho interacts differently with the isolated RBD in vitro than with full-length ROCK in vivo.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 12.   Rac(Leu61) and Rho(Leu63) mutations can override other GTPase mutations. Rho(WT) and Rac(WT) (upper panels) or activated Rac(Q61L) and Rho(Q63L) mutants (lower panels) were loaded with GTP (RhoA), GTPgamma S (Rac1), or GDP as indicated and incubated with MBP-HR1 in the Rho precipitation assay. Several of the mutationally activated GTPases bound more efficiently than the WT proteins, and this interaction was not always GTP-dependent. Data are representative of at least two experiments.


    DISCUSSION

It has been well established that the key molecular determinants for Ras-effector protein binding are the switch 1 domain (residues 32-40) and surrounding sequences, together with the switch 2 domain (for review, see Ref. 3). The switch 1 domain is also important for dictating Rac-effector specificity because Rac to Rho point mutations in the extended effector domain (12) or mutation of the core switch 1 region (13, 14) selectively disrupted signaling to various downstream effectors. However, a complete swap of residues 1-75 of Rho for Rac residues 1-73 did not disrupt the ability of Rho to induce stress fiber and focal adhesion complex formation in Swiss 3T3 cells (16). In the present study, we set out to determine what sequences in Rho are required to confer effector binding specificity to two protein kinases that contain distinct Rho-binding domains (18). We found that Rac residues 1-73 could replace the N terminus of Rho for binding to both PRK2-HR1 and ROCK-RBD. In contrast, mutating switch 1 residues that are conserved between Rho and Rac, e.g. Thr37 and Phe39 (this study), Tyr34 (40), Val38, Asp40/Glu40, and Tyr42 (41) disrupted Rho-effector binding. Therefore, it appears that whereas Rac relies on unique N-terminal residues for effector interaction, Rho utilizes the residues that it shares with Rac for this purpose. Although PRK2 and ROCK must interact with the switch 1 domain to sense whether the GTPase is in its active GTP-bound state, an additional region(s) in the C-terminal two-thirds of Rho must be required to confer its effector binding specificity. Because residues 1-75 encompass both switch 1 and 2, the specificity determinant appears to reside in a region of Rho that does not change conformation upon GTP binding. This is consistent with the ability of Rho-GDP to, albeit weakly, bind to PRK1 and PRK2 in vitro (5, 20, 28).

In addition to the switch 1 domain, a region located between residues 143 and 175 of Rac is essential for its association with the NADPH oxidase component, p67 (16). The insert domain, a 13-amino acid sequence that replaces loop 8 of Ras with an amphipathic helix, may also be required for Rac-induced oxidase activation (15, 32, 33, 42). Furthermore, a region encompassing residues 74-90 has been shown to be required for Rac to interact with the GAP domain of BCR (16). Using a series of Rho/Rac chimeras, we found that sequences C-terminal to residue 92 were not required for HR1 or RBD interaction. However, introduction of Rho residues 76-92 into Rac conferred on it Rho-effector binding ability. A similar observation was made by Fujisawa et al. (43) while the present report was under review. This sequence encompasses loop 5, beta  strand 4, loop 6, and the proximal portion of alpha  helix 3 (Fig. 6a). Only two residues within the 76-92 region (Rho residues Asp87 and Asp90) show significant divergence between Rac and Rho and are both located within the surface exposed loop 6. Swapping these residues between Rac and Rho resulted in a decrease in Rho binding and a gain of Rac binding to the PRK2-HR1 and ROCK-RBD, strongly suggesting that loop 6 is a specificity determinant for interaction with both classes of Rho effector.

All Rho/Rac chimeras that lacked an intact Rho loop 6 were unable to associate with HR1/HR1a, indicating that loop 6 is indispensable for PRK2 binding. However, the Rho75Rac (Fig. 5a) and RhoVA (Fig. 6b) chimeras, which do not contain Rho loop 6 sequences, still weakly bound to RBD. This suggests that loop 6 is not as critical a binding determinant for RBD. Nonetheless, loop 6 must contribute to RBD binding because the V85D and A88D mutations result in a gain of Rac binding and also confer upon Rac the ability to induce stress fiber formation in LLC-PK cells.

Loop 6 and switch 1 are on the same molecular surface of RhoA. It therefore seemed likely that these two loops cooperated to promote effector binding. A large, negatively charged groove is located between the insert and switch 1 domains of Rho-GTP (Fig. 8) that includes aspartate residues 13, 87, 90, and 124 (35, 44). This groove could accommodate a positively charged helix present in HR1 or RBD (which are highly charged and predicted to have considerable alpha -helical content). If so, the effector would close off the GTP-binding pocket of Rho and prevent nucleotide release. Consistent with this model, we found that the HR1a domain of PRK2, at as low as 20 molar excess over Rho, effectively inhibited guanine nucleotide release. However, the Ras-binding domain of Raf (residues 51-131) can also inhibit GTP release from Ras, implying that interaction of an effector with the switch1 domain of Ras may be sufficient to block nucleotide exchange (45). Although we cannot exclude the possibility that association of PRK2 with switch 1 alone may also be sufficient to block nucleotide exchange on Rho, preliminary data indicated that a 20-fold molar excess of RBD over Rho could not block GTP release.3 Furthermore, the HR1 domain of PKN(30) but not the ROCK-RBD (29) was found to inhibit the intrinsic GTPase activity of Rho, again suggesting differential interaction of these two effectors with Rho. Therefore, due to their stronger interaction with loop 6, PRK2 and PKN may more effectively occlude the GTP-binding pocket.

During the completion of this study, it was reported that loop 6 of Rho also confers sensitivity to p190 Rho GAP (46). Introduction of Rho residue Asp90 into CDC42 resulted in a gain of Rho GAP sensitivity, whereas the reciprocal swap of residue Ser88 from CDC42 into Rho reduced its responsiveness to p190 (46). Therefore, similarly to Ras, it appears that Rho GAPs and effector proteins interact with common domains of their cognate GTPases. In contrast, Bae et al. (40) reported that mutation of the Asp residues in loop 6 to the equivalent CDC42 residues did not disrupt the ability of Rho to activate phospholipase D (40). Instead, a D76Q mutation in loop 5 partially attenuated choline generation. There is no apparent homology between phospholipase D and the HR1 or RBD domains, suggesting that there are at least three Rho-binding structures.

It is not clear how the F78S mutation in beta -strand 4 of Rac promotes weak Rho-effector binding. Because residue 78 is partially buried in the core of Rac, and Ser78 makes contact with Thr108 and Pro109 in loop 7, located between alpha 3 and beta 5 (34), this mutation could have several subtle effects on Rac biochemistry. It has been suggested from computer modeling studies that the F78S mutation will not significantly perturb Rac structure, and it did not affect GAP-stimulated GTPase activity (34). Nonetheless, the weak interaction of Rac(F78S) with PRK2 may explain the discrepancy in Rac-PRK2 binding data observed in previous studies that may have used this mutant form of Rac (5, 20). Not knowing the source of Rac used in other studies, it is not possible to predict whether this also explains differences in Rac-ROCK interaction (12-14).

The insert domain of Rac is not required for binding to the phagocyte NADPH oxidase component p67, but its presence may be essential for oxidase activation, possibly by interacting with cytochrome b558 (15, 32, 33). Additionally it has been proposed that although switch 1 might dictate GTP- versus GDP-dependent binding of effectors, the insert domain might be responsible for conferring effector specificity (35, 47). Although swapping the RhoA insert domain with Ras loop 8 resulted in partial loss of effector binding, it did not impair binding to full-length PRK2. Furthermore, exchanging the Rho insert domain for that of Rac (RhoDelta Rac) or using a Rho92Rac chimera did not prevent the association of Rho with either HR1 or RBD. This suggested that the insert domain is not a specificity determinant for these Rho targets. A similar conclusion was made regarding Rho activation of phospholipase D (40). Due to the inability to detect robust activation of PRK2 by Rho using in vitro kinase or transcription assays and the low yield of GST-RhoDelta Ras in E. coli, it was not possible to determine whether the insert was required for PRK2 activation or stress fiber formation. However, the insert helix of Rho was essential for it to induce cellular transformation. So, as previously reported for Rac (15, 32), the insert domain in Rho may be involved in effector activation rather than being a binding determinant.

The recently solved solution structure of CDC42-GDP revealed a close contact between residues 120-129 (the insert helix) and residues 84-89 (loop 6) of CDC42 (47). It was also reported that a hydrophobic patch is formed between loop 6 (Pro89) and the insert domain (residues 127, 131, and 138) of Rho (44). It is therefore possible that deletion of the insert domain may destabilize the conformation of loop 6, making it unable to effectively interact with and/or activate Rho effectors. Alternatively, the insert domain may mediate the association of Rho-GTP with a novel effector(s) that is essential for certain Rho functions. Deletion of the insert domain of CDC42 in a similar manner to our RhoDelta Ras chimera also disrupts its ability to induce cell growth and transformation (48). This effect was independent of JNK and PAK activation or actin cytoskeletal regulation, suggesting that the insert domain couples CDC42 to a novel effector signaling pathway. The insert domain also appears to be essential for the mitogenic activity of Rac (42). Again, deletion of the insert domain did not affect JNK activation or actin polymerization but instead abrogated mitogenesis by blocking Rac-induced superoxide generation. Whether Rac- or CDC42-induced transformation pathways are shared with Rho or whether the Rho insert domain interacts with additional target proteins will require further investigation.

Our initial interpretation of data was complicated by the use of activating Leu61/Leu63 mutants. For example, Rac(Q61L) was found to bind to Rho effectors even in the absence of Rho sequences, whereas a Rho(Q63L) mutation compensated for the loss of binding by RhoVA. Q61L mutants have previously been shown in various Ras family GTPases to have higher affinity for their targets and to functionally override the effects of other mutations. For example, 1) Ras(Q61L) has a significantly higher affinity for p120 GAP than Ras(WT) or Ras(G12V) (49); 2) a Rac(D38A, Q61L) double mutant restored the ability of a Rac(D38A) effector-binding domain mutant to induce NADPH oxidase activation and membrane ruffling (50); 3) Ras(Q61L) has also been reported to bind to Raf even in a GDP-bound state (51), suggesting that this mutation influences the global conformation of Ras, thus leading to altered effector binding properties. Therefore caution should be taken in interpreting Rho interaction data, and if possible, wild type proteins should be used in in vitro assays. It should be noted that the presence of a Rac(Q61L) mutation alone did not appear to result in ROCK-induced stress fiber formation in vivo despite binding to the isolated RBD domain.

In summary, the interaction of Rho with its downstream effectors, PRK2 and ROCK, appears to involve multiple binding sites. Sequences conserved between the switch 1 domains of Rac and Rho rather than their unique residues contribute to the binding of Rho-GTP to both PRK2 and ROCK-I. However, unique residues in loop 6 of Rho enable these effectors to distinguish it from other Rho family GTPases. The insert domain may provide an additional feature for recognition of other, unknown, effectors. These domains are in close proximity in the Rho three-dimensional structure and may represent the effector-binding surface (Fig. 8). Therefore, we propose that upon GTP binding, the switch 1 domain moves closer to loop 6, enabling effectors to bind concomitantly with both domains. Upon GTP hydrolysis, the cooperation between switch 1 and loop 6 is lost, resulting in only low affinity, GDP-dependent effector binding. An alternative hypothesis is that the negatively charged residues in loop 6 induce the switch domains of Rac or Rho to adopt a Rho-like conformation to promote specific interact with Rho targets. In both models, Rho loop 6 is the key effector binding specificity determinant. Because PRK2 and ROCK-I both bound to the same Rho-Rac chimeras, it appears that both classes of Rho-binding domain have evolved to recognize similar molecular determinants. Stronger binding of RBD versus HR1 to Rho loop 6 mutants suggests that Rho-ROCK-I interaction relies less heavily on loop 6. However, because Rac(Leu61)DD could induce stress fiber formation, it appears that an interaction with Rho loop 6 is required to promote ROCK activation. A more detailed picture of Rho-effector interaction awaits the determination of a co-crystallized structure.

    ACKNOWLEDGEMENTS

We are grateful to A. Hall for generously providing Rho constructs, Z. Derewenda for providing the Protein Data Bank coordinates for the Rho-GDP crystal structure prior to general release, D. Timm for assistance with computer modeling, and C. Bi for technical assistance.

    FOOTNOTES

* This work was supported by Research Project Grant 97-007-01-BE from the American Cancer Society (to L. A. Q.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., MS-4053, Indianapolis, IN 46202-5122. Tel.: 317-274-8550; Fax: 317-274-4686; E-mail: lquillia{at}iupui.edu.

    ABBREVIATIONS

The abbreviations used are: HR, homology region; GST, glutathione S-transferase; MBP, maltose-binding protein; RBD, Rho binding domain of ROCK-1; PCR, polymerase chain reaction; HA, hemagglutinin; WT, wild type; GTPgamma S, guanosine 5'-O-(thiotriphosphate).

2 N. Raman and S. J. Atkinson, submitted for publication.

3 H. Zong and L. A. Quilliam, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
References

  1. Macara, I. G., Lounsbury, K. M., Richards, S. A., McKiernan, C., and Bar-Sagi, D. (1996) FASEB J. 10, 625-630[Abstract/Free Full Text]
  2. Hall, A. (1998) Science 279, 509-514[Abstract/Free Full Text]
  3. Campbell, S. L., Khosravi-Far, R., Rossman, K. L., Clark, G. J., and Der, C. J. (1998) Oncogene 17, 1395-1413[CrossRef][Medline] [Order article via Infotrieve]
  4. Hill, C. S., Wynne, J., and Treisman, R. (1995) Cell 81, 1159-1170[Medline] [Order article via Infotrieve]
  5. Quilliam, L. A., Lambert, Q. T., Mickelson-Young, L. A., Westwick, J. K., Sparks, A. B., Kay, B. K., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and Der, C. J. (1996) J. Biol. Chem. 271, 28772-28776[Abstract/Free Full Text]
  6. Olson, M. F., Ashworth, A., and Hall, A. (1995) Science 269, 1270-1272[Medline] [Order article via Infotrieve]
  7. Wittinghofer, A., Franken, S. M., Scheidig, A. J., Rensland, H., Lautwein, A., Pai, E. F., and Goody, R. S. (1993) Ciba Found. Symp. 176, 6-27[Medline] [Order article via Infotrieve]
  8. Stone, J. C., Colleton, M., and Bottorff, D. (1993) Mol. Cell. Biol. 13, 7311-7320[Abstract]
  9. White, M. A., Nicolette, C., Minden, A., Polverino, A., Van Aelst, L., Karin, M., and Wigler, M. H. (1995) Cell 80, 533-541[Medline] [Order article via Infotrieve]
  10. Khosravi-Far, R., White, M. A., Westwick, J. K., Solski, P. A., Chrzanowska-Wodnicka, M., Van Aelst, L., Wigler, M. H., and Der, C. J. (1996) Mol. Cell. Biol. 16, 3923-3933[Abstract]
  11. Joneson, T., White, M. A., Wigler, M. H., and Bar-Sagi, D. (1996) Science 271, 810-812[Abstract]
  12. Westwick, J. K., Lambert, Q. T., Clark, G. J., Symons, M., Van Aelst, L., Pestell, R. G., and Der, C. J. (1997) Mol. Cell. Biol. 17, 1324-1335[Abstract]
  13. Lamarche, N., Tapon, N., Stowers, L., Burbelo, P. D., Aspenstrom, P., Bridges, T., Chant, J., and Hall, A. (1996) Cell 87, 519-529[Medline] [Order article via Infotrieve]
  14. Joneson, T., McDonough, M., Bar-Sagi, D., and Van Aelst, L. (1996) Science 274, 1374-1376[Abstract/Free Full Text]
  15. Freeman, J. L., Abo, A., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 19794-19801[Abstract/Free Full Text]
  16. Diekmann, D., Nobes, C. D., Burbelo, P. D., Abo, A., and Hall, A. (1995) EMBO J. 14, 5297-5305[Abstract]
  17. Self, A. J., Paterson, H. F., and Hall, A. (1993) Oncogene 8, 655-661[Medline] [Order article via Infotrieve]
  18. Narumiya, S. (1996) J. Biochem. 120, 215-228[Abstract]
  19. Palmer, R. H., Ridden, J., and Parker, P. J. (1995) Eur. J. Biochem. 227, 344-351[Abstract]
  20. Vincent, S., and Settleman, J. (1997) Mol. Cell. Biol. 17, 2247-2256[Abstract]
  21. Yu, W., Liu, J., Morrice, N. A., and Wettenhall, R. E. (1997) J. Biol. Chem. 272, 10030-10034[Abstract/Free Full Text]
  22. Cryns, V. L., Byun, Y., Rana, A., Mellor, H., Lustig, K. D., Ghanem, L., Parker, P. J., Kirschner, M. W., and Yuan, J. (1997) J. Biol. Chem. 272, 29449-29453[Abstract/Free Full Text]
  23. Narumiya, S., Ishizaki, T., and Watanabe, N. (1997) FEBS Lett. 410, 68-72[CrossRef][Medline] [Order article via Infotrieve]
  24. Cepko, C. L., Roberts, B. E., and Mulligan, R. C. (1984) Cell 37, 1053-1062[Medline] [Order article via Infotrieve]
  25. Self, A. J., and Hall, A. (1995) Methods Enzymol 256, 3-10[Medline] [Order article via Infotrieve]
  26. Quilliam, L. A., Der, C. J., Clark, R., O'Rourke, E. C., Zhang, K., McCormick, F., and Bokoch, G. M. (1990) Mol. Cell. Biol. 10, 2901-2908[Medline] [Order article via Infotrieve]
  27. Clark, G. J., Cox, A. D., Graham, S. M., and Der, C. J. (1995) Methods Enzymol. 255, 395-412[Medline] [Order article via Infotrieve]
  28. Flynn, P., Mellor, H., Palmer, R., Panayotou, G., and Parker, P. J. (1998) J. Biol. Chem. 273, 2698-2705[Abstract/Free Full Text]
  29. Leung, T., Chen, X. Q., Manser, E., and Lim, L. (1996) Mol. Cell. Biol. 16, 5313-5327[Abstract]
  30. Shibata, H., Mukai, H., Inagaki, Y., Homma, Y., Kimura, K., Kaibuchi, K., Narumiya, S., and Ono, Y. (1996) FEBS Lett. 385, 221-224[CrossRef][Medline] [Order article via Infotrieve]
  31. John, J., Rensland, H., Schlichting, I., Vetter, I., Borasio, G. D., Goody, R. S., and Wittinghofer, A. (1993) J. Biol. Chem. 268, 923-929[Abstract/Free Full Text]
  32. Nisimoto, Y., Freeman, J. L. R., Motalebi, S. A., Hirshberg, M., and Lambeth, J. D. (1997) J. Biol. Chem. 272, 18834-18841[Abstract/Free Full Text]
  33. Toporik, A., Gorzalczany, Y., Hirshberg, M., Pick, E., and Lotan, O. (1998) Biochemistry 37, 7147-7156[CrossRef][Medline] [Order article via Infotrieve]
  34. Hirshberg, M., Stockley, R. W., Dodson, G., and Webb, M. R. (1997) Nat. Struct. Biol. 4, 147-152[Medline] [Order article via Infotrieve]
  35. Wei, Y., Zhang, Y., Derewenda, U., Liu, X., Minor, W., Nakamoto, R. K., Somlyo, A. V., Somlyo, A. P., and Derewenda, Z. S. (1997) Nat. Struct. Biol. 4, 699-703[Medline] [Order article via Infotrieve]
  36. McCallum, S. J., Wu, W. J., and Cerione, R. A. (1996) J. Biol. Chem. 271, 21732-21737[Abstract/Free Full Text]
  37. Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatsu, A., Fujita, A., Watanabe, N., Saito, Y., Kakizuka, A., Morii, N., and Narumiya, S. (1996) EMBO J. 15, 1885-1893[Abstract]
  38. Amano, M., Chihara, K., Kimura, K., Fukata, Y., Nakamura, N., Matsuura, Y., and Kaibuchi, K. (1997) Science 275, 1308-1311[Abstract/Free Full Text]
  39. Der, C. J., Finkel, T., and Cooper, G. M. (1986) Cell 44, 167-176[Medline] [Order article via Infotrieve]
  40. Bae, C. D., Min, D. S., Fleming, I. N., and Exton, J. H. (1998) J. Biol. Chem. 273, 11596-11604[Abstract/Free Full Text]
  41. Sahai, E., Alberts, A. S., and Treisman, R. (1998) EMBO J. 17, 1350-1361[Abstract/Free Full Text]
  42. Joneson, T., and Bar-Sagi, D. (1998) J. Biol. Chem. 273, 17991-17994[Abstract/Free Full Text]
  43. Fujisawa, K., Madaule, P., Ishizaki, T., Watanabe, G., Bito, H., Saito, Y., Hall, A., and Narumiya, S. (1998) J. Biol. Chem. 273, 18943-18949[Abstract/Free Full Text]
  44. Ihara, K., Muraguchi, S., Kato, M., Shimizu, T., Shirakawa, M., Kuroda, S., Kaibuchi, K., and Hakoshima, T. (1998) J. Biol. Chem. 273, 9656-9666[Abstract/Free Full Text]
  45. Herrmann, C., Martin, G. A., and Wittinghofer, A. (1995) J. Biol. Chem. 270, 2901-2905[Abstract/Free Full Text]
  46. Li, L., Zhang, B., and Zheng, Y. (1997) J. Biol. Chem. 272, 32830-32835[Abstract/Free Full Text]
  47. Feltham, J. L., Dotsch, V., Raza, S., Manor, D., Cerione, R. A., Sutcliffe, M. J., Wagner, G., and Oswald, R. E. (1997) Biochemistry 36, 8755-8766[CrossRef][Medline] [Order article via Infotrieve]
  48. Wu, W. J., Lin, R., Cerione, R. A., and Manor, D. (1998) J. Biol. Chem. 273, 16655-16658[Abstract/Free Full Text]
  49. Vogel, U. S., Dixon, R. A., Schaber, M. D., Diehl, R. E., Marshall, M. S., Scolnick, E. M., Sigal, I. S., and Gibbs, J. B. (1988) Nature 335, 90-93[CrossRef][Medline] [Order article via Infotrieve]
  50. Xu, X., Barry, D. C., Settleman, J., Schwartz, M. A., and Bokoch, G. M. (1994) J. Biol. Chem. 269, 23569-23574[Abstract/Free Full Text]
  51. Moodie, S. A., Paris, M., Villafranca, E., Kirshmeier, P., Willumsen, B. M., and Wolfman, A. (1995) Oncogene 11, 447-454[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.