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J. Biol. Chem., Vol. 278, Issue 41, 39921-39930, October 10, 2003

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Identification of Dominant Negative Mutants of Rheb GTPase and Their Use to Implicate the Involvement of Human Rheb in the Activation of p70S6K^*

Angel P. Tabancay, Jr.  , Chia-Ling Gau  ¶, Iara M. P. Machado  ||, Erik J. Uhlmann **, David H. Gutmann **, Lea Guo  and Fuyuhiko Tamanoi  

From the Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California 90095-1489 and the ^**Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, June 20, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Rheb GTPases represent a unique family of the Ras superfamily of G-proteins. Studies on Rheb in Schizosaccharomyces pombe and Drosophila have shown that this small GTPase is essential and is involved in cell growth and cell cycle progression. The Drosophila studies also raised the possibility that Rheb is involved in the TOR/S6K signaling pathway. In this paper, we first report identification of dominant negative mutants of S. pombe Rheb (SpRheb). Screens of a randomly mutagenized SpRheb library yielded a mutant, SpRhebD60V, whose expression in S. pombe results in growth inhibition, G₁ arrest, and induction of fnx1⁺, a gene whose expression is induced by the disruption of Rheb. Alteration of the Asp-60 residue to all possible amino acids by site-directed mutagenesis led to the identification of two particularly strong dominant negative mutants, D60I and D60K. Characterization of these dominant negative mutant proteins revealed that D60V and D60I exhibit preferential binding of GDP, while D60K lost the ability to bind both GTP and GDP. A possible use of the dominant negative mutants in the study of mammalian Rheb was explored by introducing dominant negative mutations into human Rheb. We show that transient expression of the wild type Rheb1 or Rheb2 causes activation of p70S6K, while expression of Rheb1D60K mutant results in inhibition of basal level activity of p70S6K. In addition, Rheb1D60K and Rheb1D60V mutants blocked nutrient- or serum-induced activation of p70S6K. This provides critical evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Rheb defines a unique family within the Ras superfamily of G-proteins (1–3). Initially Rheb was found as a gene whose expression was induced in the rat brain by the treatment involved in the long term potentiation scheme (4), but later studies with human Rheb showed that it is ubiquitously expressed with a high level of expression observed in heart and skeletal muscle (5). Rheb homologues are also found in a number of organisms including mouse, fruit fly, zebra fish, slime mold, and at least three fungi, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Aspergillus fumigatus (1, 6–9). Two Rheb genes, Rheb1 and Rheb2, are present in human and mouse, while other organisms have only one Rheb (10). Rheb proteins share features unique to this family including the presence of arginine at the position corresponding to the 12th residue of Ras and the conservation of the effector domain sequence. In addition, all known Rheb proteins end with the CAAX motif (C is cysteine, A is an aliphatic amino acid, and X is the C-terminal amino acid usually a Cys, Ser, or Met residue) required for farnesylation. The yeast and mammalian Rheb proteins have been shown to be farnesylated (1, 11, 12).

Rheb is essential in S. pombe (6, 7). Inhibition of S. pombe Rheb (SpRheb)¹ expression leads to growth inhibition and accumulation of cells in the G₀/G₁ phase (6, 7). When Rheb expression is blocked, the cells become small and round, reminiscent of cells accumulated after nitrogen starvation (6, 7). In addition, mei2⁺ and fnx1⁺, two genes that are induced by nitrogen starvation, are induced when SpRheb expression is inhibited (6). SpRheb also affects nutrient uptake as Rheb is involved in the suppression of arginine uptake (11). This function is conserved in S. cerevisiae Rheb (1) and in A. fumigatus Rheb (9). In Drosophila, Rheb is essential as larvae that are homozygous for Rheb loss of function mutation die before molting into the second instar (10). Overexpression of Rheb in the developing fly causes dramatic overgrowth of multiple tissues (10). In Drosophila S2 cells, inhibition of Rheb expression by siRNA leads to small cells arrested at the G₁ phase, while overexpression of Rheb causes an increase in cell size and accumulation of S phase cells (10). These results suggest that Rheb regulates both cell cycle and cell growth. From experiments utilizing rapamycin (13), an inhibitor of TOR, we suggested that these effects of Rheb are mediated by TOR/S6K signaling (10). Involvement of Drosophila Rheb in the TOR/S6K signaling pathway has also been suggested by Saucedo et al. (14) and by Stocker et al. (15). In mammalian cells, Rheb is reported to interact with c-Raf and B-Raf (12, 16, 17). The interaction with B-Raf causes inhibition of B-Raf activity (16).

To further characterize Rheb function in a variety of organisms, we sought to identify dominant negative mutants of Rheb. This type of mutant has been valuable in examining the function of Ras superfamily GTPases including Ras (18), RhoA (19, 20), and Rac (21). A widely used dominant negative mutation for Ras superfamily GTPases is the one that corresponds to RasS17N (18, 22). However, introduction of an analogous mutation, S20N, into S. pombe and S. cerevisiae Rheb did not confer a dominant negative property (6).² In addition, expression of mammalian RhebS20N did not exhibit any phenotype in mammalian cells (12). Therefore, taking advantage of the yeast system we undertook a screen to identify dominant negative mutants of S. pombe Rheb. We first developed an assay to identify such mutants based on the findings that the inhibition of S. pombe Rheb induces expression of fnx1⁺, a gene that is activated during the entry into the G₀ phase of the cell cycle (6, 23). Using this assay, we screened a randomly mutagenized SpRheb expression library and found a mutant, D60V, that exhibits dominant negative properties. Changing the Asp-60 residue to all possible amino acids identified dominant negative mutants with increased potency. Biochemical characterization of these dominant negative mutant proteins revealed that they have altered guanine nucleotide binding properties. We further demonstrate that introduction of analogous mutations into human Rheb leads to inhibition of p70S6K activation, providing the first evidence that Rheb plays a role in the mTOR/S6K signaling pathway in mammalian cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Strains and Reagents—SP223 (h^– ura4 leu1–32 ade6) and JU101p1 have been described previously (7). JU101p1 was derived from SP223 and contains a disruption of the chromosomal rhb1⁺ gene. JU101p1 also carries the wild type rhb1⁺ gene under the control of nmt81 promoter (24). Growth of S. pombe cells in EMM media with appropriate nutrient supplement was carried out as described by Moreno et al. (25). All yeast transformations were carried out by lithium acetate method (26). X-gal and isopropyl--D-thiogalactopyranoside were purchased from Invitrogen. Thiamine was obtained from Sigma.

Construction of SpRheb Random Mutant Library—PCR-mediated generation of random mutant library was carried out essentially as described previously (27). Diversify^TM PCR random mutagenesis kit (Clontech) was used to generate a random mutant library of the rhb1⁺ gene. The template used for the PCR was pWHASpRheb (7). The 5' primer corresponded to the hemagglutinin (HA) sequence and contained a SalI site. The 3' primer corresponded to the sequence encompassing the stop codon of rhb1⁺ and a BamHI site. The condition for mutagenesis was optimized by changing Mn²⁺ and dGTP concentrations to give at least one base change per 1000 base pairs. The amplified fragments were cloned into pREP1 vector, which provides the nmt1⁺ promoter for the expression of mutant Rheb. The resulting plasmid was called pREP1HArhb1m. The wild type version of this plasmid, pREP1HASpRheb, was constructed by introducing a HA epitope-tagged rhb1⁺ gene into pREP1.

Screen for Dominant Negative Mutants of SpRheb—The SP223-127 strain used for the screen is SP223 carrying a plasmid containing lacZ under the control of fnx1⁺ promoter (pIM127). pIM127 was constructed as follows. S. pombe cosmid clone 21D10 (provided by The Sanger Center, Cambridge, UK) contains the entire fnx1⁺ gene. We amplified 1556 base pairs upstream of the start codon of this gene by PCR using primers that contained PstI and NdeI sites. This fragment containing the fnx1⁺ promoter was inserted into pREP2 (25) that had been digested with PstI and NdeI, thus replacing the nmt1⁺ promoter. The lacZ⁺ gene was PCR-amplified from the plasmid pBI-GL (Clontech) using primers containing SalI and BamHI sites. This product was inserted downstream of the fnx1⁺ promoter resulting in the production of pIM127. SP223-127 was transformed with the random mutant library of rhb1⁺, and transformants were grown on EMM plates containing adenine and 150 µM thiamine. Transformants were replica-plated on plates lacking thiamine but containing 0.1 mg/ml X-gal. Small blue colonies were picked, and the plasmids were recovered by a yeast miniprep kit (Qiagen) followed by transformation into Escherichia coli KC8 (hsdR leuB600 trpC9830 pyrF::Tn5 hisB463 lac 74 strA galU galK) using leucine selection. The recovered plasmids were sequenced to identify the mutation in rhb1⁺.

Construction of sprhb1⁺ D60X Mutants—Residue Asp-60 of SpRheb was converted to all possible amino acids by site-directed PCR mutagenesis. pREP1HArhb1⁺ was used as the template in the PCR. Primers containing NNN (N is any nucleotide) at the codon 60 of rhb1⁺ were used in a two-step PCR mutagenesis to generate the D60X mutant fragments. The resulting PCR fragment was digested with SalI and BamHI and inserted into pREP1 digested with the same enzymes. This resulted in pREP1HArhb1⁺D60X. Subsequent clones were sequenced and resulted in Asp-60 changed to 18 of the 20 possible amino acids. The last two amino acids, methionine and tryptophan, were constructed by PCR mutagenesis using primers encoding the exact codon changes to methionine and tryptophan. All pREP1HArhb1⁺D60X plasmids were transformed into SP223 to screen for dominant negative properties.

Characterization of S. pombe Cells—SP223 transformed with pREP1HArhb1⁺D60X constructs were grown overnight in EMM + Ade. Cells were diluted to A₆₀₀ of 0.1 in EMM + Ade, and growth was followed over time. For spotting assays, cells from the same overnight cultures were diluted to A₆₀₀ of 1.0 and serially diluted 4-fold. 5 µlofthe diluted samples were spotted onto EMM + Ade and EMM + Ade + thiamine plates and allowed to grow 3 days at 30 °C. The ability of mutant SpRheb to complement the loss of SpRheb expression was examined in the following manner. First, the mutant rhb1 gene recovered from the screen was used to replace the wild type rhb1⁺ gene in the plasmid pRPUmycSpRhebpt described previously (11). The resulting plasmid contained the mutant rhb1 gene under the control of the rhb1⁺ promoter. The vector, pREP2myc, was made by introducing a Myc tag downstream of the NdeI site of pREP2. These plasmids were transformed into JU109p1, and the transformants were grown overnight in the absence of thiamine. The overnight culture was diluted to A₆₀₀ of 1, serially diluted 4-fold, and spotted onto a plate containing 150 µM thiamine. The plate was incubated for 72 h at 30 °C. Flow cytometric analysis of S. pombe cells was carried out as described by Yang et al. (11). In brief, cells were collected at 24 h and then fixed in 70% ethanol. After RNase digestion, propidium iodide was added, and cells were sonicated briefly. Cells were processed immediately on a BD Biosciences FACScan.

Purification of SpRheb Protein—SpRheb proteins were purified as His-tagged proteins. In brief, wild type and mutant forms (SpRhebD60V, -D60K, and -D60I) of the rhb1⁺ gene were cloned into the vector pET28a (Novagen) by PCR amplification followed by ligation into pET28a digested with NheI and BamHI. These constructs were transformed into E. coli BL21(DE3). SpRheb was induced by the addition of 1 mM isopropyl--D-thiogalactopyranoside for 4 h at 37 °C. Cells were harvested by centrifugation, suspended in buffer, and sonicated. Cell lysates were centrifuged, and the resulting supernatant was incubated with ProBond^TM nickel-chelating resin (Invitrogen). After binding at 4 °C, bound beads were washed several times followed by elution of the bound protein with buffer containing 350 mM imidazole. The resulting elution fractions were concentrated by Centricon columns (Amicon Bioseparations). The purity was checked by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Brilliant Blue, which revealed a single band of 22 kDa. Protein concentration of purified fractions was determined by Bradford protein assay (28) (Bio-Rad).

Biochemical Activities of SpRheb—The guanine nucleotide binding assay was carried out as described previously (29). In brief, 1.5 µg of His-SpRheb as purified above was incubated with 0.2 µCi of [³⁵S]GTPS (1250 Ci/mmol, American Radiolabeled Chemicals) in binding buffer containing 20 mM Tris, pH 7.4, 50 mM NaCl, 0.1% Triton X-100, 1 mM dithiothreitol, 40 µg/ml bovine serum albumin, 1 mM EDTA, 2 µM GTP, and 1 mM MgCl₂ at 30 °C, and the binding was examined by filtering through 0.45 µm nitrocellulose filters (Schleicher & Schuell). Nucleotide specificity was assessed by performing the guanine nucleotide binding assay as described above with the addition of excess unlabeled competing nucleotides (GDP, GTP, CTP, ATP, or UTP) in the binding buffer. The GTPase assay was carried out as described previously (29, 30). In brief, 1.5 µg of SpRheb was incubated at 37 °C for 10 min in the binding buffer as described above containing [-³²P]GTP (6000 Ci/mmol, American Radiolabeled Chemicals) and either 4 mM UTP or 2 mM UTP plus 2 mM GTP. After the binding, GTPase assay was initiated by the addition of 20 mM MgCl₂ followed by incubation at 37 °C. 2-µl aliquots were taken at various time points and spotted onto polyethyleneimine cellulose thin layer chromatography (TLC) plates (Selecto Scientific). TLC plates were developed in 1 M LiCl, 1 M formic acid. The amount of [-³²P]GDP and [-³²P]GTP was determined by PhosphorImager analysis (Model 445 SI, Amersham Biosciences). The migration of unlabeled GDP and GTP standards was detected by UV light (254 nm). GDP dissociation was examined by prebinding [³H]GDP (11.5 Ci/mmol, Amersham Biosciences) for 10 min at 30 °C in the buffer used for guanine nucleotide binding and then adding excess unlabeled GTP after adjusting the Mg²⁺ concentration to 10 mM, and the amount of radioactivity remaining bound was determined by nitrocellulose filter assay.

Mammalian Cell Culture and Transfection—HEK293 and COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and penicillin/streptomycin. Cells were transfected using Polyfect (Qiagen) according to the manufacturer's instructions, and cells were collected 36 h after transfection. In brief, the indicated DNA was incubated in serum-free media with Polyfect reagent for 10 min, diluted in serum-free media, and added to cells with fresh media. COS-7 cells were starved for nutrients by incubating in Dulbecco's modified phosphate-buffered saline for 30 min. Cells were stimulated with nutrients by changing to Dulbecco's modified Eagle's medium alone for 30 min or with growth factors by adding 20% fetal bovine serum for 15 min. The mTOR inhibitor, rapamycin, was used to treat cells at 20 nM for 1 h.

Western Blot Analysis—Total cell lysates were prepared as follows. Cells were collected from the plate in lysis buffer: 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 1% Triton X-100, 10 mM MgCl₂, 1 mM Na₃VO₄, 100 mM NaF, 20 mM -glycerophosphate, 10 mM NaPP_i, and 1x Protease Inhibitor Mixture (Roche Applied Science). Extracts were centrifuged at 13,000 x g for 10 min. Equal amounts of protein were then loaded onto a 12% polyacrylamide gel and transferred to polyvinylidene difluoride membrane. For detection of phosphorylated p70S6K, membranes were immunoblotted with anti-phospho-p70S6K (Thr-389) antibody (Cell Signaling). For detection of total p70S6K protein, membranes were stripped and immunoblotted with anti-p70S6K antibody (Santa Cruz Biotechnology). For detection of HA-SpRheb or HA-HsRheb, membranes were immunoblotted with anti-HA (12CA5) (Roche Applied Science).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Screen for Dominant Negative Mutants of S. pombe Rheb— Rheb has recently emerged as an important class of the Ras superfamily G-proteins that is critical for cell growth and cell cycle progression possibly through its involvement in the TOR/S6K signaling pathway (6, 7, 10, 14, 15). However, studies on Rheb have been hindered by the lack of dominant negative mutants. We first introduced S20N, a mutation analogous to the widely used S17N dominant negative mutation of Ras, into SpRheb. SpRhebS20N was expressed in the wild type strain, SP223, and growth of the cells was examined. No significant difference was observed compared with the growth of cells overexpressing the wild type protein (data not shown). This is in agreement with the results reported by Mach et al. (6). Therefore, we decided to randomly mutagenize SpRheb and identify dominant negative mutants. The overall scheme for the assay we devised is shown in Fig. 1A. This assay seeks to identify SpRheb mutants whose expression leads to fnx1⁺ induction since the inhibition of SpRheb expression causes the induction of this gene (6). A strain, SP223-127, carrying lacZ under the control of the fnx1⁺ promoter was constructed. This strain was transformed with a random PCR-generated library of S. pombe rhb1⁺ mutants under the control of the thiamine-repressible nmt1⁺ promoter. Transformants were first grown on a plate containing thiamine. The presence of thiamine represses the expression of mutant Rheb enabling all the transformants to grow. Transformants were replica-plated onto a plate containing X-gal but lacking thiamine. The absence of thiamine induces expression of the mutant SpRheb. The expression of a dominant negative SpRheb would cause growth arrest and induction of the fnx1-lacZ reporter construct. Therefore, colonies expressing a dominant negative SpRheb are expected to be smaller and blue. These colonies were picked, and the pREP1HASpRhebm plasmid was recovered. After retransforming the recovered plasmid into SP223-127 to confirm the induction of fnx1⁺, the plasmid DNA was sequenced to identify the mutations.

View larger version (30K): [in this window] [in a new window]   FIG. 1. A, overall scheme to screen for dominant negative mutants of SpRheb. A randomly mutagenized rhb1+ library was transformed into SP223-127, a wild type S. pombe strain expressing lacZ under the control of the fnx1+ promoter. Transformants were plated on EMM + Ade (A) + thiamine and then replica-plated onto EMM + Ade + X-gal to screen for growth arrest and -galactosidase expression. B, demonstration of lacZ expression by the dominant negative mutant. SP223-127 cells carrying mutant 22 plasmid or vector were serially diluted 4-fold and spotted on a plate containing X-gal. Cells expressing mutant 22 turned blue and grew less than the vector control. C, growth of SP223-127 cells expressing wild type SpRheb () or mutant 22 () were grown overnight in media containing thiamine. The culture was diluted into fresh media lacking thiamine to A600 of 0.05, and the growth was followed. D, mutant 22 and the wild type cells grown in C were collected at 24 h after the start of growth and subjected to flow cytometry as described under "Experimental Procedures." E, Western blot of SP223-127 cells carrying the vector, the wild type Rheb plasmid, or mutant 22. Expression of SpRheb was examined using anti-HA antibody. WT, wild type.

Two independent screens covering 25,000 transformants were carried out. This led to the identification of a mutant (mutant 22) that carries two amino acid changes, D60V and I98M. Expression of mutant 22 resulted in growth inhibition as well as the development of blue color, indicating the induction of the fnx1-lacZ reporter (Fig. 1B). Fig. 1C compares the growth of cells expressing mutant 22 to that of cells expressing wild type SpRheb. As can be seen, expression of mutant 22 significantly affected growth; the cells expressing mutant 22 grew only to approximately A₆₀₀ of 1, while the cells expressing wild type SpRheb grew to more than A₆₀₀ of 7. Analysis of the cell cycle profiles of SP223-127 cells expressing mutant 22 or wild type SpRheb by flow cytometry revealed significant enrichment of cells in the G₀/G₁ phase of the cell cycle (1N) with mutant 22 (Fig. 1D). Mutant 22 was expressed at a level slightly less than that of the wild type (Fig. 1E).

D60V Mutation Is Responsible for the Dominant Negative Property—As mentioned above, mutant 22 possesses two different amino acid changes, D60V and I98M. The D60V mutation occurs on a residue that is conserved in all known Rheb and Ras proteins (Fig. 2A). This residue is part of the G3 box, one of the motifs conserved in the Ras superfamily G-proteins (31). The I98M mutation occurs on a residue that is also conserved as either isoleucine or valine is found at this residue (Fig. 2A). To investigate which amino acid change is responsible for the dominant negative property, we separated the mutations and constructed two single mutants, one containing the D60V mutation and the other having the I98M mutation. Each mutant was examined for the ability to cause growth inhibition and cell cycle changes. Fig. 2B shows that the expression of D60V causes growth inhibition of SP223-127 cells, while the expression of I98M has only a minor effect. In addition, D60V expression led to significant enrichment of G₀/G₁ phase cells, while the cell cycle profile of cells expressing I98M was similar to that of the cells expressing the wild type SpRheb (Fig. 2C). Therefore, the D60V mutation is responsible for the dominant negative phenotypes of the double mutant.

View larger version (31K): [in this window] [in a new window]   FIG. 2. D60V mutation is responsible for the dominant negative effects. A, alignment of Rheb sequences from a variety of organisms. Sequences of Rheb proteins from S. pombe (SpRheb), human (HsRheb1), S. cerevisiae (ScRheb), and Drosophila melanogaster (DmRheb) are shown. G1–G5 boxes involved in the recognition of guanine nucleotides are indicated. The conserved Arg-15 residue is shown in bold type. Asp-60 and Ile-98 are indicated by arrows and bold type. B, left panel, growth of SP223-127 cells expressing SpRhebD60V mutant () or the wild type SpRheb () protein in the absence of thiamine. Right panel, growth of SP223-127 expressing SpRhebI98M () or SpRheb () in media lacking thiamine. Cultures were first grown overnight in media containing thiamine. The cultures were then diluted into fresh media lacking thiamine to A600 of 0.05, and the growth was followed. C, cell cycle profile of SP223-127 expressing the wild type SpRheb, SpRhebD60V, or SpRhebI98M. Cells were collected at 24 h after diluting into media without thiamine and analyzed by flow cytometry as described under "Experimental Procedures." WT, wild type.

Mutating Asp-60 to All 20 Possible Amino Acids Identifies Strong Dominant Negative Mutants—It is probable that stronger dominant negative mutants were not uncovered in our initial screens because they were not represented in the rhb1 mutant library. Therefore, to obtain more potent dominant negative mutants, we changed the aspartic acid 60 to all possible amino acids. This was carried out by PCR mutagenesis replacing codon 60 with NNN (N = A, C, G, or T) (see "Experimental Procedures"). For the change to methionine or tryptophan, we needed to carry out separate mutagenesis using primers having codons corresponding to these amino acids. D60X mutants were transformed individually into SP223-127 cells, and the transformants were grown in the absence of thiamine to express the mutant proteins. The doubling times of SP223-127 expressing the mutant proteins were compared against that of the wild type protein. As can be seen in Fig. 3A, we found that the expression of D60I or D60K mutant leads to a strong growth inhibition, roughly a 3-fold increase in doubling time. Because the growth stops with the expression of D60K or D60I mutant, the difference with D60V is even more pronounced when we compare their absorbances 48 h after induction (data not shown). We also found that growth inhibition similar to that seen with the expression of the D60V mutant is observed with a number of mutants such as D60R, D60L, D60P, and D60T. On the other hand, some mutants such as D60F, D60Y, and D60W did not exhibit any growth inhibition. We have confirmed expression of all these dominant negative mutants by Western blot analysis (Fig. 3B). It is important to point out that the D60K and D60I mutants are expressed at a level lower than that observed with the wild type or the D60V mutant, yet they exhibit strong dominant negative phenotypes.

View larger version (33K): [in this window] [in a new window]   FIG. 3. Changing Asp-60 residue to all possible amino acids. A, doubling times of D60X mutants in comparison to wild type. SP223 transformed with pREP1HASpRhebD60X encoding all possible amino acids were grown overnight in EMM + Ade. Cultures were diluted to A600 of 0.05 in EMM + Ade, and growth was monitored. Measurements of A600 were taken at the specified time points. The doubling time was calculated between the 24- and 48-h time points when cultures are in logarithmic phase. The ratio of the mutant doubling time to that of wild type was taken. B, expression of D60X mutants. Extracts of SP223 expressing various D60X mutants were prepared and separated by SDS-PAGE. Western blot analysis was performed using anti-HA antibody. C, inhibition of cell growth by the expression of D60V, D60I, or D60K mutant in wild type S. pombe cells. SP223 transformed with plasmids carrying SpRhebD60V, -D60I, or -D60K were grown in EMM + Ade overnight and diluted to A600 of 1.0. A 4-fold serial dilution was performed and spotted onto EMM + Ade plates. Plates were incubated at 30 °C for 3 days. D, Asp-60 mutants arrest in G1 phase of the cell cycle. FACS analysis was performed on SP223 expressing D60V, D60I, and D60K mutants. After 24 h growth in EMM + Ade, cells were taken and analyzed by FACS. FACS was performed according to the "Experimental Procedures." WT, wild type.

Mutants D60K, D60I, and D60V were further characterized. Fig. 3C shows growth inhibition by spotting cells on a plate without thiamine. Expression of the D60V mutant led to significant growth inhibition. Growth of cells expressing D60K was almost completely blocked. Fig. 3D shows FACS analyses of cells expressing these dominant negative mutants. As can be seen, expression of D60V leads to significant accumulation of G₀/G₁ phase cells. Expression of D60K or D60I causes more dramatic accumulation of G₀/G₁ phase cells. Thus, the expression of D60I or D60K causes strong inhibition accompanied by G₁ arrest.

Dominant Negative Rheb Proteins Exhibit Altered Guanine Nucleotide Binding Properties—To gain insight into the potential mechanism of the dominant negative effect, we decided to examine biochemical activities of the mutant proteins. We first established basic biochemical activities of the wild type Rheb protein. S. pombe Rheb protein was His-tagged and purified using a nickel column as described under "Experimental Procedures." Fig. 4A shows that SpRheb binds GTP as examined using [³⁵S]GTPS. The binding was stimulated by the addition of Mg²⁺ as the addition of 0.1–1 mM Mg²⁺ gave GTP binding severalfold higher than that seen without Mg²⁺ (data not shown). The ratio between SpRheb and GTP was calculated to be 1–0.7, and the binding was specific to guanine nucleotides as demonstrated by the addition of excess unlabeled nucleotides (Fig. 4B). Although both GTP and GDP compete with the binding of [³⁵S]GTPS, GTP had a greater effect in inhibiting the binding of [³⁵S]GTPS. Fig. 4C demonstrates that Rheb has an intrinsic GTPase activity. For this experiment, SpRheb was first bound to [-³²P]GTP, and then Mg²⁺ concentration was adjusted to 20 mM, and the incubation was continued. Appearance of radioactivity at a spot corresponding to GDP on polyethyleneimine cellulose in a time-dependent manner was observed. In this experiment, excess UTP was used to inhibit any nonspecific phosphatases. To confirm that the GDP is generated by hydrolysis of bound GTP, excess GTP was included in the reaction mixture. The excess GTP would block binding of [-³²P]GTP to Rheb. In this case, the appearance of GDP was no longer observed (Fig. 4C). GTPase activity was also demonstrated using [-³²P]GTP bound to SpRheb and examining for the hydrolysis of -phosphate by using a nitrocellulose filter assay (data not shown). Finally, dissociation of the bound nucleotides was demonstrated as shown in Fig. 4D. In this experiment, we first bound [³H]GDP by incubating SpRheb in the presence of 1 mM Mg²⁺. Then excess GTP was added after adjusting the Mg²⁺ concentration to 10 mM. As can be seen, without the addition of excess GTP, the bound GDP stays bound to SpRheb and is not dissociated. On the other hand, release of [³H]GDP was observed when an excess amount of unlabeled GTP was added to SpRheb.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Biochemical activities of the wild type SpRheb protein. A, GTP binding activity of SpRheb. His-tagged SpRheb protein () as well as boiled protein () were incubated with [35S]GTPS, and the binding was assayed using nitrocellulose filter as described under "Experimental Procedures." B, the specificity of guanine nucleotide binding of SpRheb. SpRheb protein was incubated with [35S]GTPS in the presence of 20-fold excess GTP, GDP, CTP, UTP, or ATP, and the bound radioactivity was examined by nitrocellulose filter assay. The amount of radionucleotide bound in the absence of unlabeled nucleotide was set as 100% (Control). All values are representative of experiments repeated at least three times. C, intrinsic GTPase activity of SpRheb. SpRheb protein was incubated with [-32P]GTP in the presence of either 4 mM unlabeled UTP or 2 mM unlabeled UTP plus 2 mM unlabeled GTP, and the reaction mixture was spotted on polyethyleneimine cellulose as described under "Experimental Procedures." D, GDP dissociation of SpRheb. SpRheb protein was first incubated with [3H]GDP in the presence of 1 mM Mg2+ for 10 min at 30 °C. Mg2+ concentration was adjusted to 10 mM in the presence () or absence () of 20-fold excess unlabeled GTP. [3H]GDP radioactivity remaining bound to SpRheb was examined by nitrocellulose filter assay. All values are representative of experiments repeated at least three times. Nuc, nucleotide.

To compare biochemical activities of the dominant negative mutants with those of the wild type, D60K, D60I, and D60V proteins were purified as His-tagged proteins. The purity of these His-tagged proteins is shown in Fig. 5A. First we realized that the dominant negative mutants exhibit significantly decreased GTP binding compared with the wild type protein. As shown in Fig. 5B, binding of GTP to the dominant negative mutant proteins was less than 7% of that seen with the wild type protein. Similar results were obtained with [³⁵S]GTPS (data not shown). Among the three dominant negative mutants, the level of GTP binding was the lowest with D60K, while D60V showed the highest level of binding. We next examined binding of GDP. As shown in Fig. 5B, GDP binding to D60V and D60I was comparable to that observed with the wild type protein. On the other hand, little binding of GDP was detected with the D60K mutant. The dominant negative mutants appear to retain specific binding of guanine nucleotides as GDP and GTP competed with the binding of radiolabeled GDP to the D60V mutant, while ATP, CTP, or UTP did not (data not shown). These results suggest that the D60K mutant has lost the binding of both GTP and GDP, while the D60V and D60I mutants lost the binding of GTP only. The preferential binding of GDP to the D60V and D60I mutants is further supported by the result of the GDP dissociation experiment shown in Fig. 5C. In this experiment, [³H]GDP was bound to the proteins and then challenged with 10-fold excess GTP. While GDP bound to the wild type Rheb dissociated under this condition, GDP bound to the D60V and D60I mutants stayed bound to the protein. When 20-fold excess GTP was used, GDP dissociated more readily from the D60V mutant than from the D60I mutant as shown in Fig. 5C. These results suggest that, while both D60V and D60I mutant proteins exist preferentially as a GDP-bound form, D60I mutant is more likely to remain in a GDP-bound form.

View larger version (21K): [in this window] [in a new window]   FIG. 5. Guanine nucleotide binding and GDP dissociation activities of the dominant negative SpRheb mutants. A, 2 µg of each His-tagged protein was separated on an SDS gel and visualized by staining with Coomassie Brilliant Blue. B, left panel, GTP binding of dominant negative mutants and the wild type protein was examined by incubating 1.5 µg of each protein with [-32P]GTP for 10 min at 30 °C. Right panel, GDP binding was examined by incubating 1.5 µg of each protein with [3H]GDP for 10 min at 30 °C. C, dissociation of [3H]GDP from wild type () and dominant negative mutants D60V () and D60I (). Proteins (1.5 µg) were incubated with [3H]GDP for 10 min at 30 °C. Dissociation was initiated by adding 10-fold (left panel) and 20-fold (right panel) excess non-radiolabeled GTP and 10 mM MgCl2. Time points were taken, and the percentage of remaining bound [3H]GDP was determined by scintillation counting. All values are representative of experiments repeated at least three times. WT, wild type.

Use of Dominant Negative Human Rheb Implicates Rheb in the Activation of p70S6K—To begin to explore the use of dominant negative Rheb, we focused on the recent development suggesting that Rheb is involved in the insulin/TOR/S6K pathway (10, 14, 15). We have found that Drosophila Rheb regulates both cell growth and cell cycle (10). These effects appear to be mediated by the TOR signaling pathway as we find that mutant flies with heterozygous loss of function are hypersensitive to rapamycin (13), an inhibitor of TOR, and effects of overexpression of Rheb in a Drosophila tissue culture cell line are blocked by the treatment with rapamycin (10). Saucedo et al. (14) have shown that the inhibition of Rheb expression in Drosophila tissue culture cell line by the addition of siRNA inhibits the activation of p70S6K, and Stocker et al. (15) detected activation of p70S6K in flies that overexpress Rheb. We sought to extend these observations to mammalian cells.

We first established that transient expression of Rheb leads to the activation of p70S6K in mammalian cells. As described previously (10), there are two Rheb genes, Rheb1 and Rheb2,in mammalian cells. Both these genes are expressed in a variety of mammalian cells as detected by reverse transcription-PCR, and Northern analysis showed that most tissues express both Rheb1 and Rheb2, although Rheb2 appears to exhibit a more limited expression profile.³ Fig. 6A shows that transient expression of human Rheb1 or Rheb2 in human embryonic kidney HEK293 cells results in the activation of p70S6K as detected by the use of an antibody specific for Thr(P)-389 S6K. The total amount of S6K is unchanged. Since we used an antibody specific for the phosphorylation dependent on mTOR, we believe that mTOR is involved in the Rheb-induced activation of p70S6K. This idea was confirmed by the use of rapamycin. Stimulation of S6K by Rheb1 or Rheb2 as well as the basal level S6K activity was inhibited by the addition of rapamycin. Stimulation of S6K phosphorylation by the expression of Rheb1 was also detected using other cell lines including COS-7, HCT116, and mouse embryonic fibroblasts (data not shown).

View larger version (32K): [in this window] [in a new window]   FIG. 6. Rheb enhances basal level of S6K phosphorylation, and dominant negative Rheb can inhibit stimulated levels of phosphorylation. A, Rheb1 and Rheb2 can enhance basal levels of S6K phosphorylation in HEK293 cells. HEK293 cells were transiently transfected with Myc-rat S6K and the indicated plasmid. S6K phosphorylation and expression levels were detected by immunoblotting with the indicated antibodies. The enhancement of S6K phosphorylation is blocked by treatment with the mTOR inhibitor, rapamycin. 24 h after transfection, cells were treated with either Me2SO or 20 nM rapamycin (Rapa) for 1 h before lysing cells. B, dominant negative mutants can inhibit the basal level of S6K phosphorylation in HEK293 cells. HEK293 cells were transfected with Myc-rat S6K and the indicated Rheb1 constructs. Lysates were collected 48 h after transfection, and the level of S6K phosphorylation was examined. C, dominant negative mutants of Rheb1 can inhibit nutrient- and serum-stimulated S6K phosphorylation. COS-7 cells were transfected with Myc-rat S6K and the indicated dominant negative constructs. The cells were starved for 30 min in Dulbecco's phosphate-buffered saline 36 h after transfection. The cells were then stimulated with nutrients (Dulbecco's modified Eagle's medium without serum) for 30 min or with 20% fetal bovine serum for 15 min, lysates were collected, and the level of S6K phosphorylation was examined. p-S6K, phosphorylated S6K; IB, immunoblot.

To examine whether dominant negative human Rheb is capable of inhibiting p70S6K activation, D60K and D60V mutations were introduced into human Rheb1, and their effects on S6K activity was examined. Fig. 6B shows that transient transfection of the dominant negative mutant D60K leads to significant inhibition of p70S6K phosphorylation in HEK293 cells. This is in contrast to the wild type Rheb1 protein that exhibits stimulation of p70S6K. Transient transfection of D60V caused a slight decrease of p70S6K, but this effect was much weaker than that of D60K. Western analysis confirmed the expression of D60V and D60K mutants (data not shown). Expression of D60K, on the other hand, did not affect Akt activity as examined by the use of antibody specific to phospho-Akt (data not shown). These results show that the dominant negative Rheb mutants have the ability to suppress basal level S6K activity.

We also examined whether the dominant negative Rheb1 mutants inhibit S6K activity induced by the addition of amino acids. Monkey kidney COS-7 cells were starved for amino acids by incubating in phosphate-buffered saline. Normal media were added, and the activation of S6K was examined as before. As can be seen in Fig. 6C, S6K activity was induced significantly by the addition of nutrients. However, expression of dominant negative D60K mutant abolished this increase. A similar inhibition of the induction of S6K was observed with D60V mutant. We also assessed serum induction of S6K activity. Again D60K and D60V mutants were capable of inhibiting this serum-induced S6K activity. These results point to significant roles Rheb plays in the mTOR/S6K signaling pathway in mammalian cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have succeeded in obtaining dominant negative Rheb mutants by random mutagenesis of S. pombe rhb1⁺ gene. Expression of the mutants causes growth inhibition, arrest of the cell cycle at the G₁/S boundary, and induction of fnx1-lacZ expression. These properties closely resemble those that are observed when the expression of Rheb is inhibited in S. pombe (6, 7). Our screens first identified aspartic acid 60 changed to valine, which resulted in dominant negative properties. Alteration of Asp-60 to all possible amino acids yielded dominant negative mutants with enhanced potency, D60I and D60K. These, as well as other mutants, with different levels of dominant negative effects should provide valuable tools for the study of Rheb protein in higher organisms.

Biochemical characterization of these dominant negative mutant proteins revealed altered guanine nucleotide binding properties of the mutants compared with the wild type protein. The D60K mutant exhibits a loss of binding of both GTP and GDP likely causing the protein to exist as a nucleotide-free form. The D60I mutant exhibits dramatically decreased binding of GTP, while the binding of GDP remains similar to that of the wild type protein. The net result would be the preferential binding of GDP to this mutant protein. In fact, GDP, once bound to the mutant protein, appears to stay bound even when challenged with excess GTP. The D60V mutant also exhibits decreased binding of GTP; however, the decrease of GTP binding is less pronounced compared with the D60I mutant. In addition, dissociation assays have suggested that the D60V mutant releases bound GDP more readily than the D60I mutant in the presence of excess GTP. These differences may explain why the D60I mutant is more potent than the D60V mutant in its dominant negative property. These results suggest that the Asp-60 residue of Rheb is critical for the recognition of GTP and GDP. The analogous residue in Ras, Asp-57, binds a catalytic Mg²⁺ through an intervening water molecule and is critical for both GDP and GTP binding (31, 32). Presumably changing the Asp-60 residue to valine or isoleucine results in the loss of recognition of GTP but not GDP. On the other hand, having lysine at this position interferes with the recognition of both GTP and GDP.

Dominant negative mutants of the Ras superfamily G-proteins generally act by sequestering their GDP/GTP exchange factors (18). This is due to an increased interaction of GDP-bound or nucleotide-free protein with its guanine nucleotide exchange factor. The nucleotide-free protein has a particularly strong interaction with guanine nucleotide exchange factor as this form resembles an intermediate in the guanine nucleotide exchange factor reaction (33, 34). Our results showing that the dominant negative Rheb mutants exist in either a GDP-bound form or a nucleotide-free form support the idea that the mechanism of action of these mutants involves sequestering a GDP/GTP exchange factor for Rheb. This idea is also in line with our observation that the nucleotide-free D60K mutant is more potent than the D60V mutant, which is GDP-bound. Further experiments to identify exchange factors for Rheb may provide deeper insight into the mechanism of dominant negative properties of these mutants. It is also important to point out that our mutants may provide valuable reagents to identify Rheb exchange factors in assays such as the yeast two-hybrid assay.

The ability to use a dominant negative mutant may be particularly important to elucidate the involvement of Rheb in signal transduction pathways in a variety of systems as dominant negative mutants have been successful in elucidating the signaling pathways of the Ras superfamily G-proteins (18). To explore this idea, we introduced the D60K or D60V mutation into human Rheb and examined the ability of these mutants to influence the mTOR/S6K signaling pathway in mammalian cells. This was possible since the Asp-60 residue we identified is highly conserved in all Rheb proteins (1). We provide the first evidence that both human Rheb1 and Rheb2 are capable of activating p70S6K and that the Rheb-induced activation of p70S6K is inhibited by rapamycin, an inhibitor of mTOR. Dominant negative Rheb1 proteins, RhebD60K or RhebD60V, are capable of inhibiting basal as well as induced level of p70S6K activity, while Rheb appears not to affect the level of Akt activation as determined by the use of an antibody against phosphorylated Akt (data not shown). Akt is upstream of mTOR in the insulin signaling pathway (35). These results suggest that Rheb functions upstream of mTOR but downstream of Akt. Epistasis analysis of Drosophila Rheb in the insulin/dTOR/S6K signaling pathway suggested that Drosophila Rheb functions upstream of dTOR but downstream of TSC1/2, a negative regulator of mTOR (14). Interestingly mutations in TSC1 or TSC2 genes are found in patients with tuberous sclerosis, a genetic disease associated with the development of benign tumors (hamartomas) (36). We are currently using other inhibitors of the mTOR signaling pathway to further investigate the mechanism of p70S6K activation.

Our finding that Rheb is involved in the mTOR/S6K signaling pathway is significant as this pathway is an important regulator of cell growth in mammalian cells and is up-regulated in a number of human cancers (37). Rheb may be an important contributor for cancers that exhibit an activated mTOR/S6K signaling pathway. It has been reported that the expression of Rheb is up-regulated in transformed cells (5). Further analysis to examine expression and activation of Rheb as well as Rheb-induced S6K activation in a variety of cancer cells may be revealing. Another issue concerning mammalian Rheb is its possible involvement in the Ras/Raf/mitogen-activated protein kinase signaling pathway. It has been reported that Rheb interacts with c-Raf and B-Raf (12, 16, 17), and the interaction with B-Raf causes inhibition of B-Raf activity (12, 17). Rheb expression has been shown to antagonize transformation of NIH3T3 cells by activated Ras (12). Our finding that human Rheb is involved in the activation of mTOR/S6K signaling may suggest that Rheb is capable of affecting both the mTOR/S6K and the Ras/Raf signaling pathways in mammalian cells. Further experiments using our dominant negative mutants as well as siRNA for Rheb may provide insight into these questions.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants CA41996 and CA32737 (to F. T.), a Tuberous Sclerosis Alliance grant (to D. H. G.), and National Institutes of Health Grant F32-CA94665 (to E. J. U). Flow cytometry performed in the UCLA Flow Cytometry Core Facility was supported by National Institutes of Health Grants CA16042 and AI28697. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by the Eugene Cota Robles Fellowship.

^¶ Supported by the Pauley Fellowship.

^|| Supported by CNPq (Brasilia, DF, Brazil) and Universidade Federal do Parana (Curitiba, PR, Brazil). Present address: Departamento de Farmacia, Universidade Federal do Parana, Curitiba, Parana, Brazil 80210-170.

To whom correspondence should be addressed: Dept. of Microbiology, Immunology & Molecular Genetics, 1602 Molecular Sciences Bldg., UCLA, Los Angeles, CA 90095-1489. Tel.: 310-206-7318; Fax: 310-206-5231; E-mail: fuyut{at}microbio.ucla.edu.

¹ The abbreviations used are: SpRheb, Schizosaccharomyces pombe Rheb; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; HA, hemagglutinin; TLC, thin layer chromatography; Ade, adenine; GTPS, guanosine 5'-3-O-(thio)triphosphate; FACS, fluorescence-activated cell sorter; TOR, target of rapamycin; S6K, S6 Kinase; siRNA, small interfering RNA; EMM, Edinburgh minimal media; TSC, tuberous sclerosis complex.

² A. P. Tabancay, Jr. and F. Tamanoi, unpublished observation.

³ C.-L. Gau and F. Tamanoi, unpublished observation.

	ACKNOWLEDGMENTS

 
We thank Dr. Jun Urano, Dr. Nitika Thapar, Dr. Juran Kato-Stankiewicz, and Chen Jiang for discussion and critical reading of our manuscript. We also thank Dr. Junji Yamauchi for the p70S6K construct.

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