©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Switching Nucleotide Specificity of Ha-Ras p21 by a Single Amino Acid Substitution at Aspartate 119 (*)

Jie-Ming Zhong (1), Mo-Chou Chen-Hwang (1), Yu-Wen Hwang (1) (2)(§)

From the (1) Molecular Biology Department, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314 and (2) CSI/IBR Center for Developmental Neuroscience and Graduate Program in Biology, City University of New York, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We examined c-Ha-Ras harboring an aspartate to asparagine substitution at position 119 (mutation D119N). The Asp-119 is part of the conserved NK XD motif shared by members of the regulatory GTPase family. This asparagine residue has been proposed to participate in direct bonding to the guanine ring and to determine the guanine-nucleotide binding specificity. The D119N mutation was found to alter nucleotide specificity of Ha-Ras from guanine to xanthine, an observation that directly supports the essential role of hydrogen bonding between the side chain of the aspartic acid residue and the guanine ring in nucleotide binding specificity. Besides nucleotide binding specificity, the D119N mutation has little or no effect on the interaction of Ha-Ras with SDC25C, SOS1, GAP, or Raf. Neither does it affect the hydrolysis of nucleotide triphosphate. Like xanthine-nucleotide-specific EF-Tu, xanthine-nucleotide-specific Ras and related proteins will be useful tools for elucidating cellular systems containing multiple regulatory GTPases.


INTRODUCTION

The Ha-Ras is a member of the regulatory GTPase family (1, 2) . Regulatory GTPases bind guanine-nucleotide and function as molecular switches in regulating cellular processes. The activity of a regulatory GTPase is dependent on the phosphorylation state of the bound guanine-nucleotide. GTP binding activates the regulatory GTPase, promotes effector interaction, and elicits subsequent biological effects. This interaction leads to the hydrolysis of the -phosphate and returns the regulatory GTPase to the inactive GDP-bound state. The inactive regulatory GTPase can then be reactivated by exchanging the bound GDP with an exogenous GTP (1, 2) . Thus, the activity of regulatory GTPase is controlled through the balance of GTP hydrolysis and guanine-nucleotide exchange (1, 2) . Factors that interact with and regulate the activity of Ras p21 include a group of CDC25-related proteins (such as SDC25C and SOS), which stimulate guanine-nucleotide exchange reaction; GAP,() which promotes GTP hydrolysis; and the putative effector Raf protein (3, 4, 5, 6, 7, 8, 9) . Recently, phosphatidylinositol 3-kinase has also been suggested as a possible target of Ras (10) .

Despite functional diversity, regulatory GTPases share distinct patterns of sequence homologies (11) . Three most prominent conserved motifs are G XXXXGK(S/T), D XXG and NK XD. These consensus sequences lie in the vicinity of the bound guanine-nucleotide and form direct contacts either with the moieties of guanine-nucleotide or with the essential cofactor magnesium ion (12, 13, 14, 15, 16, 17, 18, 19) . It is likely that the conserved sequences serve as the foundation for the conserved mechanistic functions of regulatory GTPase, such as guanine-nucleotide binding, conformational changes, GTP hydrolysis, and nucleotide exchange (1, 2) .

Aspartate 119 of Ha-Ras is part of the conserved motif NK XD, the domain interacting with guanine base. The x-ray structures of regulatory GTPases have proposed that the carboxylate side chain of the aspartic acid in the NK XD domain interacts with the endocyclic N-1 and the exocyclic 2-amino group through hydrogen bonding, and such interactions provide the specificity for nucleotide binding (12, 13, 14, 15, 16, 17, 18, 19) . This hypothesis was confirmed in EF-Tu by the finding that the EF-Tu(D138N) mutant preferentially binds and uses xanthine-nucleotide for its activities including aminoacyl-tRNA binding and peptide elongation in the ribosomes (20, 21, 22) . The observation of the EF-Tu(D138N) mutant activities suggests that the interaction between the aspartic acid residue and guanine ring can be fully substituted by the interaction of asparagine and xanthine ring, a base containing a 2-carbonyl group. The nucleotide substrate specificity of adenylosuccinate synthetase also appears to be achieved through the same aspartic acid-guanine ring interaction. Similarly to EF-Tu, the substitution of an aspartic acid to asparagine at position 333 of adenylosuccinate synthetase converts substrate specificity from GTP to XTP (23) . In this study, we examined the nucleotide specificity of Ha-Ras harboring an Asp-119 to Asn substitution, the corresponding mutation of EF-Tu(D138N). This mutant was isolated and partially characterized earlier by Feig et al. (24) as a GTP-binding deficient mutant.


MATERIALS AND METHODS

Mutant Construction and Protein Purification

The Ha-Ras(D119N) was constructed by oligonucleotide site-directed mutagenesis as described by Kunkel and co-workers (25) . Single-stranded DNA from M13 phage clone containing the entire c-Ha-Ras was used as the template for mutagenesis. The following oligonucleotide, GGCCAGGTTACACTTGTT, was used for mutant construction. The Ha-Ras(D119N) was subsequently constructed as a GST-fusion protein using a modified pGEX3 vector (26) . The Ha-Ras(D119N) was also cloned into an Escherichia coli expression vector containing the T7 RNA polymerase promoter for in vitro labeling as described (27) . The procedures for preparing GST fusion proteins Ras and Raf were described elsewhere (26) . SDC25C was expressed from the vector pTTQ-SDC25 (28) and purified as described by Créchet et al. (29) . Preparation of [-P]XTP-[-P]XTP was synthesized according to a modified procedure of Glynn and Chappell (30) . Briefly, reaction in 1 ml of mixture containing 50 mM Tris-HCl, pH 8.0, 3 mM MgCl, 5 mM dithiothreitol, 0.5 mM XTP, 0.5 mM NAD, 0.5 mM 1,3-diphosphoglycerate, 50 mg/ml glyceraldehyde-3-phosphate dehydrogenase, 5 mg/ml phosphoglycerate kinase, and 500 µCi/ml phosphorus-32 (specific activity 285 Ci/mg of phosphorus) was allowed to proceed at room temperature for 15 h. The incorporation of P into XTP was monitored by a charcoal adsorption assay and is usually equal to 60-65% of total input phosphorus-32. [-P]XTP was partially purified from the reaction mixture using ion exchange resin Q-Sepharose. The elution of phosphate and XTP was performed using 50 mM NaCl and 300 mM NaCl, respectively.

Preparation of GST-mSOS Fusion Protein

The GST-fusion protein containing residues 539 to 1046 of mouse SOS1 catalytic domain was constructed similarly as described by Liu et al. (31) . The mouse SOS1 catalytic domain DNA was generated from the full-length cDNA clone by polymerase chain reaction using the following oligonucleotides, CCTTTATCGATGTCAGCTGAAGAGAAAAACAAC and GGAACTCGAGTCATGGATTTGATGGACGAACCC, respectively, as the 5` and the 3` primer. The 5` primer contains a ClaI site, and the 3` primer contains a XhoI site. In addition, a stop codon in-frame with the SOS1 coding sequence was included in the 3` primer. The resulting 1550-base pair polymerase chain reaction product was digested with ClaI and XhoI and cloned into the unique ClaI- XhoI site of the modified pGEX3 vector (26) to yield the GST fusion clone. The procedures for expression and purification of GST-SOS1 were as described (26) .

Nucleotide Binding and Exchange Assay

The GTP and XTP dissociation constants were determined by equilibrium binding and calculated from the Scatchard plot as described (32) . The equilibrium binding was performed in a reaction mixture containing 50 mM Tris-HCl, pH 8.0, 2 mM MgCl, 50 mM KCl, 1 mM dithiothreitol, 1 µM (100 pmol) GST-Ras protein, 4 mM EDTA, and varying concentrations of [H]GTP (specific activity 1500 cpm/pmol) or [-P]XTP (specific activity 1400 cpm/pmol) for 10 min at 30 °C. The concentrations of nucleotide triphosphate used were in the range of 10to 10 M for the bonding pairs and 10to 10 M for the nonbonding pairs. The time course SDC25C- and SOS1-stimulated guanine-nucleotide exchange reaction was performed using 1 µM (200 pmol) GST-Ras protein, 60 nM SDC25C or 300 nM GST-SOS1, and 5 µM [H]GTP (specific activity 1500 cpm/pmol) or [P]XTP (specific activity 1400 cpm/pmol) as described (27) . The kinetic parameters of SDC25C- and SOS1-stimulated reactions were determined in 100 µl of reaction mixture using 0.25, 05, 1, and 2 µM substrate and either 20 nM SDC25C or 100 nM GST-SOS1 as described previously (27) .

In Vitro Ha-Ras p21 Labeling and SDC25C Binding Assay

The intact wild type and D119N Ha-Ras were labeled in vitro with [S]methionine as described (27) . The Ha-RasDC25C complex formation and dissociation were performed in a Pharmacia Superdex 75 HR 10/30 column attached to a high performance liquid chromatography column as described (27) .

Measurement of Nucleotide Triphosphate Hydrolysis

Nucleotide triphosphate hydrolysis was measured by monitoring the release of the -phosphate group from [-P]GTP or [-P]XTP as described below. GST-Ras(WT) and GST-Ras(D119N) were labeled in the presence of EDTA to equilibrium with [-P]GTP and [-P]XTP, respectively. The [Mg] in the reaction mixture was brought up to 10 mM, and then the free nucleotide in the reaction mixture was removed by passing through a G-25 column. The column was eluted with a buffer containing 20 mM Na-HEPES, pH 7.5, 2 mM MgCl. GTPase reaction was performed in 200 µl of reaction mixture containing 20 mM Na-HEPES, pH 7.5, 2 mM MgCl, 0.125 µM labeled GST-Ras. The reaction was initiated by the addition of the full-length human GAP (final concentration 0.4 nM) and incubated at 30 °C. At the indicated time interval, an aliquot of 25 µl was withdrawn, and the amounts of free phosphate were measured as described (33) and used to calculate nucleotide hydrolysis. The apparent Kand kwere determined in two sets of reactions which contained four different substrate concentrations (0.125, 025, 0.4, and 0.5 µM concentrations of either GST-Ras[-P]GTP or GST-Ras(D119N)[-P]XTP) in 200 µl of HEPES-Mgbuffer. One set contained only substrate and the other contained substrate and GAP. The reactions were initiated by adding GAP (final concentration 0.2 nM) and allowed to proceed for 5 min at 30 °C. The extent of GTP hydrolysis was determined as described above and used for calculating kinetic parameters. The intrinsic kof nucleotide triphosphate hydrolysis was determined similarly as described above using 0.25 µM GST-fusion protein and 2 to 20 µM nucleotide triphosphate.

Raf and Ras Binding

S-labeled Ha-Ras p21 was charged with nucleotide triphosphate (GTP or XTP) or diphosphate (GDP or XDP) at 30 °C to equilibrium in 35 µl of binding solution containing 25 mM Tris, pH 7.5, 2 mM MgCl, 100 mM NHCl, 0.2 mg/ml bovine serum albumin, 20 µM nucleotide, and 5 mM EDTA for 5 min. The reaction was terminated by the addition of 20 mM MgCl. Complex formation between Ras and Raf was determined by mixing the GTP- or XTP-equilibrated S-labeled Ras (5.6 pmol) with 2.8 nmol of GST-Raf fusion protein in a 200 µl of Raf binding buffer composed of 50 mM Na-HEPES, pH 7.5, 100 mM KCl, 20 µM ZnCl, 5 mM MgCl, 1 mg/ml bovine serum albumin. After incubation on ice for 1 h, 20 µl of glutathione-Sepharose beads (12.5% gel slurry) were added to the reaction mixture, and the incubation was continued with gentle shaking for another hour at 4 °C. The free Ras and GST-Raf were removed by washing the beads 5 times with 1 ml of ice-cold washing buffer (50 mM Na-HEPES, pH 7.5, 100 mM KCl, 5 mM MgCl, 20 µM ZnCl, and 0.1% Triton X-100). RasGST-Raf complexes were eluted from the washed beads by resuspension in 30 µl of polyacrylamide gel electrophoresis loading buffer. The coprecipitated S-labeled Ras protein was identified by autoradiography following electrophoresis on a 10% polyacrylamide gel. The amounts of coprecipitated Ras was determined in a liquid scintillation counter and used to calculate the ratio of Ras coprecipitated by Raf. The number of coprecipitated Ras represented the average of at least two trials.


RESULTS

The intact Ha-Ras(D119N) has proved to be very difficult to purify by the nondenaturing protocol we routinely used (27) . The majority of the expressed Ha-Ras(D119N) is in the insoluble fraction after cell disruption, and repeated chromatography of the soluble fraction always resulted in small amounts of protein of variable purity. This problem was largely eliminated by using GST-fusion protein; therefore, GST-fusion proteins were used in this study except when stated otherwise.

GTP and XTP Binding

The dissociation constants of the GST-fusion Ras proteins for GTP and XTP were determined by the equilibrium binding method (). The Kfor GST-Ras(WT) was calculated to be 1.6 10 M. This value is similar to that of the intact protein (24, 32) , a result indicating that the introduction of the GST moiety does not perturb nucleotide binding. As expected, the affinity for GTP is drastically reduced by the D119N mutation. The Kis 1.9 10 M for the D119N mutant, which is about 3 orders of magnitude weaker than that of the wild type. This difference in the GTP affinity reported here is about 10-fold greater than that observed by others (24) . On the other hand, the protein's affinity for XTP was significantly enhanced by the D119N mutation. The Kis 4.1 10 M for the wild type Ras and 3.9 10 M for the D119N mutant, an enhancement in XTP affinity of about 2 orders of magnitude.

Kinetics of Nucleotide Exchange

The intrinsic and SDC25C-stimulated nucleotide exchange was measured. As shown in Fig. 1 , regardless of the nucleotide and Ras species, SDC25C readily stimulates the nucleotide exchange to reach equilibrium. This observation shows that SDC25C interaction is not disrupted by the D119N mutation and SDC25C does not distinguish XTP from GTP. However, the level of nucleotide binding at equilibrium for the bonding pairs (GTP with the wild type and XTP with D119N mutant) is much higher than that of the nonbonding pairs. Under the assay condition (5 µM nucleotide), about 8% GST-Ras(D119N) binds GTP and about 30% GST-Ras(WT) binds XTP at equilibrium while the binding was nearly completed for both the bonding pairs. The level of nucleotide binding reflects protein's affinity for nucleotide, since the concentration of nucleotide in the assay is not sufficient to support the saturation binding for the nonbonding pairs. The higher dissociation rate with nonbonding nucleotide also results in a higher intrinsic exchange rate, particularly, GTP exchange of the D119N mutant (Fig. 1, inset).


Figure 1: The intrinsic and SDC25C-stimulated nucleotide exchange. A, GTP exchange; B, XTP exchange. The experiments were performed using 1 µM GST-Ras protein, 60 nM SDC25C, and 5 µM [H]GTP or [P]XTP as described (27). The inset in A is an expansion of the region corresponding to lower y axis value of A. Symbols used in the plots are GST-Ras(WT) alone (), GST-Ras(WT) with SDC25C (), GST-Ras(D119N) alone (), and GST-Ras(D119N) with SDC25C ().



The kinetic parameters for the SDC25C-stimulated reaction were subsequently determined for the bonding pairs (). The apparent Kof the SDC25C-stimulated reaction was 1.8 µM for the wild type and 1.6 µM for the D119N mutant (). This result supports the conclusion that the interaction of Ras with SDC25C was not altered by the D119N mutation. However, the D119N mutant displays a SDC25C-stimulated exchange rate which is about 25% faster than that of the wild type ().

The SOS1-stimulated nucleotide exchange was also examined Fig. 2). Similarly to SDC25C, GST-SOS1 readily promotes nucleotide exchange to GST fusion Ras proteins regardless of the nucleotide (Fig. 2). The nucleotide exchange is not the outcome of the GST and GST interaction as GST by itself has no detectable nucleotide exchange stimulating activity on GST-Ras (data not shown). The time course of the wild type and GTP exchange is nearly identical with that of the D119N mutant and XTP exchange (Fig. 2). In fact, the steady state kinetics reveals little difference between the wild type and the D119N mutant in the SOS1-stimulated nucleotide exchange (). Again, similarly to SDC25C, SOS1 does not differentiate between XTP and GTP. Interestingly, although SOS1 and SDC25C appear to interact with Ras with similar affinity, the rate of nucleotide exchange stimulated by SOS1 was significantly lower than that stimulated by SDC25C ().


Figure 2: The time course of SOS1-stimulated nucleotide exchange. The experiments were performed on the bonding pairs (GTP exchange for the wild type and XTP exchange for the D119N mutant) using 1 µM GST-Ras protein, 300 nM GST-SOS1, and a 5 µM concentration of either [H]GTP or [P]XTP as described (27). , GST-Ras(WT) alone; , GST-Ras(WT) with SOS1; , GST-Ras(D119N) alone; and (), GST-Ras(D119N) with SOS1.



SDC25C Binding

The SDC25C-stimulated XTP exchange suggests that the D119N mutant is able to form a complex with SDC25C similar to that of the wild type (27) . Thus, we examined the ability of the intact Ras(D119N) to interact with SDC25C and nucleotide. As with the wild type Ras, the addition of SDC25C to Ras(D119N) converts Ras(D119N) into the Ras(D119N)SDC25C complex which can be distinguished from the uncomplexed Ras in a gel filtration column (Fig. 3). However, Ras(D119N) appears to bind SDC25C more efficiently than the wild type Ras because a 10-fold molar excess of SDC25C causes quantitative conversion of Ras(D119N) to the complex form but not Ras(WT) (Fig. 3). The lower yield of complex formation with wild type Ras has been attributed to the presence of guanine-nucleotide in the translation mixture as well as to the bound guanine-nucleotide; therefore, a reduction in guanine-nucleotide affinity such as the D119N mutation, will improve the efficiency of RasSDC25C complex formation (27) . We subsequently measured the nucleotide concentration which promotes complete complex dissociation (I). The concentration of nucleotide for dissociating the Ras(WT)SDC25C complex was 0.6 µM for GDP, 0.2 µM for GTP, or 10 µM for XDP. In contrast, it requires 25 µM GDP, 0.5 µM XTP, or 0.8 µM XDP to dissociate the Ras(D119N)SDC25C complex. These results show that the amounts of nucleotide required for complex dissociation parallels the protein's affinity for each nucleotide.


Figure 3: The interaction of the wild type Ras and D119N with SDC25C and nucleotide. The complex formation and dissociation was performed using S-labeled intact Ras in a gel filtration column as described previously (26). A, the wild type with SDC25C and GTP; B, D119N mutant with SDC25C and XTP. Symbols used for both plots are: , 0.5 pmol of labeled Ras alone; , labeled Ras with 5 pmol of SDC25C; , labeled Ras with 5 pmol of SDC25C in the presence of 0.5 µM GTP ( A) or XTP ( B).



Nucleotide Triphosphate Hydrolysis and GAP Interaction

In the normal cycle of its function, Ras utilizes and hydrolyzes GTP; therefore, we analyzed XTP hydrolysis of the D119N mutant. GST-fusion Ras proteins were used in this study. GST-Ras(WT) exhibits a slow intrinsic rate of GTP hydrolysis which can be greatly enhanced by the addition of GAP (Fig. 4 A). Apparently, GTP hydrolysis is not affected by the presence of the GST moiety. Similarly, GST-Ras(D119N) also exhibits a low rate of XTP hydrolysis which can be accelerated by the addition of GAP (Fig. 4 B). The kvalues of GAP-stimulated nucleotide triphosphate hydrolysis at 0.2 nM GAP were estimated to be 6.45 and 5.08 sfor GST-Ras(WT) and GST-Ras(D119N), respectively (). This represents an approximately 5 10-fold stimulation ratio for both GST-Ras(WT) and GST-Ras(D119N). The Kvalues of GAP-stimulated hydrolysis are also similar for both wild type and D119N mutant (). These observations support the conclusion that the D119N mutation dose not affect the mechanism of nucleotide triphosphate hydrolysis of Ha-Ras as well as its interaction with GAP.


Figure 4: The intrinsic and GAP-stimulated hydrolysis of nucleotide triphosphate. The experiments were performed using GST-fusion Ras and the full length human GAP as described under ``Materials and Methods.'' A, GTP hydrolysis of GST-Ras(WT); B, XTP hydrolysis of GST-Ras(D119N). , GST-Ras(WT) without GAP; , GST-Ras(WT) with GAP; , GST-Ras(D119N) without GAP; and , GST-Ras(D119N) with GAP.



Raf Binding

Ras was found to interact with Raf in a GTP-dependent manner (4, 5, 6, 7, 8, 9) . Subsequently, we examined XTP-dependent Raf binding activity of D119N mutant. The intact forms of wild type and D119N mutant were labeled in vitro with [S]methionine, charged with nucleotide, reacted with GST-Raf, and then precipitated by glutathione-Sepharose resin as described under ``Materials and Methods.'' As expected, GTP induces the interaction of Ras(WT) with GST-Raf (Fig. 5). Similar to the effect of GTP on Ras(WT), XTP promotes the interaction of Ras(D119N) with GST-Raf. The amounts of coprecipitated Ras(D119N) (9.6%) were close to that of Ras(WT) (10.7%), an observation suggesting that the interaction of Ras with Raf is not altered by D119N mutation. Compared to nucleotide triphosphates, nucleotide diphosphates were much less effective for promoting the Ras and Raf interaction (Fig. 5). The nonbonding nucleotide triphosphate is also able to promote substantial Raf binding if sufficient amounts of that are provided. For example, XTP at 0.5 mM produces 8.5% coprecipitated Ras(WT); while GTP at 0.5 mM yields 7.2% coprecipitated Ras(D119N).


Figure 5: Nucleotide triphosphate-dependent Raf interaction. The Raf interaction was analyzed using a coprecipitation assay consisting of S-labeled intact Ras and GST-fusion Raf N-terminal domain as described (26). The nucleotide concentration used in the assay was 20 µM. Lane 1, wild type Ras precharged with GDP; lane 2, wild type Ras precharged with GTP; lane 3, D119N mutant precharged with XDP; lane 4, D119N mutant precharged with XTP.




DISCUSSION

The x-ray structures of regulatory GTPases suggest that the specificity of guanine-nucleotide binding is accomplished through the interaction of the conserved NK XD domain with the guanine ring, specifically, through the hydrogen bond between the carboxylate side chain of the aspartic acid residue and the endocyclic N-1 as well as the exocyclic 2-amino group of the guanine ring (12, 13, 14, 15) . This hypothesis has been confirmed in EF-Tu and adenylosuccinate synthetase by using an Asp to Asn substitution mutation, which converts nucleotide specificity from GTP to XTP (20, 21, 22, 23) . In Ha-Ras, the importance of the aspartic acid-guanine ring interaction to the nucleotide binding specificity also has been implicated using an Asp to Ala substitution mutant in which Ras(D119A) was found to greatly affect the binding of guanine-nucleotide but not inosine-nucleotide (34) . In this study, we examined the biochemical properties of the D119N mutant, a mutant expected to utilize xanthine-nucleotide.

We first analyzed the binding of GTP and XTP to the wild type and the D119N Ras. As expected from the x-ray model of Ha-Ras, the D119N mutation drastically weakens GTP binding but greatly enhances XTP binding (). The difference in binding strength is in the neighborhood of 2 to 3 orders of magnitude, a difference equivalent to about 3 to 4 kcal/mol. The nonbonding pair is predicated to have one fewer hydrogen bond than the bonding pair, but the actual difference in energy is much greater than one would anticipate from a single hydrogen bond in aqueous solution. We cannot explain this discrepancy; however, it is possible that the guanine ring binding pocket of Ha-Ras may provide a hydrogen bonding environment which is very different from that of aqueous solution. The GST-Ras(WT) appears to bind nucleotide (either GTP or XTP) with greater strength than the D119N mutant binds its counterpart. For example, there is an approximately 5-fold difference between the wild type and the D119N mutant in binding to the nonbonding nucleotide (). This phenomenon may be due to the ability of the charged carboxylate group of aspartic acid to form stronger bonds with the guanine ring than the uncharged carbonyl side chain of asparagine in the D119N mutant. The nucleotide binding strength is reflected in the intrinsic nucleotide exchange rates as the nonbonding pairs, particularly GTP exchange of the D119N mutant, have faster rates of intrinsic exchange than the bonding pairs (Fig. 1).

Both SDC25C and SOS1 promote XTP exchange to the D119N mutant to about the same extent that they stimulate GTP exchange to the wild type Ras (Figs. 1 and 2); therefore, we conclude that the D119N mutation does not affect the interaction of Ha-Ras with either SDC25C or SOS1. This conclusion is in agreement with the findings of the steady state kinetics determinations (). The D119N mutant appears to have a higher SDC25C-stimulated exchange rate than the wild type; however, a difference of about 25% may not be significant. Another interesting finding of our study is that the nucleotide specificity is strictly the property of Ha-Ras, and neither SDC25C nor SOS1 distinguishes between XTP and GTP. Despite the fact that GST-SOS1 has a similar affinity as SDC25C for RasNTP, it exhibits much lower activity in stimulating nucleotide exchange than SDC25C (). This lower stimulating activity is likely to be the inherent property of GST-SOS1 since it also has a lower activity than SDC25C in stimulating guanine-nucleotide exchange onto the intact Ha-Ras (data not shown).

The D119N mutant appears to have higher affinity for binding SDC25C than the wild type Ras under equilibrium conditions (Fig. 2); however, this phenomenon is likely to be due to the reduced guanine-nucleotide binding affinity associated with the D119N mutation. Nucleotide and SDC25C compete in binding to Ha-Ras; the reduction in binding affinity for one would appear as an enhancement of affinity for the other. Therefore, the ratio of nucleotide required for complex dissociation () to the nucleotide dissociation constant () is a better assessment of SDC25C binding affinity. The XTP ratio for the D119N mutant is similar to the GTP ratio for the wild type Ras. Thus, we conclude that the interaction with SDC25C is not significantly altered by the D119N mutation, a conclusion in agreement with the steady-state kinetics study.

D119N mutant is capable of hydrolyzing XTP. In fact, the intrinsic XTP hydrolysis rate of D119N is similar to the intrinsic GTP hydrolysis rate of wild type Ras ( Fig. 4and ). GAP greatly enhances the rate of GTP hydrolysis in the wild type Ras as well as that of XTP hydrolysis in the D119N mutant, and the ratios of GAP stimulation appear to be similar ( Fig. 3and ). These observations suggest that the mechanism of GTP hydrolysis is not affected by the D119N mutation. Nevertheless, we found that the GAP stimulation ratio was a few fold lower than that reported by others (35) . Furthermore, GAP-stimulated XTP (in D119N mutant) and GTP hydrolysis (in the wild type) reactions have similar apparent Kvalues, a result indicating that the interaction of Ha-Ras and GAP is not altered by the D119N mutation. As with SDC25C and SOS1, we found that GAP does not differentiate XTP from GTP.

The D119N mutation also does not perturb the ability of Ras to interact with Raf; however, the interaction is XTP-dependent in the D119N mutant while it is GTP-dependent in the wild type (Fig. 5). This observation, together with the finding of GAP interaction, suggests that nucleotide triphosphate-induced conformational changes are not affected by the D119N mutation. Similarly to SDC25C, SOS1, and GAP, Raf does not differentiate between XTP and GTP.

Overall, our results suggest that the primary role of the Asp-119 residue in Ha-Ras is to determine nucleotide binding specificity. They also indicate that Asp-119 is not directly involved in binding SDC25C, SOS1, GAP, or Raf. Asp-119 also is not involved in nucleotide triphosphate hydrolysis or nucleotide triphosphate-induced activities of Ha-Ras. Therefore, Ras(D119N) is predicated to behave like the wild type Ras if sufficient guanine-nucleotide or xanthine-nucleotide is provided. The reduction in the guanine-nucleotide affinity by D119N mutation is in the neighborhood of three orders of magnitude, a reduction that should be readily overcome by the intracellular guanine-nucleotide. This explains why Ha-Ras(D119N) still possesses transforming activity similar to that of the wild type (24) .

The xanthine-nucleotide binding mutant of EF-Tu, D138N, has been used to probe the involvement of EF-Tu in the elongation cycle of protein synthesis (21, 22) . The advantage of using such a mutant is that the action of EF-Tu in the peptide elongation cycle can be monitored in the presence of EF-G (the peptide translocation factor), a necessary factor for protein biosynthesis, which also utilizes and hydrolyzes GTP (36) . This approach led to the intriguing finding that not one but two molecules GTP are hydrolyzed by EF-Tu in each step of peptide elongation (37) . Since the homologous mutations in EF-Tu (20) , adenylosuccinate synthetase (23) , and Ha-Ras produce similar XTP binding properties, the conversion to XTP binding preference is likely to be a general phenomenon for regulatory GTPases. There are many cellular processes involving multiple regulatory GTPases. One example is the vesicular transport system (38) . We believe that mutation to the XTP-specific mutant will be an invaluable tool for elucidating the role of specific regulatory GTPases in these complex systems.

  
Table: The apparent GTP/XTP dissociation constant of GST-fusion Ras

The dissociation constants were determined by the equilibrium binding method in the presence of EDTA as described under ``Materials and Methods.''


  
Table: The apparent Kand kof SDC25C- and SOS1-stimulated nucleotide exchange

The parameters were determined for the bonding pairs only. The concentrations of exchange factor used were 20 nM SDC25C or 100 nM GST-SOS1. The concentration of NTP used in the assay was 5 µM.


  
Table: Nucleotide concentration for promoting RasSDC25C complex dissociation

All experiments were performed using 0.5 pmol of labeled intact Ras and 5 pmol of SDC25C. ND, not determined.


  
Table: Kinetic parameters of intrinsic and GAP-stimulated nucleotide hydrolysis

The concentration of GAP used in the GAP-stimulated assays was 0.2 nM.



FOOTNOTES

*
This work was supported in part by the New York State Office of Mental Retardation and Developmental Disabilities and by National Institutes of Health Grant CA53782 (to Y. W. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Molecular Biology Department, NYS Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY 10314. Tel.: 718-494-5337; Fax: 718-494-5287.

The abbreviations used are: GAP, GTPase activating protein; c-Ha-Ras, cellular Ha-Ras; D119N, Asp-119 to Asn mutation; D138N, Asp-138 to Asn mutation; EF-G, elongation factor G; EF-Tu, elongation factor Tu; GST, glutathione S-transferase.


ACKNOWLEDGEMENTS

We thank Drs. Frank McCormick and Gideon Bollag of Onyx Pharmaceuticals for providing Ras GAP, Andrea Parmeggiani for supplying SDC25C, Ulf R. Rapp for the human Raf clone, and David Bowtell for the mouse SOS1 DNA. We also thank Drs. Carl Dobkin and David Miller for critical reading of this manuscript.


REFERENCES
  1. Bourne, H. R., Sanders, D. A., and McCormick, F. (1990) Nature 348, 125-132 [CrossRef][Medline] [Order article via Infotrieve]
  2. Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127 [CrossRef][Medline] [Order article via Infotrieve]
  3. Boguski, M. S., and McCormick, F. (1993) Nature 366, 643-654 [CrossRef][Medline] [Order article via Infotrieve]
  4. Moodie, S. A., Willumsen, B. M., Weber, M. J., and Wolfman, A. (1993) Science 260, 1588-1590 [Medline] [Order article via Infotrieve]
  5. Koide, H., Satoh, T., and Kaziro, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8683-8686 [Abstract/Free Full Text]
  6. Van Aslet, L., Barr, M., Marcus, S., Polverino, A., and Wigler, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6213-6217 [Abstract]
  7. Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Cell 74, 205-214 [Medline] [Order article via Infotrieve]
  8. Warne, P. H., Viciana, P. R., and Downward, J. (1993) Nature 364, 352-355 [CrossRef][Medline] [Order article via Infotrieve]
  9. Zhang, X., Settleman, J., Kyriakis, J. M., Takeuchi-Suzuki, E., Elledge, S. J., Marshall, M. S., Bruder, J. T., Rapp, U. R., and Avruch, J. (1993) Nature 364, 308-313 [CrossRef][Medline] [Order article via Infotrieve]
  10. Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M. J., Waterfield, M. D., and Downward, J. (1994) Nature 370, 527-532 [CrossRef][Medline] [Order article via Infotrieve]
  11. Dever, T. E., Glynias, M. J., and Merrick, W. C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1814-1818 [Abstract]
  12. la Cour, T. F. M., Nyborg, J., Thirup, J., and Clark, B. F. C. (1985) EMBO J. 4, 2385-2388 [Abstract]
  13. Jurnak, F. (1985) Science 230, 32-36 [Medline] [Order article via Infotrieve]
  14. Pai, E. F., Kabach, W., Krengel, U., Holmes, K. C., John, J., and Wittinghofer, A (1989) Nature 341, 209-214 [CrossRef][Medline] [Order article via Infotrieve]
  15. Tong, L., De Vos, A. M., Milburn, M. V., and Kim, S.-H. (1991) J. Mol. Biol. 217, 503-516 [Medline] [Order article via Infotrieve]
  16. Berchtold, H., Reshetnikova, L., Reiser, C. O., Schirmer, N. K., Sprinzl, M., and Hilgenfeld, R. (1993) Nature 365, 126-132 [CrossRef][Medline] [Order article via Infotrieve]
  17. Kjeldgaard, M., Nissen, P., Thirup, S., and Nyborg, J. (1993) Structure 1, 35-50 [Medline] [Order article via Infotrieve]
  18. Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663 [CrossRef][Medline] [Order article via Infotrieve]
  19. Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 369, 621-628 [CrossRef][Medline] [Order article via Infotrieve]
  20. Hwang, Y. W., and Miller, D. L. (1987) J. Biol. Chem. 262, 13081-13085 [Abstract/Free Full Text]
  21. Weijland, A., and Parmeggiani, A. (1993) Science 259, 1311-1314 [Medline] [Order article via Infotrieve]
  22. Weijland, A., Parlato, G., and Parmeggiani, A. (1994) Biochemistry 33, 10711-10717 [Medline] [Order article via Infotrieve]
  23. Kang, C., Sun, N., Honzatko, R. B., and Fromm, H. J. (1994) J. Biol. Chem. 269, 24046-24049 [Abstract/Free Full Text]
  24. Feig, L. A., Pan, B.-T., Roberts, T. M., and Cooper, G. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4607-4611 [Abstract]
  25. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382 [Medline] [Order article via Infotrieve]
  26. Sung, Y. J., Carter, M., Zhong, J. M., and Hwang, Y. W. (1995) Biochemistry 34, 3470-3477 [Medline] [Order article via Infotrieve]
  27. Hwang, Y. W., Zhong, J. M., Poullet, P., and Parmeggiani, A. (1993) J. Biol. Chem. 268, 24692-24698 [Abstract/Free Full Text]
  28. Mistou, M. Y., Jacquet, E., Poullet, P., Rensland, H., Gideon, P., Schlichting, I., Wittinghofer, A., and Parmeggiani, A. (1992) EMBO J. 11, 2391-2397 [Abstract]
  29. Créchet, J.-B., Poullet, P., Mistou, M.-Y., Parmeggiani, A., Camonis, J., Boy-Marcotte, E., Damak, F., and Jacquet, M. (1990) Science 248, 866-868 [Medline] [Order article via Infotrieve]
  30. Glynn, I. M., and Chappell, J. B. (1964) Biochem. J. 90, 147-149 [Medline] [Order article via Infotrieve]
  31. Liu, B. X., Wei, W., and Broek, D. (1993) Oncogene 8, 3081-3084 [Medline] [Order article via Infotrieve]
  32. Manne, V., Yamazaki, S., and Kung, H.-F. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 6953-6957 [Abstract]
  33. Crystal, R. G., Elson, N. A., and Anderson, W. F. (1974) Methods Enzymol. 30, 101-127 [Medline] [Order article via Infotrieve]
  34. Sigal, I. S., Gibbs, J. B., D'Alonzo, J. S., Temeles, G. L., Wolanski, B. S., Socher, S. H., and Scolnick, E. M. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 952-956 [Abstract]
  35. Gideon, P., John, J., Frech, M., Lautwein, A., Clark, R., Scheffler, J. E., and Wittinghofer, A. (1992) Mol. Cell Biol. 12, 2050-2056 [Abstract]
  36. Kaziro, Y. (1978) Biochim. Biophys. Acta 505, 95-127 [Medline] [Order article via Infotrieve]
  37. Weijland, A., and Parmeggiani, A. (1994) Trends Biochem. Sci. 19, 188-193 [Medline] [Order article via Infotrieve]
  38. Nuoffer, C., and Balch, W. E. (1994) Annu. Rev. Biochem. 63, 949-990 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.