Effector Recognition by the Small GTP-binding Proteins Ras and Ral*

Bettina BauerDagger §, Gladys Mirey§parallel **, Ingrid R. VetterDagger , Juan A. García-RaneaDagger Dagger , Alfonso ValenciaDagger Dagger , Alfred WittinghoferDagger , Jacques H. Camonisparallel , and Robbert H. CoolDagger §§

From the Dagger  Max-Planck-Institut für Molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany, the parallel  Institut Curie, INSERM 248, 26 rue d'Ulm, F-75248 Paris, Cedex 05, France, and the Dagger Dagger  Protein Design Group, Centro Nationale de Biotecnología, Campus de la Universidad Autónoma, Cantoblanco, Madrid M-28049, Spain

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The Ral effector protein RLIP76 (also called RIP/RalBP1) binds to Ral·GTP via a region that shares no sequence homology with the Ras-binding domains of the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors. Whereas the Ras-binding domains have a similar ubiquitin-like structure, the Ral-binding domain of RLIP was predicted to comprise a coiled-coil region. In order to obtain more information about the specificity and the structural mode of the interaction between Ral and RLIP, we have performed a sequence space and a mutational analysis. The sequence space analysis of a comprehensive nonredundant assembly of Ras-like proteins strongly indicated that positions 36 and 37 in the core of the effector region are tree-determinant positions for all subfamilies of Ras-like proteins and dictate the specificity of the interaction of these GTPases with their effector proteins. Indeed, we could convert the specific interaction with Ras effectors and RLIP by mutating these residues in Ras and Ral. We therefore conclude that positions 36 and 37 are critical for the discrimination between Ras and Ral effectors and that, despite the absence of sequence homology between the Ral-binding and the Ras-binding domains, their mode of interaction is most probably similar.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ral is a small GTP-binding protein belonging to the subfamily of Ras proteins (1, 2), which function as molecular switches in signal transduction pathways by alternating between an active GTP-bound and an inactive GDP-bound conformation. The ratio between the active and inactive form of the Ras proteins is regulated by the action of two types of proteins (3): GTPase activating proteins (GAPs),1 which inactivate GTPases by stimulating the slow GTPase reaction, and guanine nucleotide exchange factors (GEFs), which activate GTPases by stimulating the slow GDP dissociation rate, allowing the protein to rapidly come at equilibrium with the cellular pool of guanine nucleotide (4). The guanine nucleotide affinities and the intrinsic GTPase activity of Ral are very similar to that of Ras (5). Ral is ubiquitously expressed, but it is especially abundant in brain, testis, and platelets (6-10). The protein was found both in the plasma membrane and in cytoplasmic vesicles (11, 12).

The cellular function of Ral remained elusive for a long time. Recently, however, progress was made via the discovery of Ral-specific regulatory activities. Multiple Ral-specific guanine nucleotide exchange factors (RalGEFs) have been isolated (13-18). Furthermore, although so far the corresponding genes have not been isolated, Ral-specific GAP activities were identified in brain, testis, and platelets (19-20). Strikingly, in addition to a Ral-specific GEF domain, the RalGEFs contain a C-terminally located Ras-binding domain (RBD), which is able to bind the GTP-bound forms of Ras and Rap proteins, so that the RalGEFs were recognized as one of the new families of Ras protein effectors (Ref. 21 and references therein). The RBDs of RalGDS (22, 23) and Rlf (24) were shown to be structurally similar to the RBD of the Ser/Thr protein kinase c-Raf-1 (25-27), a well characterized Ras-effector protein. Interestingly, a Ras-dependent stimulation of Ral was proposed to function parallel to the Ras-Raf-Mek-Erk pathway in several types of cells (28-35), but in platelets, Ral appears to be stimulated in a fashion similar to Rap1A, suggesting that also Rap1A can function upstream of Ral (36). In addition to the regulatory proteins, a putative Ral effector protein was identified: RLIP76, also named RalBP1 or RIP (37-39). Its Ral-binding domain (RalBD) was identified by deletion studies (37) and was used successfully as a probe in a pull-down assay to measure the activation of endogenous Ral upon cellular stimulation (35, 36). Finally, Ral was shown to be involved in the phorbol ester-stimulated phospholipase D activity, apparently through a direct interaction of Ral with phospholipase D 1 (28, 30, 40-42). Additional complexity in the Ral pathway(s) emerged with the isolation of the RLIP76-binding protein Reps1, which can also bind to the adaptor proteins Crk and Grb2 (43), and with the observation that calmodulin binds to RalA (44) and that both Ral and Arf are needed for phospholipase D activation (45).

Even though Ral interacts with RLIP76, it is largely unknown how this interaction takes place. Secondary structure prediction of RLIP76 indicated that the structure of RalBD comprises a coiled-coil region (37) and thus differs from the ubiquitin-like fold of the RBDs. In this work, we have investigated the effects of several mutations on the interaction between Ral and RLIP76 by double hybrid analysis and biochemical methods. Moreover, the specificity of the interaction of Ras and Ral proteins with their effectors was studied by sequence space analysis of the Ras-like protein sequences. The foremost result is that Lys-47 and Ala-48 (corresponding to Ras residues Ile-36 and Glu-37) in the effector region enable Ral to discriminate between RLIP76 and Ras effector molecules. Ral(K47I) is able to interact almost as potently as Ras with RalGEF-RBDs, whereas mutation E37A in Ras is sufficient for a significantly interaction with RLIP76. We thus conclude that, as opposed to the other members of the Ras-subfamily, Ral is unable to interact with Ras effector molecules due to critical differences in the effector region. Furthermore, introduction of mutations in the effector region may induce erroneous binding to effectors specific for other GTP-binding proteins. Finally, the fact that mutation of only two residues changes binding specificity suggests that the modes of protein-protein interaction in the Ral·RLIP and in the Ras·RBD complexes are similar.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Isolation of C-terminally Truncated sRalA and hRalB Proteins-- Using PCR, we cloned in the pGEX-4T3 vector (Amersham Pharmacia Biotech) the fragment of the simian RalA gene encoding amino acids 1-178 (sRalA1-178; hereafter called sRalA) via restriction sites BamHI/EcoRI and the fragment of the human RalB gene encoding amino acids 1-179 (hRalB1-179; hereafter called hRalB) via BamHI/SalI sites. For overproduction of recombinant proteins, we used the protease-negative Escherichia coli strain AD202 (46). Cultures were grown in standard I-medium (Merck) containing 100 mg/liter ampicillin and 10 mg/liter kanamycin. Synthesis of recombinant proteins was induced at an A600 of 0.9 by addition of 100 nM isopropyl-beta -D-thiogalactopyranoside and subsequent incubation at 30 °C overnight. Bacteria cells were harvested by centrifugation; resuspended in phosphate-buffered saline containing 10% glycerol, 5 mM MgCl2, 5 mM dithioerythritol, 14 mM beta -mercaptoethanol, 25 µM Pefabloc SC (Merck), and 10 µM GDP; and lysed by sonication. After centrifugation for 1 h at 40,000 × g, the supernatant was loaded on a glutathione-Sepharose column and washed with Buffer A (50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM MgCl2, 5 mM dithioerythritol, 10 µM GDP, 10% glycerol). Subsequently, we loaded the column with thrombin (Serva; 2 NIH units per estimated mg of GST-fused protein) in Buffer A containing 5 mM CaCl2 and incubated overnight at 4 °C. The cleaved Ral protein was eluted with Buffer A, concentrated in Vivaspin vials (Vivascience), and further purified using a Superdex 75-Sepharose column (Amersham Pharmacia Biotech) using 50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 5 mM MgCl2, 5 mM dithioerythritol, 10 µM GDP. After concentration of the pooled fractions, the Ral proteins were dialyzed against Buffer B (50 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 5 mM dithioerythritol), aliquoted, shock-frozen in liquid nitrogen, and stored at -80 °C.

Ral and Ras proteins were loaded with Gpp(NH)p or mGpp(NH)p (the derivative of 5'-guanylylimidodiphosphate, carrying the fluorescent N-methylanthraniloyl-group at the 2'- or 3'-position of the ribose (mixture of both isoforms)) by incubation with a 4-fold excess of (m)Gpp(NH)p in 50 mM Tris-HCl, pH 7.6, 200 mM (NH4)2SO4, 10 mM EDTA, 5 mM dithioerythritol, and 5 units of alkaline phosphatase per mg of Ral or Ras for 1 h at room temperature. Subsequently, separation of the Ral or Ras protein from unbound nucleotides was obtained by gel filtration in Buffer B. Thereafter, the protein was concentrated using Vivaspin vials, and the concentration of Ral·(m)Gpp(NH)p was determined by high pressure liquid chromatography analysis.

Mutagenesis of the Effector Region of hRalB and of Ha-Ras-- The effector mutants of hRalB were made by PCR-based site-directed mutagenesis (71) using template pGEX-4T3-hRalB1-179, upstream primer 5'-CGCGTGGATCCATGGCT-3' (the BamHI restriction site is underlined), and downstream primer 5'-TCCCTGAATTCGGCTG-3' (the EcoRI restriction site is underlined). The downstream primer hybridizes to a sequence of hRalB comprising an internal EcoRI restriction site located in the triplets encoding amino acids Glu-106/Phe-107 of hRalB. The mutagenic primers were 5'-GTTGGCAAGAACGCCCTGACG-3' for S28N, 5'-GAGTTTGTAAAAGACTATGA-3' for E41K, 5'-GTTTGTAGAAAAGTATGAACC-3' for D42K, 5'-TGAACCTACCATCGCTGACAGTTAT-3' for K47I, 5'-ACCTACCAAAGAAGACAGTTAT-3' for A48E, 5'-TATGAACCTACCATCGAAGACAGTTATAG-3' for the double mutant K47I/A48E, 5'-CTACCAAAGCTGAGAGTTA-3' for D49E, and 5'-CTACCAAAGCTAACAGTTATAG-3' for D49N (the mutated codons are underlined). The BamHI/EcoRI-cleaved PCR fragments were ligated into the BamHI/EcoRI-cleaved pGEX-4T3-hRalB1-179 vector.

The Ras mutants were made as described above for the Ral mutants using upstream primer 5'-CGCGGATCCATGACAGAATACAAGCTTGTTGTTGTTG-3' (the BamHI restriction site is underlined), downstream primer 5'-CCACAGTGCGTGCAGCCAGGTCAC-3' (Ras residues Cys-118 to Val-125), and mutagenesis primers 5'-GACCCCACTAAAGAGGATTCCTACC-3' for I36K, 5'-GACCCCACTATAGCGGATTCCTACC-3' for E37A, and 5'-GACCCCACTAAAGCGGATTCCTACC-3' for I36K/E37A (mutated codons underlined). The plasmid pGEX-4T3-Ras, a kind gift of Dorothee Vogt, was used as a template. The mutated BamHI/NcoI-fragments were switched with the corresponding fragment in the pGEX-4T3-Ras wild type plasmid.

Isolation of RalBD-- The Ral-binding domain (RalBD) of human RLIP76, comprising amino acids 397-518 (37), was cloned by PCR in the BamHI and SalI sites of pGEX-4T3. Synthesis, isolation, and storage were performed basically as described for Ral but using buffers without GDP. GST-fused RalBD was eluted from the column with Buffer A containing 10 mM of reduced glutathione and subsequently dialyzed against Buffer C (50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 15% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 25 nM Pefabloc, 1% (v/v) Igepal CA-630 (Sigma), and 2 mM MgCl2).

Isolation of the Complex sRalA·Gpp(NH)p·RalBD-- To prepare the complex sRalA·Gpp(NH)p·RalBD, the lysate of 20 g of GST-RalBD-expressing AD202 cells were loaded on a GSH-Sepharose column and washed with phosphate-buffered saline containing 14 mM 2-mercaptoethanol, 10 mM dithioerythritol, 10 nM Gpp(NH)p, 25 nM Pefabloc, 2 mM MgCl2, and 10% glycerol. Thereafter, 20 mg of Gpp(NH)p-bound sRalA were loaded on the column. The GST fusion was cleaved with 40 NIH units of thrombin at 4 °C overnight, after which the complex was eluted. The complex was separated from noncomplexed RalBD by two successive chromatographic steps on a Superdex 75 gel filtration column in 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 14 mM 2-mercaptoethanol, 10 mM dithioerythritol, 10 µM Gpp(NH)p, 25 µM Pefabloc, 2 mM MgCl2, and 10% glycerol.

Two-hybrid Analysis-- The wild type and mutated Ral and Ras genes were subcloned by BamHI/SalI fragments into pVJL10, a derivative of pBTM116 (47). Fragments containing the RalBD or the RBD of c-Raf-1 (amino acids 51-131) were subcloned by PCR in the pGAD3S2X vector. The Ras-binding domain of mRalGDS (amino acids 702-852) or mRlf (amino acids 607-778) was expressed from pGAD1318. Plasmids pGAD3S2X and pGAD1318 are derivatives of the two-hybrid vector pGAD-GH.

2H assays were performed as described (37). L40 yeast cells transformed with plasmids allowing expression of LexA-fused proteins were mated with AMR70 cells transformed with plasmids allowing expression of proteins fused to the GAL4 activation domain. Diploids were tested for histidine prototrophy and LacZ expression. Quantification of beta -galactosidase activity was performed as described (48).

GST Pull-down Experiment-- 400 µg of GST-RalBD was bound to 400 µl of GSH-Sepharose beads in 3 ml of Buffer C. Beads were collected by centrifugation and washed with the same buffer. For each assay 5 µM of sRalA wt, hRalB wt, or hRalB mutants, loaded with GDP or Gpp(NH)p, were incubated in 1 ml with 3.5 µM GST-RalBD bound to GSH-Sepharose beads for 1 h at 4 °C. Beads were collected by centrifugation, washed three times with Buffer C, and resuspended with 15 µl of SDS-sample buffer. The proteins were separated by 15% SDS-polyacrylamide gel electrophoresis and detected by Coomassie Brilliant Blue staining.

Fluorescence Measurements-- The interaction of hRalB wt and the hRalB(K47I/A48E) mutant with the Rlf-, Rgl-, and RalGDS-RBDs was characterized with a Perkin-Elmer fluorescence spectrometer LS50B as described before (24, 49). Measurements were performed in Buffer B containing 5% glycerol at 37 °C; excitation wavelength was 366 nm, and emission wavelength was 450 nm. The determined dissociation constants were corrected with the percentage of the activity of the effector, which was specified by an active site titration as described (49).

Sequence and Structural Analysis-- An assembly of 476 Ras sequences was collected from different data bases with the help of the Genequiz software (50). A comprehensive nonredundant alignment of these sequences was build with CLUSTALW (51) using the BLOSUM62 matrix (52). Only sequences with maximal 80% similarity were accepted, with the exception of Ral: only 8 Ral sequences were found in the data bases, all of which were accepted in the alignment. The final multiple sequence alignment that was obtained after some hand editing included 179 sequences, corresponding to 98 Rab, 36 Rho, 37 Ras, and 8 Ral sequences. This alignment can be found at http://www.cnb.uam.es/~cnbprot/ral.dir.

The detection of those residues that allot specificity to a subfamily, i.e. those residues that are conserved within a given subfamily and differ from the other subfamilies, which are named tree-determinant residues, was carried out with SequenceSpace (53). These tree-determinant residues are likely to be responsible for the functional differences between protein subfamilies. This approach was used in different systems, including the alpha -subunits of the trimeric G-proteins (54), SH2 domains (55), protein kinases,2 and alcohol dehydrogenases (56), among others. In all these cases, the positions of the tree-determinant residues fit nicely with known protein interaction sites, e.g. the peptide substrate binding sites in the SH2 domains.

Surface accessibility of residues in the GDP-bound conformation of sRalA,3 and the GDP- and the GTP-bound conformation of Ras (Protein Data Bank codes 4q21 and 5p21, respectively) was calculated with the program DSSP (57).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of the Complex of sRalA·Gpp(NH)p with RLIP76

The first trials to isolate the complex by incubating the purified proteins sRalA·Gpp(NH)p and RalBD, and subsequent purification over gel filtration column in 50 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 150 mM NaCl, 5 mM dithioerythritol, were unsuccessful. Under these conditions, Ral behaved as a dimer and RalBD as a multimer (not shown). Consequently, a proper separation of the complex from the single proteins was not possible, and we could not be certain that a complex was formed.

Therefore, we modified our strategy. We bound GST-RalBD to a GSH-Sepharose column, loaded sRalA·Gpp(NH)p to the column, and, after washing in order to remove unbound Ral, cleaved with thrombin and eluted the complex from the column. Only with sRalA·Gpp(NH)p could a complex be eluted (Fig. 1); one could not be eluted with sRalA·GDP (not shown), demonstrating that the interaction between Ral and RalBD is GTP-specific, consistent with earlier findings with different techniques (37-39).


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Fig. 1.   Isolation of the complex sRalA1-178·Gpp(NH)p·RalBD. A, SDS gel of fractions of the first gel filtration. GSH, pooled fractions of the GSH-Sepharose elution with a mixture of the complex and some unbound RalBD (band with apparent molecular mass of 20 kDa); M, molecular mass marker (Sigma); numbered lanes, eluted fractions of the gel filtration (fraction numbers above). Fractions 18-22 of this first step were pooled and used for the second gel filtration chromatography. B, SDS gel showing eluted fractions 19-27 of the second gel filtration. Fractions 20-22 show a 1:1 complex of sRalA1-178·Gpp(NH)p and RalBD.

The complex-containing fractions of the GSH-Sepharose elution were further purified by gel filtration to remove noncomplexed proteins and to determine the ratio in which the complex is build. A partial separation of noncomplexed RalBD, which here behaved as a dimer, from the complex could be obtained by a first Superdex-75 chromatography step (Fig. 1A). The pooled fractions containing the complex (fractions 18-22) were subjected to a second gel filtration, after which a nearly pure complex (fractions 20-22) could be obtained (Fig. 1B). The complex eluted as a 33-kDa protein, showing it to be a 1:1 complex of the Ral protein (20.4 kDa) and RalBD (14.7 kDa). It was noted that the nonfused form of RalBD runs with a too high apparent molecular mass (Fig. 1), whereas GST-fused RalBD runs normally on SDS-polyacrylamide gel electrophoresis (Fig. 2) (molecular mass, 40.9 kDa).


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Fig. 2.   In vitro binding of RalBD to Ral wild type and Ral effector mutants. GST-fused RalBD (indicated upper band) was incubated with the Gpp(NH)p- and the GDP-bound form of sRalA1-178 wt (RalA wt), hRalB1-179 wt (RalB wt), the hRalB effector mutants Y43W full-length (RalBf.l. Y43W), hRalB1-179(K47I) (RalB K471), hRalB1-179(A48E) (RalB A48E), hRalB1-179(K47I/A48E) (RalB K47I/A48E), and Ras1-171 wt (Ras wt).

In Vitro Characterization of the Ral-RLIP76 Interaction

It was shown by fluorescence measurements for several effector molecules that they can inhibit the intrinsic dissociation rate of the GTP-bound complex of Ras-like proteins in a concentration-dependent manner (15, 49, 58). However, addition of up to 10 µM RalBD did not lead to any effect on the intrinsic dissociation rate of sRalA·mGpp(NH)p or hRalB·mGpp(NH)p (not shown), nor to a change in emission spectrum.

Because fluorescence measurements did not allow the characterization of the interaction, we used a pull-down assay to analyze the binding of Ral to RLIP in vitro. We used a GST-fused RalBD and full-length RLIP76 as baits and tested the binding to Ral preloaded with either GDP or Gpp(NH)p. Partially purified GST-coupled full-length Rlip76 was able to specifically bind the Gpp(NH)p-bound forms of sRalA and hRalB (not shown). Also, the truncated form, RalBD, can interact specifically with the Gpp(NH)p-bound forms of sRalA and hRalB (Fig. 2). In comparison, Ras does not bind to RalBD.

What Makes the Specificity of the Interaction with Effector Molecules

A Theoretical Approach The complete functional interchange between a Ras protein and a Ral protein requires a large number of changes equal to the number of differences between the sequences. In order to select among them those residues that will affect the specificity of the interaction of Ras and Ral with their regulating proteins, we have chosen a general approach described before (53). After building a representative alignment of Ras-like proteins (see under "Experimental Procedures"), we could define those positions that correspond to good tree-determinants, i.e. that are conserved within a subfamily and differ from the other subfamilies. In addition, tree-determinants that are in the protein interior were eliminated as putative participants in the binding site. Finally, because we were mostly interested in the specific interaction with effector proteins, the exposed tree-determinants positions that do not change their surface exposition between the GTP- and GDP-bound states were eliminated.

Sequence space analysis was performed as described (53). A comparison of the Ras, Rho, and Rab subfamilies and of the Ras-Ral (comprising the Ras and Rap proteins, but not Ral), Ral, and Rho subfamilies is depicted in Fig. 3. Only 14 of the completely conserved residues in the eight known Ral sequences are different in the other subfamilies (Ral-tree-determinants), namely positions 7, 24, 25, 33, 36, 37, 43, 46, 53, 67, 70, 92, 93, and 160 (Fig. 3A; note that we used the Ras numbering in these comparisons). When comparing the Ras-Ral, Ral, Rho, and Rab subfamilies, only position 37 is conserved but different in each subfamily (Fig. 3): Glu in Ras-Ral (approximately 90% conserved), Ala in Ral (100%), Phe in Rho (nearly 100%), and Gly in Rab (nearly 100%). Position 36 is also conserved in Ral (Lys; 100%) and Rho (Val; >95%), and conserved but shared in Ras-Ral and Rab (Ile; > 95% conserved). These two positions thus are the best tree-determinants for each of these subfamilies. When comparing the Ral, Ras-Ral, and Rho subfamily, other tree-determinants specific of different subsets of families can be detected, e.g. position 92, which is conserved but different in each subfamily: Glu/Asp in Ras-Ral (approximately 70% conserved), Ala in Ral (100%), and Asn in Rho (approximately 80%). A residue that is shared by the Ras-Ral and Rho subfamily and different from Ral, is position 53: Leu in Ras-Ral and Rho (nearly 100% conserved) and Ile in Ral (100%). When comparing only the Ras-Ral and Ral subfamilies, several positions are conserved in each subfamily but different: 24 (Ile/Val in Ras-Ral, Met in Ral), 25 (Gln in Ras-Ral, Tyr in Ral), 33 (Asp in Ras-Ral, Glu in Ral), 43 (Gln in Ras-Ral, Lys in Ral), 67 (Met in Ras-Ral, Ile in Ral), and 70 (Gln in Ras-Ral, Asn in Ral) (>65% conserved in Ras-Ral, 100% in Ral). Finally, positions 7, 46, 93, and 160 are only conserved in Ral, not in the other subfamilies.


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Fig. 3.   Sequence space analysis of the different Ras subfamilies and alignment of the consensus sequences in the switch I region. A, sequence space analysis of the Ras-Ral (comprising Ras and Rap proteins but not Ral), Ral and Rho subfamilies basically as described (53). The sequence of each subfamily is represented as a vector point in a multidimensional space (sequence space), with residue positions and types as the basic dimensions. Directions in sequence space represent specific sequence patterns and are depicted as planar projections. The tree-determinant residues for each subfamily can be recognized as the extreme positions on the subfamily axis and are here indicated by circled clusters. In addition, the positions of the tree-determinant residues are numbered near these clusters. Positions 53 and 130 are conserved but shared by the Rho and Ras-Ral subfamilies and are located between these subfamily axes. Positions shared with Ral were not considered due to the low number of Ral sequences. The positions depicted in boldface are tree-determinant residues for each subfamily or shared by two subfamilies. B, sequence space analysis of the Ras, Rho, and Rab subfamilies. Note that whereas position 37 is conserved for all subfamilies and located within the tree-determinant cluster of each subfamily, position 36 is conserved for all subfamiles but shared between the Ras and Rab subfamilies. Consequently, it is located between the clusters of these subfamilies. C, sequence alignment of four Ras subfamilies (Rab, Rho, Ras-Ral, and Ral). The number of protein sequences used for the analysis are indicated in parentheses. The numbering of the Ras residues is indicated above the alignment. Positions conserved in each subfamily are represented by the corresponding letter(s). The two best tree-determinants in positions 36 and 37 are framed according to chemical similarity. X indicates a variable position. The mutated positions described in this work are indicated by arrows.

The analysis of the structural characteristics (surface exposition and change of solvent accessibility upon the transition of the GDP- to GTP-bound conformation) extends the tree-determinant analysis. Five of the 14 Ral tree-determinant residues (positions 7, 24, 46, 53, and 160) represent conservative substitutions between apolar residues in different subfamilies, and all of them are part of the protein interior. Position 93 is occupied mostly by small apolar residues in all the subfamilies, except Ral, in which position 93 is a Thr. However, it does not seem to be a good candidate because it is neither exposed in Ral·GDP nor in the GDP- and GTP-bound conformation of Ras. Position 92, which is an Ala in Ral but a polar residue in the other families, is also not very exposed in Ral or Ras. The solvent accessibility of most of the remaining Ral tree-determinant positions that are exposed is affected by the change between the GDP and GTP states. Position 25 is exposed but has a similar solvent accessibility in the GDP- and the GTP-bound state. Moreover, position 25 is occupied by a polar residue in all subfamilies. These characteristics make the participation of position 25 in the specific interaction with the effector less likely. Position 43 has a larger solvent accessibility in the Ras·GDP conformation, whereas positions 33, 36, 37, 67, and 70 have a larger solvent accessibility in the Ras·GTP conformation. The latter positions thus appear to offer more interacting surface in the GTP-bound state than in the GDP-bound conformation and are consequently, based on the sequence and structure analysis, good candidates for the interaction with the effector.

Taken together, five positions fit to our criteria: positions 33, 36, 37, 67, and 70. When limiting our view to Ras-Ral and Ral, positions 33 (Asp in Ras-Ral and Glu in Ral), 67 (Met in Ras-Ral and Ile in Ral), and 70 (Gln in Ras-Ral and Asn in Ral) contain conservative changes between Ras-Ral and Ral, whereas positions 36 (Ile in Ras-Ral and Lys in Ral) and 37 (Glu in Ras-Ral and Ala in Ral) differ dramatically. Thus, the latter two residues are our best candidates for the functional conversion between Ras and Ral. Both residues 36 and 37 are exposed and change substantially between the GDP- and GTP-bound conformations of Ras and are part of the interaction between Ras and the Raf-RBD (27). Remarkably, these positions also appear to be the best tree-determinant positions for all subfamilies.

A Practical Approach

Mutational Analysis of the Ral-RalBD Interaction-- A series of hRalB variants carrying mutations in the effector region was created in order to analyze the interaction between Ral and RLIP. Fig. 3C shows the alignment between the effector regions of the diverse subfamilies of small GTP-binding proteins and indicates which mutations were introduced in Ral. The effects of the mutations were tested by 2H analysis and by the pull-down assay.

As became clear from the 2H analysis shown in Fig. 4, mutations in Ral residues 41, 42, 43, and 47 did not affect significantly the interaction of Ral with RalBD. Weaker binding to RalBD was observed with mutants hRalB(A48E) and hRalB(D49N), whereas double mutant hRalB(K47I/A48E) did not show any binding to RalBD. Similarly, in our pull-down assay, we observed that mutation A48E strongly affects the interaction, whereas the double mutation K47I/A48E practically abrogates the binding of hRalB to RalBD (Fig. 2).


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Fig. 4.   Double hybrid analysis of Ral effector mutants. L40/AMR70 diploid cells expressing various combinations of LexA-fused proteins (rows) and GAL4 activation domain-fused proteins (columns) were tested for beta -galactosidase activity using a filter method.

As expected, the dominant-negative mutant hRalB(S28N), which is homologous to Ras(S17N), did not show any interaction with the effector molecule RLIP in the 2H analysis (Fig. 4).

The Nature of Ral Residues 47 and 48 Determines the Interaction with Effector Molecules-- Our 2H analysis of the effector mutants of Ral shows that the double mutation K47I/A48E abrogates the interaction of Ral with RalBD. In addition, the double mutation enables Ral to bind RBDs of several Ras-specific effector molecules, e.g. the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors RalGDS and Rlf (Fig. 4). Apparently, these residues are essential to hinder the interaction of Ral with Ras effectors. As can be seen in Fig. 3, hRalB(Lys-47) and hRalB(Ala-48) are homologous to Ras(Ile-36) and Ras(Glu-37), respectively. Thus, the double mutation K47I/A48E turns Ral into a Ras-like protein with respect to the amino acid sequence in the effector region and to the interaction with effector molecules. Interestingly, mutation K47I is sufficient to enable Ral to interact with the RalGEF-RBDs, whereas the extra mutation A48E is necessary in order to induce interaction with Raf-RBD.

We have determined the affinity of the mutant protein hRalB(K47I/A48E) for the RBDs of RalGDS, Rgl, and Rlf by a fluorescence assay as described (15, 49, 58). This fluorescence assay is based on the observation that RBDs are capable to decrease the dissociation rate of the GTP-bound form of Ras proteins and uses the fluorescently labeled GTP analogue mGpp(NH)p (Fig. 5). Whereas RalGDS- and Rgl-RBDs bind to the mutant with a low affinity, as shown by the dissociation constants of 7.0 and 15.2 µM, respectively, Rlf-RBD shows an affinity comparable to that of Ras and Rap1A wild type (Table I). This underlines the observation that the Ras-binding domain of Rlf differs in a number of aspects from that of the other two RalGEF proteins (24).


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Fig. 5.   Guanine nucleotide dissociation inhibitor effect of RalGEF-RBDs on Ral wt and Ral(K47I/A48E). Whereas none of the RalGEF-RBDs interacts with hRalB wt (data not shown), the three analyzed Ras effectors show a different affinity to hRalB(K47I/A48E), in which the specificity determining residues are changed to their analogous amino acids in Ha-Ras. Rlf-RBD () has the highest affinity to Ral(K47I/A48E); this affinity is comparable to its affinity toward Ha-Ras (24). RalGDS-RBD (black-triangle) and Rgl-RBD (black-diamond ) show an affinity to Ral 5 times lower than to Ha-Ras.

                              
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Table I
Dissociation constants of the complexes of hRalB(K47I/A48E)·mGpp(NH)p with the RalGEF-RBDs

We undertook the reciprocal experiment and tried to turn a Ras in a Ral. An identical 2H assay to test interaction between RalBD or Ras effectors on one hand and Ras wt, Ras(I36K), Ras(E37A), or Ras(I36K/E37A) on the other hand showed only weak interaction (not shown). Therefore, we decided to measure these interactions with the more quantitative beta -galactosidase assay in solution (59), as well with histidine prototrophy. As can be seen in Fig. 6, in this assay, the effects of mutations K47I, A48E, and K47I/A48E in hRalB on the interactions with RalBD and Ras effectors are similar, as determined with the 2H and pull-down assays (Figs. 2 and 4). The reversed mutations I36K, E37A, and I36K/E37A in Ras cause, to a large extent, the expected, opposite effects. Importantly, mutation E37A is apparently enough to induce a weak but significant interaction between Ras and RLIP. This is in agreement with the observation that mutation A48E in Ral strongly reduces the interaction with RLIP. Furthermore, Lys47 in Ral appears not to be essential for RLIP binding, but to be a requisite to prevent interaction with Ras effectors. Similarly, mutation I36K reduces the binding of Ras to RalGEFs (and to some extent to Raf), but does not induce binding of Ras to RLIP. The combination of both mutations induces in Ral a full shift in affinity from RLIP toward the Ras effectors, and in Ras an abrogation of the interaction with the Ras effectors and a weak but significant induction of binding to RLIP. These results are confirmed by the selection on His- plates (Fig. 7).


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Fig. 6.   Histogram of beta -galactosidase activity as a tool to measure the interaction between Ras and Ral mutants with Ras and Ral effector proteins. Student's t test was performed to determine the significance of the interaction of Ras(E37A) and Ras(I36K/E37A) with RLIP and Raf relative to the signal observed with the empty vector. The resulting data are indicated in the histograms (<0.05, significant; >0.05, insignificant).


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Fig. 7.   Double hybrid analysis of Ras mutants: His- selection. L40/AMR70 diploid cells expressing various combinations of LexA-fused proteins (rows) and GAL4 activation domain-fused proteins (columns) were patched on DO-WL (drop-out medium depleted of Trp and Leu) plates and replica-plated on DO-WLH (drop-out medium depleted of Trp, Leu, and His) and DO-WL plates to be tested for histidine prototrophy. All patches grew on DO-WL (data not shown). Pictures were taken of after 3 days at 30 °C.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

For several years after the discovery of Ral, overexpression of putatively activated or dominant-negative Ral variants did not lead to any obvious cellular phenotype, and thus the cellular function of Ral remained elusive. This situation changed upon the discovery that RalGEFs were potential Ras effector proteins (60, 61). Soon, it became clear that Ral was involved in Ras-dependent cellular transformation parallel to the Ras-Raf pathway (28-35). Direct detection of the GTP-form of endogenous Ral was made possible by pull-down assays using the GST-coupled form of RalBD, the Ral-binding domain of the Ral-specific effector protein RLIP76. This way, it could be demonstrated that Ral is activated by a Ras-dependent pathway in Chinese hamster ovary cells (35) and possibly by a Rap1-dependent pathway in platelets (36).

In this work, we have analyzed the interaction between Ral and the effector protein RLIP76. For the first time, we were able to isolate and purify the complex between Ral and the RalBD. Our results suggest that RalBD occurs as a homodimer that dissociates upon binding to Ral. Thus, binding to Ral induces a monomerization of RalBD. This may have a physiological function, e.g. to render the GAP region accessible to its substrate as proposed before (37). On the other hand, in vitro experiments suggested that the binding of Ral to RalBD does not affect the GAP activity of RLIP76 on Rac (62).

Earlier, it was shown that mutation T46A (for comparison, the homologous residue in Ras will be given in parentheses) (Ras Thr-35) inhibits the interaction between Ral and RLIP (37). We have further dissected the interaction between Ral and RLIP76 by testing a series of Ral mutants in a 2H assay, as shown in Fig. 4. Mutations E41K (Ras Asp-30) and D42K (Ras Glu-31) do not seem to affect the interaction of Ral with RLIP. This is different from the homologous mutations in Ras, because position 31 is of crucial importance for the interaction with Ras effectors: the wild type residue Glu-31 confers a high affinity for c-Raf-1, whereas Ras(E31K), which mimics Rap, has a high affinity for RalGDS (27, 63). Comparable to the Ras-Raf interaction (58), mutation Y43W (Ras Tyr-32) does not seem to affect the interaction with RLIP. Residue Glu-37 in Ras has been demonstrated to be important for interaction with c-Raf-1, but not with RalGEFs (24, 31-33, 64-66). In Ral, mutation A48E (Ras Glu-37) affects the interaction with RLIP, as shown by 2H analysis (Figs. 4 and 7), the pull-down assay (Fig. 2), and the beta -galactosidase assay (Fig. 6). Also, mutation D49N in Ral (Ras Asp-38) weakens the interaction with RLIP in our 2H analysis (Fig. 4). A stronger effect of this mutation was observed using immunoprecipitation with an epitope-tagged Ral-binding domain of RLIP76 (amino acids 86-626) (38). In Ras, mutation D38N affects the interaction with c-Raf-1 (67), whereas mutation D38E affects the interaction with Rlf (34) but not with c-Raf-1 (64, 65, 68).

Interestingly, mutation K47I (Ras Ile-36) hardly affects the interaction of Ral with RLIP but enables Ral to interact with Ras effector proteins RalGDS and Rlf (Fig. 4). In combination with mutation A48E, which strongly reduces the Ral-RLIP interaction, double mutant hRalB(K47I/A48E) has practically no affinity for RLIP (Figs. 2, 4, 6, and 7). At the same time, this double mutant is able to interact with the Ras-specific effector molecules, of which at least the RalGEFs are bound with affinities that are comparable to those of Ras (Table I). On the other hand, introduction of mutations I36K and E37A abrogates the interaction of Ras with its effector molecules but enables Ras to interact with RLIP, even though the interaction is still weak (Fig. 6 and 7).

Sequence space analysis shows that Ras positions 36 and 37 (Ral residues 47 and 48) are tree-determinant positions for the Ras, Ral, Rho, and Rab subfamilies because they are conserved in each subfamily but differ between the subfamilies (position 36 is shared by the Rab and Ras subfamilies) (Fig. 3b). Thus, in agreement with our experimental data, this analysis indicates these positions to be the most likely candidates that determine the specificity of the interaction of Ras and Ral with their effector(s).

The crystal structure of sRalA·GDP, which will be described in detail elsewhere,3 shows that the three-dimensional structures of Ras and Ral are quite similar, as expected from the high sequence similarity. The main structural differences between Ral and Ras are located in the switch II region, which shows a shift of the alpha -helix comprising residues 78-85 of sRalA (Ras 67-74). In contrast, the switch I regions of Ral and Ras superimpose very well, with a root mean square deviation of 0.3 143 for 11 Calpha -atoms. In Fig. 8, we have depicted the electrostatic potentials of the surfaces of Ha-Ras (in the GDP- and GTP-bound conformation) with that of sRalA and the modeled structure of the double mutant RalA(K47I/A48E), with the switch I region toward the reader. When comparing the GDP-bound structures of Ras and Ral, it becomes evident that the positive charge of Lys-47 in Ral and the negative charge of Glu-37 in Ras represents the main difference in the switch I region between these proteins, in accordance with our mutational analysis.


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Fig. 8.   Comparison of the crystal structures of Ha-Ras and Ral. The electrostatic potentials of the crystal structures of Ha-Ras1-166·Gpp(NH)p (Protein Data Bank code 5p21) Ha-Ras1-171·GDP (Protein Data Bank code 4q21), sRalA1-178·GDP (Footnote 3) and the modeled structure of the double mutant sRalA(K47I/A48E)1-178·GDP were calculated with GRASP (70). Negative potentials are indicated with red and positive potentials with blue; scale is -15 to +15 kT/e. Selected residues in the switch I region are indicated with numbers.

Remarkably, Ral and its partners RalGDS and RLIP do not exist in Saccharomyces cerevisiae or in any other unicellular eukaryotes (as far as genomic sequences are known), an evident discrepancy with Ras and the Ras-MAP kinase signaling modules. The Ras-Ral signaling module is thus of late appearance in evolution, and one is tempted to correlate the appearance of this Ras pathway with the emergence of multicellularity---perhaps at the same time as the receptor tyrosine kinase appearance in evolution. This aspect underlines the uniqueness of Ral within the Ras subfamily, as shown in this work by theoretical and experimental means.

In conclusion, our analysis shows that Lys-47 in Ral, even though not essential for interaction with RLIP76, is a requisite to prevent interaction of Ral with Ras effector proteins. The neighboring residue, Ala-48, in its turn, is important for the interaction with RLIP. Mutation of these residues to the Ras-like amino acids enables Ral to interact with the Ras effector proteins Raf and RalGEFs, whereas the opposite mutations enables Ras to interact with RLIP. Our results thus indicate that effects of mutations in the effector region of small GTPases should be interpreted with care, because interactions with other effector proteins may be induced. In this light, it seems possible that the remarkable dominant negative effects of Rac(Q61L/F37A) may not be caused by unproductive interactions with Rac effectors (69) but by productive or unproductive interactions with effectors of other small GTPases. Last but not least, despite a differently predicted secondary structure for RLIP, our results suggest that the mode of interaction between RLIP76 and Ral is similar to the mode of the interaction between RBD and Ras.

    ACKNOWLEDGEMENTS

We thank Çetin Koç, Beate Drawe, and Markus Dettenberg for their help in producing the Ral and Ras mutants, Christiane Theiß and Andreas Arndt for excellent technical assistance and Christian Herrmann for supplying RalGDS-RBD. 2H plasmids expressing RalGDS- and Rlf-RBDs were kindly provided by Vanessa Nancy and Jean de Gunzburg. J. A. G.-R. and A. V. acknowledge the interesting discussions and technical support provided by the members of the Protein Design Group CNB-CSIC, in particular Antonio del Sol and Florencio Pazos.

    FOOTNOTES

* This work was partially supported by Institut Curie, Ligue contre le Cancer, Association pour la Recherche sur le Cancer, and Grant BIO4-CT96-1110 of the European Community.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.

§ These authors contributed equally.

Supported by Grant BIO4-CT96-1110 of the European Community.

** Supported by a Ministère de l'Education Nationale de la Recherche et de la Technologie Ph.D. fellowship.

§§ To whom correspondence should be addressed. Tel.: 49-231-133-2105; Fax: 49-231-133-2199; E-mail: robbert.cool{at}mpi-dortmund.mpg.de.

2 P. Bork, C. Sander, and A. Valencia, unpublished results.

3 I. R. Vetter, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: GAP, GTPase activating protein; GDS, guanine nucleotide dissociation stimulator; GEF, guanine nucleotide exchange factor; Gpp(NH)p, 5'-guanylylimidodiphosphate; GST, glutathione S-transferase; RBD, Ras-binding domain; PCR, polymerase chain reaction; wt, wild type; RalBD, Ral-binding domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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