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¶

¶ Dept. of Microbiology, Immunology and Molecular Genetics, Jonsson Comprehensive

Cancer Center, Molecular Biology Institute, University of California, Los Angeles,

Los Angeles, CA 90095-1489

* Dept. of Neurology, Washington University School of Medicine, St. Louis, MO 63110

Send correspondence to:

Fuyuhiko Tamanoi, Dept. of Microbiology, Immunology & Molecular Genetics,

1602 Molecular Sciences Bldg., UCLA, Los Angeles, CA 90095-1489

Tel: 310-206-7318, Fax: 310-206-5231

Email: fuyut@microbio.ucla.edu

Running title: Identification of Dominant Negative Rheb

§Present address: Departamento de Farmacia, Universidade Federal do Parana, Brazil

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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Summary

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 D60 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 is the first

evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells.

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Introduction

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 and 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 C, S, or M residue) required for farnesylation. The yeast and

mammalian Rheb proteins have been shown to be farnesylated (1, 11, 12).

Rheb is essential in Schizosaccharomyces 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.

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cerevisiae Rheb (1) and in A. fumigatus Rheb (9). In Drosophila, Rheb is essential, as

larvae which 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 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

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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 D60 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.

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Experimental Procedures

Strains and reagents - SP223 (h- ura4 leu1-32 ade6) and JU101p∆1 have been described

previously (7). JU101p∆1 was derived from SP223 and contains a disruption of the

chromosomal rhb1+ gene. JU101p∆1 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 (5-bromo-

4-chloro-3-indolyl-β-D-galactosidase) and IPTG (isopropyl-β-D-thiogalactopyranoside)

were purchased from Gibco/Invitrogen. Thiamine was obtained from Sigma.

Construction of SpRheb random mutant library - PCR-mediated generation of random

mutant library was carried out essentially as previously described (27). DiversifyTM 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.

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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 yeast miniprep kit (Qiagen) followed by transformation

into E. coli KC8 (hsdR leuB600 trpC9830 pyrF::Tn5 hisB463 lac∆φ74 strA galU galK)

using leucine selection. The recovered plasmids were sequenced to identify the mutation

in rhb1+.

Construction of sprhb1+ D60X mutants - Residue D60 of SpRheb was converted to all

possible amino acids by site-directed PCR mutagenesis. pREP1HArhb1+ was used as the

template in the PCR reaction. 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

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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 D60 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 O.D.600 0.1 in

EMM+Ade and growth followed over time. For spotting assays, cells from the same

overnight cultures were diluted to O.D.600 1.0 and serially diluted 4-fold. 5µl of the

diluted samples were spotted onto EMM+Ade and EMM+Ade+Thiamine plates and

allowed to grow 3 days at 30oC. 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 JU109p∆1 and the transformants were grown overnight in the

absence of thiamine. The overnight culture was diluted to O.D.600 of 1, serially diluted

four-fold and spotted onto a plate containing 150 µM thiamine. The plate was incubated

for 72 hours at 30 ºC. Flow cytometric analysis of S. pombe cells was carried out as

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described by Yang et al. (11). Briefly, cells were collected at 24h and then fixed in 70%

ethanol. After RNase digestion, propidium iodide was added and cells were sonicated

briefly. Cells were processed immediately on a Becton Dickinson FACScan.

Purification of SpRheb protein - SpRheb proteins were purified as His-tagged proteins.

Briefly, 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 IPTG for 4 hrs at

37 ºC. Cells were harvested by centrifugation, suspended in buffer, and sonicated. Cell

lysates were centrifuged and the resulting supernatant was incubated with ProBondTM

nickel-chelating resin (Invitrogen). After binding at 4 ºC, 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 approximately 22 kDa. Protein concentration of purified fractions was

determined by Bradford protein assay (28) (Bio-Rad).

Biochemical activities of SpRheb - Guanine nucleotide binding assay was carried out as

previously described (29). Briefly, 1.5 µg of His-SpRheb purified above was incubated

with 0.2 µCi [35S]GTPγS (1250 Ci/mmol, American Radiolabeled Chemicals) in binding

buffer containing 20 mM Tris pH 7.4, 50 mM NaCl, 0.1% TritonX-100, 1 mM

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dithiothreitol (DTT), 40 µg/ml bovine serum albumin (BSA), 1 mM EDTA, 2 µM GTP

and 1 mM MgCl2 at 30 ºC, and the binding examined by filtering through 0.45 µm

nitrocellulose filters (Schleicher & Schuell). Nucleotide specificity was assessed by

performing 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. GTPase assay was carried out as previously described (29, 30). Briefly, 1.5 µg of

SpRheb was incubated at 37 ºC for 10 min in the binding buffer as described above

containing α-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 ºC. 2 µl aliquots were

taken at various time points and spotted onto polyethyleneimine (PEI) cellulose thin layer

chromatography (TLC) plates (Selecto Scientific). TLC plates were developed in 1M

LiCl/1M Formic Acid. The amount of [α-32P]GDP and [α-32P]GTP was determined by

PhosphorImager analysis (Model 445 SI, Molecular Dynamics). The migration of

unlabeled GDP and GTP standards was detected by UV (254 nm). GDP dissociation was

examined by prebinding [3H]GDP (11.5 Ci/mmol, Amersham Biosciences) for 10 min at

30 ºC in the buffer used for guanine nucleotide binding and then adding excess

unlabelled 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 Medium (DMEM) supplemented with 10% heat-inactivated

fetal bovine serum (FBS) and penicillin/streptomycin. Cells were transfected using

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Polyfect (Qiagen) according to manufacturer’s instructions, and cells were collected 36

hours after transfection. Briefly, the indicated DNA was incubated in serum free media

with Polyfect reagent for 10 minutes, diluted in serum free media, and was added to cells

with fresh media. COS-7 cells were starved for nutrients by incubating in Dulbecco’s

Modified PBS for 30 minutes. Cells were stimulated with nutrients by changing to

DMEM alone for 30 minutes or with growth factors by adding 20% FBS for 15 minutes.

The mTOR inhibitor, rapamycin, was used to treat cells at 20 nM for 1 hour.

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 β-

glycerophosphate, 10 mM NaPPi, and 1xProtease Inhibitor Cocktail (Roche). Extracts

were centrifuged at 13,000xg for 10 minutes. Equal amounts of protein were then loaded

onto a 12% polyacrylamide gel and transferred to PVDF membrane. For detection of

phosphorylated p70S6K, membranes were immunoblotted with anti-phospho p70S6K

(T389) antibody (Cell Signaling). For detection of total p70S6K protein, membranes

were stripped and immunoblotted with anti-p70S6K antibody (Santa Cruz). For detection

of HA-SpRheb or HA-HsRheb, membranes were immunoblotted with anti-HA (12CA5)

(Roche).

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Results

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

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reporter construct. Therefore, colonies expressing a dominant negative SpRheb are

expected to be smaller and blue. These colonies were picked and the

pREP1HASpRhebm plasmid recovered. After retransforming the recovered plasmid

into SP223-127 to confirm the induction of fnx1+, the plasmid DNA was sequenced to

identify the mutations.

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 O.D.600 1, while the

cells expressing wild type SpRheb grew to more than O.D.600 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 slighly 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

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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.

Mutating D60 to all twenty 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 Figure 3A, we found that the expression of D60I or D60K mutant leads to a strong

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growth inhibition; rougly 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 O.D.s 48 hours 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 (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.

Mutants D60K, D60I and D60V were further characterized. Figure 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. Figure 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 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 Rheb protein. S. pombe Rheb protein was His-

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tagged and purified using a nickel column as described in the Experimental Procedures.

Figure 4A shows that SpRheb binds GTP, as examined using [35S]GTPγS. The binding

was stimulated by the addition of Mg2+, as the addition of 0.1 to 1 mM Mg2+ gave GTP

binding several-fold higher than that seen without Mg2+ (data not shown). The ratio

between SpRheb and GTP was calculated to be approximately 1 to 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]GTPγS, GTP had a greater effect in inhibiting the binding of [35S]GTPγS. Figure 4C

demonstrates that Rheb has an intrinsic GTPase activity. For this experiment, SpRheb

was first bound to [α-32P]GTP, and then Mg2+ concentration was adjusted to 20 mM and

further incubated. Appearance of radioactivity at a spot corresponding to GDP on PEI

cellulose in a time dependent manner was observed. In this experiment, excess UTP was

used to inhibit any non-specific 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 [α32P]GTP to Rheb. In this case, the appearance of GDP

was no longer observed (Fig. 4C). GTPase activity was also demonstrated using [γ-

32P]GTP bound to SpRheb and examining for the hydrolysis of γ-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

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hand, release of [3H]GDP was observed when excess amount of unlabeled GTP was

added to SpRheb.

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 Figure 5A. First, we realized that the

dominant negative mutants exhibit significantly decreased GTP binding compared with

the wild type protein. As shown in Figure 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]GTPγS (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 Figure 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 GDP dissociation experiment shown in

Figure 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

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mutant than from the D60I mutant as shown in Figure 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.

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

devlopment 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) showed

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 RT-PCR and Northern analysis showed that most tissues

express both Rheb1 and Rheb2, although Rheb2 appears to exhibit a more limited

expression profile.3 Figure 6A shows that transient expression of human Rheb1 or Rheb2

in human embryonic kidney HEK293 cells results in the activation of p70S6K, as

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detected by the use of an antibody specific for phospho(Thr-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).

To examine whether dominant negative human Rheb is capable of inhibiting

p70S6K activaiton, D60K and D60V mutations were introduced into human Rheb1 and

their effects on S6K activity was examined. Figure 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 (PBS). Normal media

were added and the activation of S6K was examined as before. As can be seen in Figure

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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.

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Discussion

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

D60 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 to

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.

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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 D60 residue of

Rheb is critical for the recognition of GTP and GDP. The analogous residue in Ras, D57,

binds a catalytic Mg2+ through an intervening water molecule and is critical for both GDP

and GTP binding (31, 32). Presumably, changing D60 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 (GEFs) (18). This is due to an increased

interaction of GDP-bound or nucleotide-free protein with its GEF. The nucleotide-free

protein has a particularly strong interaction with GEF, as this form resembles an

intermediate in the GEF 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

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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 D60 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 upregulated 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 upregulated in transformed cells (5). Further

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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/MAP 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.

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Acknowledgements

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. This work was supported by grants from NIH, CA41996 and

CA32737 to FT, a grant from Tuberous Sclerosis Alliance to DHG, and NIH F32 grant to

EJU. Flow cytometry performed in the UCLA Flow Cytometry Core Facility was

supported by NIH CA16042 and AI28697. IMPM was supported by CNPq (Brasilia, DF,

Brazil) and Universidade Federal do Parana (Curitiba, PR, Brazil). AT and C-L, G. were

supported by the Eugene Cota Robles Fellowship and the Pauley Fellowship,

respectively.

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Footnotes

1. The abbreviations used are: SpRheb, Schizosaccharomyces pombe Rheb; PCR,

polymerase chain reaction; X-gal, 5-bromo-4-chloro-3-indolyl-1-β-D-galactoside; GEF,

guanine nucleotide exchange factor; GAP, GTPase activating protein; HA,

hemagglutinin; PEI, polyethyleneimine; TLC, thin layer chromatography.

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

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

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Figure legends

Figure 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 + A (adenine) + thiamine and then replica plated onto EMM + A +

X-gal to screen for growth arrest and β-galactosidase expression. B: Demonstration of

lacZ expression by the dominant negative mutant. SP223-127 cells carrying mutant 22

plasmid or vector were serially diluted four-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 (p) or mutant 22 (¿) were grown

overnight in media containing thiamine. The culture was diluted into fresh media lacking

thiamine to OD600 of 0.05 and the growth was followed. D: Mutant 22 and the wild type

cells grown in C were collected at 24 hours after the start of growth and subjected to flow

cytometry as described in 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.

Figure 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 R15 residue is shown in bold type. D60 and

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I98 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 OD600 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 hr after

diluting into media without thiamine and analyzed by flow cytometry as described in

Experimental Procedures.

Figure 3. Changing D60 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 OD600 0.05 in EMM+Ade and growth was monitored. Measurements of OD600

were taken at the specified time points. The doubling time was calculated between the

24- and 48-hour 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 on

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 OD600 1.0. Four-fold serial

dilution was performed and spotted onto EMM+Ade plates. Plates were incubated at

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30oC for 3 days. D: D60 mutants arrest in G1 phase of the cell cycle. FACS analysis

was performed on SP223 expressing D60V, D60I, and D60K mutants. After 24 hour

growth in EMM+Ade, cells were taken and analyzed by FACS. FACS was performed

according to Experimental Procedures.

Figure 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]GTPγS and the binding was assayed using nitrocellulose filter as

described in Experimental Procedures. B: The specificity of guanine nucleotide binding

of SpRheb. SpRheb protein was incubated with [35S]GTPγS 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 [α-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 spotted

on PEI cellulose as described in 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 ºC. 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.

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Figure 5. Guanine nucleotide binding and GDP dissociation activities of the dominant

negative SpRheb mutants. A: 2 µg each of 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 [γ-32P]GTP for 10 min at 30 oC. Right panel, GDP

binding was examined by incubating 1.5 µg of each protein with [3H]GDP for 10 min at

30 oC. C: Dissociation of [3H]GDP from wild type (¿) and dominant negative mutants

D60V (¢) and D60I (p). Proteins (1.5 µg) were incubated with [3H]GDP for 10 min at

30 oC. 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 determined by scintillation counting. All values

are representative of experiments repeated at least three times.

Figure 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 indicated plasmid. S6K phosphorylation

and expression levels were detected by immunoblotting with indicated antibodies. The

enhancement of S6K phosphorylation is blocked by treatment with the mTOR inhibitor,

rapamycin. 24 hours after transfection, cells were treated with either DMSO or 20 nM

rapamycin (Rapa) for 1 hour 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 indicated Rheb1 constructs. Lysates were collected 48

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hours after transfection, and the level of S6K phosphorylation 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 incated

dominant negative constructs. The cells were starved for 30 minutes in D-PBS 36 hours

after transfection. They were then stimulated with nutrients (DMEM without serum) for

30 minutes or with 20% FBS for 15 minutes and lysates were collected and the level of

S6K phosphorylation examined.

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0 12 24 36 48 60 72 84 960.0

2.5

5.0

7.5

SpRheb

# 22

Time (h)

OD

600

SpRhebWT 22Vector

C

D

E# 22

Vector

1:1 1:4 1:16

1:64

1:256

EMM+A+X-gal

B

Mutant 22SpRheb

1N 2N 1N 2N

Fig.1

A

SpRheb mutant library

nmt1ppREP1HArhb1m

leu

rhb1-

Screen for growth arrestand lacZ+ expression

leu-, ura-

SP223-127

ura4

lacZfnx1p

Grow on EMM+A+Thiamine

Replica plate onto EMM+A-Thiamine + X-gal

pIM127

Transform SpRhebmutant library

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D60V

OD

600

Time (min)

0.010.1

110

100

0 12 24 36 48 60 72

WT

D60V

I98M

OD

600

Time (min)

0.010.1

110

100

0 12 24 36 48 60 72

WTI98M

A

B

C 1 50 SpRheb (1) M----------APIKSRRIAVLGSRSVGKSSLTVQYVENHFVESYYPTIENTFSKNIKYK HsRheb1 (1) M----------PQSKSRKIAILGYRSVGKSSLTIQFVEGQFVDSYDPTIENTFTKLITVN ScRheb (1) MEYATMSSSNSTHNFQRKIALIGARNVGKTTLTVRFVESRFVESYYPTIENEFTRIIPYK DmRheb (1) M----------P-TKERHIAMMGYRSVGKSSLCIQFVEGQFVDSYDPTIENTFTKIERVKConsensus (1) M SK RKIAVLGYRSVGKSSLTIQFVE FVESYDPTIENTFTKII K 51 110 SpRheb (51) GQEFATEIIDTAGQDEYSILNSKHSIGIHGYVLVYSITSKSSFEMVKIVRDKILNHTGTE HsRheb1 (51) GQEYHLQLVDTAGQDEYSIFPQTYSIDINGYILVYSVTSIKSFEVIKVIHGKLLDMVGKV ScRheb (61) SHDCTLEILDTAGQDEVSLLNIKSLTGVRGIMLCYSIINRASFDLIPILWDKLVDQLGKD DmRheb (50) SQDYIVKLIDTAGQDEYSIFPVQYSMDYHGYVLVYSITSQKSFEVVKIIYEKLLDVMGKKConsensus (61) QDY L IIDTAGQDEYSIFP YSIDI GYILVYSITSRKSFEMIKIIYDKLLD LGK 111 158 SpRheb (111) WVPIVVVGNKSDLH-----MQRAVTAEEGK-------ALANEWKCAWTEASARHNENVAR HsRheb1 (111) QIPIMLVGNKKDLH-----MERVISYEEGK-------ALAESWNAAFLESSAKENQTAVD ScRheb (121) NLPVILVGTKADLGRSTKGVKRCVTKAEGEKLASTIGSQDKRNQAAFIECSAELDYNVEE DmRheb (110) YVPVVLVGNKIDLH-----QERTVSTEEGK-------KLAESWRAAFLETSAKQNESVGDConsensus (121) IPIVLVGNK DLH M R VS EEGK ALA W AAFLE SAK N V D

159 185 SpRheb (159) AFELIISEIEKQ----------ANPSPP-----GDGKGCVIA HsRheb1 (159) VFRRIILEAEKM----------DGAAS------QGKSSCSVM ScRheb (181) TFMLLLKQMERVEG-------TLGLDAE------NNNKCSIM DmRheb (158) IFHQLLILIENE----------NGNP-------QEKSGCLVSConsensus (181) VF LLL EIEK G S D CSIS

D60V I98M

G1 G2

G3

G4 G5

Fig. 2

1000 1000 100000

Cou

nts

Cou

nts

Cou

nts

0

0

100

010

0

012

0

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Vec

tor

WTVKIRLQPC H G S T

SpRhebD60 mutants

VectorWT

D60VD60I

D60K

1:4

A

B

C

D

Fig. 3

D60V

Vector

WT

D60I

D60K

0

0.5

1

1.5

2

2.5

3

3.5

D G A S T C V L I M P F Y W E N Q H K R

Dou

blin

g Ti

me

(fold

WT)

1:1

AEMNWY F

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0255075

100125150

None GTP GDP CTP UTP ATP

% C

ontro

l

A B

C

0

1

2

3

4

5

0 5 10 15 20

Time (min)

[35S

]-GTP

bou

nd (p

mol

) SpRheb

Boiled

0 30 90 0 30 90UTP UTP/GTP

Time (min)Unlabeled Nuc.

GDP

GTP 0

20

40

60

80

100

120

0 5 10 15 20 25 30

Time (min)

% b

ound

[3H]

-GDP

rem

aini

ng

No excess GTP

Excess GTP

D

Fig. 4

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0

1

2

3

4

5

WT D60V D60K D60I

32P-

GTP

inco

rpor

atio

n (p

mol

)

01234567

WT D60V D60K D60I

3H-G

DP in

corp

orat

ion

(pm

ol)

0

25

50

75

100

0 5 10 15 20Time (min)

% 3

H-G

DP r

emai

ning

WT

D60V

D60I

A

C

GTP Binding GDP Binding

GDP Dissociation

Fig. 5

10X GTP 20X GTP

D60WT V I K

B

25

36476081

19

0

25

50

75

100

0 5 10 15 20Time (min)

% 3

H-G

DP r

emai

ning

D60ID60VWT

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p-S6K (T389) IB:

S6K

A

20 nM Rapa

HA-Rheb1

HA-Rheb2

B

HA

p-S6K (T389) IB:

myc-S6K

HA-Rheb1D60K

HA-Rheb1D60V

HA-Rheb1

p-S6K (T389) IB:

S6K

Nutrients

Rheb1D60K

Rheb1D60V

Serum

C

Fig. 6

+ ++ +

+ + +

+

++

+ +++

+ + ++ + +

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Gutmann, Lea Guo and Fuyuhiko TamanoiAngel P. Tabancay ., Jr, Chia-Ling Gau, Iara M.P. Machado, Erik J. Uhlmann, David H.

implicate the involvement of human Rheb in the activation of p70S6KIdentification of dominant negative mutants of Rheb GTPase and their use to

published online July 17, 2003J. Biol. Chem. 

  10.1074/jbc.M306553200Access the most updated version of this article at doi:

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