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INTRODUCTION |
The p70 S6 kinase
(p70s6k)1 is a
serine/threonine kinase that is ubiquitously activated at the
G0/G1 transition of the cell cycle in mammalian
cells (1-3). This protein kinase phosphorylates 40 S ribosomal S6
protein at five serine residues near the carboxyl terminus in
vitro (4). Studies using targeted disruption of the
p70s6k gene (5) and using transfection of a
dominant negative or a rapamycin-resistant mutant of
p70s6k (6, 7) have shown that p70s6k
mediates S6 phosphorylation in vivo. S6 phosphorylation has
been proposed to be a factor regulating initiation of mRNA
translation (see review in Ref. 8), and the studies described above (5, 6) concluded that the role of p70s6k in cell proliferation
resides in specific regulation of translation of mRNAs encoding
ribosomal proteins. Thus, p70s6k activity is considered to
be a factor required for up-regulation of ribosomal biogenesis, which
facilitates G1 progression during the cell cycle (9).
In regards to the activation of p70s6k, it appears there
are at least two upstream regulatory pathways; one is through
phosphatidyl inositol-3 kinase (PI3K) and the other is through
mammalian target of rapamycin (mTOR; also termed FRAP and RAFT). Many
growth factors, such as platelet-derived growth factor and epidermal
growth factor, activate PI3K through their binding to specific
receptors, and previous studies using chemical inhibitors of PI3K,
mutant platelet-derived growth factor receptors, and mutant PI3Ks, all
indicated that PI3K is involved in the activation of p70s6k
by growth factors (10-13). Indeed, it has been demonstrated recently that PDK1, a downstream kinase of PI3K, phosphorylates
p70s6k at Thr229 and activates the kinase (14,
15).
The involvement of mTOR in the regulation of p70s6k has
been initially demonstrated by studies using the immunosuppressive drug rapamycin (see reviews in Refs. 16 and 17). Rapamycin associates with a
cellular protein FKBP12 in cells, and the rapamycin-FKBP12 complex then
binds to mTOR. It has also been demonstrated that mTOR is required for
the inhibitory action of rapamycin on p70s6k (18).
Moreover, mTOR has an intrinsic protein serine/threonine kinase
activity and regulates the activity and phosphorylation of
p70s6k in vivo in a manner that is dependent on
the kinase activity of mTOR (18). Although initial reports concluded
that mTOR did not directly phosphorylate p70s6k, it has
been demonstrated that mTOR phosphorylates p70s6k at
Thr389 in vitro (19). In addition to
p70s6k, mTOR regulates another translation regulatory
molecule, eIF-4E-binding protein 1 (4E-BP1) (20-23), independent of
p70s6k activity (5, 6). mTOR is demonstrated to
phosphorylate the translation repressor protein 4E-BP1 at its serine
and threonine residues (24). The hypophosphorylated species of 4E-BP1
binds tightly to eIF-4E (an N7-methylguanosine cap-binding
subunit of eIF-4F complex) and prevents eIF-4E from associating with
eIF-4G (a scaffolding protein in eIF-4F complex). The phosphorylation
of 4E-BP1 by mTOR is thought to release eIF-4E and facilitate
translational initiation of capped mRNA (21). Thus, mTOR regulates
1) ribosomal protein synthesis at the level of mRNA translation
through p70s6k activity, and 2) overall protein synthesis
by controlling translational initiation of capped mRNA through
4E-BP1.
In contrast to the PI3K pathway, which is activated by various growth
factors, it was unclear what factor physiologically regulated mTOR. A
study on proteolytic responses to amino acid deprivation demonstrated
that ribosomal S6 phosphorylation was induced by supplementation of
amino acids (25), suggesting that amino acid concentration in culture
media may be a regulatory factor for p70s6k. Indeed, Fox
et al. (26) demonstrated that amino acids stimulate phosphorylation of p70s6k in rat adipocytes. More recently,
Hara et al. (27) have reported that amino acid concentration
regulates p70s6k activity and phosphorylation of 4E-BP1 in
Chinese hamster ovary cells. The study demonstrated that a
rapamycin-resistant mutant (p70
2-46/
CT104) of p70s6k
was resistant to amino acid deprivation, indicating that amino acid
sufficiency and mTOR both signal to p70s6k through a common
effector, which could be mTOR itself or an mTOR-controlled downstream
element. In this study, we further explored the mechanism by which
amino acid concentration regulates p70s6k.
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EXPERIMENTAL PROCEDURES |
Cells and Reagents--
Human T-lymphoblastoid Jurkat
cells were obtained from ATCC. Human alveolar rhabdomyosarcoma Rh30
cells were established from the bone marrow of a patient with
metastatic tumor (28). Rh30 cells constitutively expressing wild type
mTOR (WT-mTOR) or a rapamycin-resistant mTOR (S2035I) were obtained by
transfection of cells with pcDNA3-AU1mTORwt or
pcDNA3-AU1mTORSI. BHK21 cells and their temperature-sensitive
mutant temperature-sensitive (ts) BN250 were obtained from RIKEN cell
bank (Tsukuba, Japan). Rapamycin and wortmannin were obtained from
Calbiochem (San Diego, CA), and stored in ethanol (1 mg/ml) and
dimethyl sulfoxide (10 mM), respectively. Cycloheximide
(Sigma) was dissolved in ethanol and stored as a 10 mg/ml stock
solution. Puromycin (Sigma) was stored in distilled water (2 mg/ml).
Amino acid alcohols (Sigma) L-leucinol, L-phenylalaninol, L-alaninol,
L-histidinol, L-tyrosinol,
L-methioninol, and D-leucinol were stored in
distilled water (5 M). Methyl 2-aminoisobutyric acid
(MeAIB) and 2-amino-2-norbornane-carboxylic acid were obtained from
Sigma and stored in distilled water at 200 and 100 mM, respectively.
Cell Culture--
Jurkat cells and Rh30 cells were maintained in
RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% (v/v)
heat-inactivated fetal calf serum (FCS) (HyClone, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies).
For amino acid deprivation, exponentially growing cells were washed
twice with amino acid-free RPMI 1640 medium (RPMI 1640 select-amine
kit, catalog no. 17402, Life Technologies) and resuspended at 5 × 105 cells/ml in the same amino acid-free RPMI 1640 medium
supplemented with 10% dialyzed FCS (catalog no. 26300, Life
Technologies) for the indicated times at 37 °C in a
5%-CO2 incubator. In some experiments, individual amino
acids were deprived instead of total amino acid deprivation. For amino
acid supplementation, cells (5 × 105 cells/ml) were
incubated in the amino acid-free RPMI 1640 medium supplemented with
10% dialyzed FCS for 3-16 h. The same volume of RPMI 1640 medium
containing 2× concentrations of amino acids +10% dialyzed FCS were
added to the culture, and cells were incubated for the indicated times.
The concentrations (in µM) of amino acids (1×) in RPMI
1640 (Life Technologies) were as follows: Gln, 2050; Asn, 380; His,
100; Trp 24; Pro, 170; Cyst, 410; Gly, 130; Val, 170; Leu, 380; Ile,
380; Ser, 290; Thr, 170; Phe, 90; Tyr, 110; Met, 100; Glu, 140; Asp
150; Arg, 1150; and Lys, 220. BHK21 and tsBN250 cells were maintained
in Dulbecco's modified Eagle's medium (Life Technologies)
supplemented with 10% (v/v) heat-inactivated FCS (HyClone) at 33 °C
in a 5%-CO2 incubator. For transition of cells to
nonpermissible temperature, culture plates were transferred to a
5%-CO2 incubator at 39 °C.
Kinase Assay--
Cells (2 × 106) were
harvested, and the activities of the kinases were measured as we
described previously (3). Briefly, cells were lysed in a buffer
containing 10 mM potassium phosphate, 1 mM
EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM
-glycerophosphate, 1 mM
Na3VO4, 2 mM dithiothreitol, 40 µg/ml phenylmethylsulfonyl fluoride, and 0.1% Nonidet P-40. The
protein kinases in 250 µg of total cellular proteins were
immunoprecipitated using specific antibodies: for p70s6k, a
rabbit polyclonal antibody raised against the carboxyl-terminal 18 amino acids of p70s6k (sc-230, Santa Cruz Biotechnology,
Santa Cruz, CA); for Akt, a goat polyclonal antibody against an epitope
corresponding to amino acids 461-480 of human Akt1 (sc-1618, Santa
Cruz Biotechnology); for p90rsk, a rabbit polyclonal
antibody raised against an epitope 682-724 of mouse Rsk1 (06-185,
Upstate Biotechnology, Lake Placid, NY); and for Cdk2, a rabbit
polyclonal antibody raised against an epitope 283-298 of human Cdk2
(sc-163, Santa Cruz Biotechnology). The kinase activity was measured by
incorporation of 32P into specific substrate peptides (for
p70s6k and p90rsk, an S6 peptide, RRRLSSLRA;
for Akt, a Gsk3 peptide, GRPRTSSFAEG; for Cdk2, a histone H1 peptide, AVAAKKSPKKAKKPA).
Immunoblot--
Cells (5 × 106) were washed
with phosphate-buffered saline and lysed at 4 °C with 25 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5% sodium
deoxycholate, 2% Nonidet P-40, 0.2% SDS, 1 µM
phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 50 µM leupeptin. Lysates were resolved by SDS 7.5% (for
p70s6k) or 15% (for 4E-BP1) polyacrylamide gels and
transferred to nitrocellulose filters. After blocking of the filters
with a solution containing 1% bovine serum albumin, the filters were
incubated with primary antibodies. The antibodies used for
p70s6k are the same as described above. For 4E-BP1, a
rabbit polyclonal antibody raised against a recombinant His-tagged rat
PHAS1 (4E-BP1) was used (24). Specific reactive proteins were detected
by the ECL method, employing a donkey anti-rabbit Ig antibody linked to
horseradish peroxidase (Amersham Pharmacia Biotech).
Amino Acid Uptake--
Cells (2 × 106) were
harvested, centrifuged at 1500 rpm for 5 min, and resuspended in 100 µl of a buffer containing 140 mM NaCl, 5.4 mM
KCl, 1.8 mM CaCl2, 0.8 mM
MgSO4, 5 mM D-glucose, 25 mM HEPES (pH 7.5), 25 mM Tris (pH 7.5). The
cell suspension was loaded on 100 µl of a buffer containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgSO4, 5 mM D-glucose, 25 mM HEPES (pH 7.5),
25 mM Tris (pH 7.5), 50 µM nonradioactive
His, and 1 µl (1 µCi) of [3H]His (1 mCi/ml) (Amersham
Pharmacia Biotech) with or without 10 mM of
L-histidinol, layered on 300 µl of silicone oil/paraffin oil (92:8) in microcentrifuge tubes (29). After the indicated time (30 s, 2 min, 5 min, 30 min, or 60 min), cells were centrifuged for 2 min
at 14,000 rpm, and water phase was discarded. After washing surface of
oil twice with 750 µl of phosphate-buffered saline, oil phases were
discarded also. Cell pellets were then lysed in 50 µl of saline with
2% Triton X-100. Radioactivity of the total lysates was measured using
a liquid scintillation counter.
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RESULTS |
Amino Acid Deprivation Inactivated and Supplementation Activated
p70s6k but Not Other Protein Serine/Threonine
Kinases--
The human T-lymphoblastoid cell line, Jurkat cells, were
transferred to an amino acid-free medium supplemented with 10%
dialyzed FCS and incubated for the indicated times (amino acid
deprivation). In addition, after culturing cells in the amino acid-free
medium with 10% dialyzed FCS for 16 h, cells were transferred to
the regular RPMI 1640 medium containing amino acids with 10% dialyzed FCS and incubated at the indicated times (amino acid supplementation). Initially, the activity of p70s6k was measured in the
system, as well as the activities of other growth-related
serine/threonine kinases, Akt, p90rsk, and Cdk2 (Fig.
1). p70s6k activity decreased
within 15 min after deprivation of amino acids and became undetectable
within 30 min; levels remained undetectable throughout the time course.
Additionally, supplementation of amino acids increased
p70s6k activity within 15 min, and the activity reached a
plateau level within 60 min. In contrast, the activity of Akt, which is
a downstream kinase of PI3K (13), was not inhibited by amino acid
deprivation, nor did it increase following amino acid supplementation.
p90rsk is a downstream kinase of extracellular
signal-regulated kinases and has the highest homology with
p70s6k in the catalytic domains (30). In contrast to
p70s6k, the activity of p90rsk transiently
increased by transferring cells to an amino acid-free medium, and then
decreased gradually. However, p90rsk remained partially
active throughout the time course of the experiments. Additionally,
amino acid supplementation transiently decreased p90rsk
activity, but overall it did not dramatically alter kinase activity for
360 min. Cdk2 is active especially at the late G1 and S
phases of the cell cycle. Cdk2 activity was partially decreased by both amino acid deprivation and by supplementation within 30 min, but the
kinase remained partially active throughout the time course.

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Fig. 1.
Effects of amino acid deprivation and
supplementation on the activities of p70s6k, Akt,
p90rsk, and Cdk2. A, amino acid
deprivation. Exponentially growing Jurkat cells were washed twice with
amino acid-free RPMI 1640 medium and resuspended at 5 × 105 cells/ml in the same amino acid-free medium
supplemented with 10% dialyzed FCS for the indicated times at 37 °C
in a CO2 incubator. B, amino acid
supplementation. Jurkat cells (5 × 105 cells/ml) were
incubated in the amino acid-free medium with 10% dialyzed FCS for
16 h. The same volume of RPMI 1640 medium containing 2×
concentrations of amino acids + 10% dialyzed FCS were added into the
culture, and cells were incubated for the indicated times. Cells
(2 × 106) were harvested, and the specific activity
of immunoprecipitated p70s6k, Akt1, p90rsk, or
Cdk2 was measured by incorporation of 32P into specific
substrate peptides as described under "Experimental Procedures."
Error bars here and in figures below represent S.D. of the
data. Results show one representative experiment out of three.
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Amino Acid Supplementation Increased Phosphorylation of
p70s6k and 4E-BP1--
The hyperphosphorylated species of
p70s6k, which correspond to the active form of the kinase,
is determined as a band(s) with a lower mobility in immunoblots (9). On
the other hand, the hypophosphorylated p70s6k, which
corresponds to the inactive form of the kinase, is determined as a band
with the highest mobility. Cells treated with an amino acid-starved
medium demonstrated only the hypophosphorylated species of
p70s6k, and supplementation of amino acids increased the
hyperphosphorylated species of p70s6k within 5-15 min
(Fig. 2). In contrast to
p70s6k, amino acid deprivation/supplementation essentially
did not alter the phosphorylation status of p90rsk,
similarly evaluated by a mobility shift in an immunoblot analysis (data
not shown).

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Fig. 2.
Effects of amino acid supplementation on the
phosphorylation of p70s6k and 4E-BP1. Jurkat cells
(5 × 105 cells/ml) were incubated in the amino
acid-free medium with 10% dialyzed FCS for 16 h. The same volume
of RPMI 1640 medium containing 2× concentrations of amino acids + 10%
dialyzed FCS were added into the culture, and cells were incubated for
the indicated times. The protein extracts from cells were separated in
7.5% (for p70s6k) or 15% (for 4E-BP1)-SDS-polyacrylamide
gel and transferred to nitrocellulose filters. The specific proteins
were detected by immunoblotting using the ECL method as described under
"Experimental Procedures." Results show one representative
experiment out of three.
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The phosphorylation status of 4E-BP1, another downstream event of mTOR,
was also examined by a gel mobility shift in an immunoblot assay
(20-23). Cells treated with an amino acid-starved medium demonstrated
bands with higher mobilities, corresponding to the hypophosphorylated
species of 4E-BP1. Amino acid supplementation induced bands with lower
mobilities, which correspond to hyperphosphorylated 4E-BP1, within 15 min. These hyperphosphorylated bands became more predominant within
1-3 h. Additionally, hyperphosphorylated species of p70s6k
and 4E-BP1 were eliminated by amino acid deprivation within 5-15 min
and 30 min, respectively (data not shown).
Rapamycin, but Not Wortmannin, Inhibited Amino Acid-induced
p70s6k Activation--
Because amino acid concentrations
affected p70s6k without affecting Akt activity, it does not
seem that amino acids regulate p70s6k through PI3K.
Furthermore, similar effects of amino acids on p70s6k and
4E-BP1 suggested that mTOR, or an unidentified factor that mediates
mTOR signals to both p70s6k and 4E-BP1, is involved in
amino acid-induced activation of p70s6k. In order to
further investigate these observations, the effects of wortmannin and
rapamycin on amino acid-induced p70s6k activation were
examined. Jurkat cells were treated in an amino acid-free medium for
16 h and then supplemented with total amino acids or none for
1 h in the presence or absence of specific inhibitors of PI3K and
mTOR, wortmannin (100 nM) and rapamycin (10 ng/ml), respectively. As shown in Fig. 3,
p70s6k activation induced by amino acid addition was
inhibited by rapamycin. In contrast, wortmannin at the concentration
that is known to inhibit PI3K activity over 95% inhibited
p70s6k only by ~25%. In contrast, Akt, which was already
active in the cell culture regardless of amino acid concentrations, was
inhibited by wortmannin by about 60%. Similar to the
p70s6k activity, 4E-BP1 phosphorylation induced by amino
acid addition was inhibited by rapamycin but not by wortmannin (data
not shown).

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Fig. 3.
Rapamycin but not wortmannin inhibits amino
acid-induced p70s6k activation. Jurkat cells (5 × 105 cells/ml) were incubated in amino acid-free medium
with 10% dialyzed FCS for 16 h. Then, the same volume of either a
medium containing 2× concentrations of amino acids + 10% dialyzed FCS
(AA(+)) or amino acid-free medium + 10% dialyzed FCS
(AA( )) were added into the culture for 1 h. Rapamycin
(RAP) (10 ng/ml) or wortmannin (WOR) (100 nM) was added to the culture 15 min prior to the addition
of AA(+) medium or AA( ) medium. Cells were harvested, and the
activities of p70s6k and Akt were measured as described
above. Results show one representative experiment out of three.
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Rapid activation of p70s6k (within 15 min) after
re-addition of total amino acids suggested that this may be independent
of newly synthesized proteins. In order to confirm this, cycloheximide was added to the culture before addition of total amino acids. Cycloheximide (100 µM), which inhibits protein synthesis
over 95%, did not inhibit amino acid-induced p70s6k
activation (data not shown). These data indicate that amino acids activate p70s6k independent of newly synthesized proteins.
Amino Acid Supplementation Induced p70s6k Activation in
the Presence of Rapamycin in Cells Expressing a Rapamycin-resistant
Mutant of mTOR--
In order to further investigate mechanisms by
which amino acids regulate the mTOR pathway, we utilized cells
constitutively expressing a rapamycin-resistant mTOR. Human
rhabdomyosarcoma Rh30 cells constitutively expressing either WT-mTOR or
a rapamycin-resistant mutant (S2035I-mTOR) were obtained by
co-transfection of the mTOR expression vectors and a neo-resistant gene
expression vector, followed by G418 selection as described previously
(31). Parental Rh30 cells, WT-mTOR Rh30 cells, or S2035I-mTOR Rh30
cells were first incubated for 3 h in an amino acid-deprived
medium. Rapamycin (10 ng/ml) was then added to the culture. Thirty min
after addition of rapamycin, total amino acids were added, and cells
were incubated for the indicated times. As shown in Fig.
4, amino acid supplementation was not
able to induce p70s6k in the presence of rapamycin in
parental Rh30 cells or WT-mTOR Rh30 cells, as shown in Jurkat cells
above. In contrast, amino acids did induce p70s6k in the
presence of rapamycin in S2035I-mTOR Rh30 cells.

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Fig. 4.
Amino acid sensitivity of p70s6k
in the presence of rapamycin in cells constitutively expressing
S2035I-mTOR. Exponentially growing Rh30 cells (parent, WT-mTOR, or
S2035I-mTOR) were first treated with amino acid-free medium for 3 h. Rapamycin (10 ng/ml) was then added to the culture. After 30 min,
the same volume of RPMI 1640 medium containing 2× concentrations of
amino acids + 10% dialyzed FCS were added into the culture (time
0). Cells were harvested at the indicated times, and
p70s6k activity was measured as described above.
WT-mTOR and S2035I-mTOR are Rh30 cells
constitutively expressing wild type mTOR and S2035I mutant of mTOR,
respectively. Results show one representative experiment out of
two.
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Effects of Selective Inhibitors for Amino Acid Transport Systems on
Amino Acid-induced p70s6k Activation--
The mechanism by
which amino acids regulate p70s6k activity was further
explored. Fox et al. (26) reported that supplementation of a
set of neutral amino acids, which were preferentially transported by
system L transporter, was able to induce p70s6k
phosphorylation. In contrast, another set of neutral amino acids preferentially transported by system A or ASC, or a set of charged amino acids, was less effective. A potential explanation for these results is that a specific amino acid transporter, such as system L, is
linked to activation of p70s6k. In order to investigate
this possibility, we examined the effects of specific inhibitors of
various amino acid transporter systems on amino acid-induced
p70s6k activation. There are three major amino acid
transporter systems known for uptake of neutral amino acids; system A,
system L, and system ASC. It is reported that over 90% of neutral
amino acids are transported through these three systems in mammalian
cells (32). Each of the three systems has a model substrate that has a
high affinity to the system. MeAIB has a high affinity to system A and
competitively inhibits transport of other amino acids through the
system. In contrast, MeAIB does not significantly affect transport of
amino acids through other transporters, including system L and system
ASC. Similarly, 2-amino-2-norbornane-carboxylic acid and cysteine can
competitively inhibit amino acids-transport through system L and system
ASC, respectively. Jurkat cells were initially incubated with amino
acid-deprived medium for 16 h, and then
concentrations
of total amino acids contained in regular RPMI 1640 medium were added
to the culture in the presence or absence of these competitive model
substrates (10 mM). This concentration of total amino acids
was able to induce approximately one-half the activity of
p70s6k when compared with addition of 1× total amino acids
of RPMI 1640 medium. As shown in Fig. 5,
all of the model substrates partially inhibit p70s6k
activation induced by amino acid supplementation. This is inconsistent with the idea that a specific neutral transporter is linked to p70s6k activation. If amino acid transporters are signaling
mediators that sense extracellular amino acid concentration and
subsequently transduce signals to p70s6k, then the data
suggest that all three systems should link to p70s6k
activation. Alternatively, the data may be explained by altered intracellular amino acid concentration.

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Fig. 5.
Effects of selective inhibitors for neutral
amino acid-transport systems on amino acid-induced p70s6k
activation. Jurkat cells (5 × 105 cells/ml)
were incubated in amino acid-free medium with 10% dialyzed FCS for
16 h. concentrations of total amino acids contained in
RPMI 1640 were added to the culture in the presence of a vehicle
(distilled water (control)) or 10 mM of
2-amino-2-norbornane-carboxylic acid, MeAIB, or Cys for 1 h. Cells
were harvested, and the activity of p70s6k was measured as
described above. Results show one representative experiment out of
three.
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Amino Acid Alcohols Inhibit p70s6k Activity--
The
mechanism by which the cell recognizes the lack of an amino acid seems
reasonably well understood in bacteria and yeast, and basically it is
through increased concentration of intracellular deacylated tRNA or, in
some cases, through reduced availability of aminoacylated tRNA (see
under "Discussion"). In order to investigate the involvement of the
tRNA aminoacylation in the regulation of p70s6k in
mammalian cells, we initially utilized amino acid alcohols. The alcohol
derivatives of amino acids inhibit their corresponding tRNA synthetases
and thus prevent aminoacyl-tRNA formation (33, 34). For example,
L-histidinol inhibits L-His binding to
tRNAHis synthetase and thereby increases deacylated
tRNAHis. Various amino acid alcohols were added to
exponentially growing Jurkat cells. As shown in Fig.
6A, addition of 2 mM of either leucinol, phenylalaninol, alaninol,
histidinol, tyrosinol, or methioninol (all L-type)
inhibited p70s6k activity to various extents in 30 min.
Addition of D-leucinol, in contrast, did not inhibit
p70s6k activity. It should also be noted that addition of
D-amino acids, including D-Leu and
D-Gln, did not increase p70s6k activity in
total amino acid-starved cells, whereas addition of L-Leu
or L-Gln did increase p70s6k activity partially
(data not shown). The dose response, time effect, and specificity of
amino acid alcohols on p70s6k was examined using
histidinol, one of the most effective amino acid alcohols in the
inhibition of p70s6k. Fig. 6B demonstrates the
dose response of histidinol in the inhibition of p70s6k.
Histidinol (10 mM) inhibited p70s6k activity
within 30 min and activity remained low for 24 h (Fig. 6C). The time course was compatible with that of
p70s6k activity upon total amino acid deprivation (Fig.
1A). For comparison, the time course of His deprivation on
p70s6k is shown in Fig. 6C as well. His
deprivation decreased p70s6k activity to an extent similar
to histidinol within 30 min, but there was a rebound of the kinase
activity within 1-6 h after deprivation of the amino acid. This
transient rebound was also observed when lower concentrations of
histidinol (0.5 or 2 mM) were added to the culture. The
effects of histidinol on p70s6k activity was considered to
be specific and not due to its toxic effects on cells, because it did
not affect p90rsk, Akt, or cdk2 within 6 h (Fig.
6D). Additionally, we confirmed that the effect of
histidinol was not mediated through inhibition of His transport (Fig.
6E). Uptake of histidine within 60 min after addition of
radiolabeled His was not affected by the presence of 10 mM
histidinol. These data suggest that blockade of tRNA aminoacylation may
inhibit p70s6k.

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Fig. 6.
Effects of amino acid alcohols.
A, effects of various amino acid alcohols. Exponentially
growing Jurkat cells were incubated with 2 mM of amino acid
alcohols for 30 min. Cells were harvested and the activity of
p70s6k was measured as described above. Amino acid alcohols
used here were L-leucinol, L-phenylalaninol,
L-alaninol, L-histidinol,
L-tyrosinol, L-methioninol, and
D-leucinol. Results here and below show one
representative experiment out of three to five. B,
dose effect of L-histidinol. Exponentially growing Jurkat
cells were incubated with 0-10 mM of
L-histidinol for 30 min. Cells were harvested, and the
activity of p70s6k was measured as described above.
C, time course of L-histidinol. Exponentially
growing Jurkat cells were incubated with 10 mM of
L-histidinol for 0-24 h. Alternatively cells were
transferred to histidine-free RPMI 1640 medium supplemented with 10%
dialyzed FCS (His deprivation). Cells were harvested at the
indicated times, and the activity of p70s6k was measured as
described above. D, effects of L-histidinol on
other growth-related kinases. Exponentially growing Jurkat cells were
incubated with 10 mM of L-histidinol for 0-6
h. Cells were harvested, and the activities of p70s6k, Akt,
p90rsk, and Cdk2 were measured as described above. The data are shown
by percentage of activity compared to the activity of each kinase in
cells before treatment of histidinol. E, effects of
L-histidinol on His uptake into cells. Exponentially
growing Jurkat cells were harvested, and [3H]His uptake
(~50 µM of total His) into cells (within 0.5-60 min)
was measured in the presence or absence of 10 mM
L-histidinol as described under "Experimental
Procedures."
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Effects of Inhibitors of Peptide Elongation--
Cycloheximide is
an inhibitor of peptidyl transferase, and puromycin interferes peptide
elongation by accepting the nascent peptide chain from the peptidyl
tRNA molecule on the ribosome (35). Because these drugs block the
utilization of aminoacylated tRNA by interfering with peptide
elongation, their action is expected to cause a shift in the
aminoacylation equilibrium (36). The ratio of deacylated tRNAs
decreases by addition of cycloheximide or puromycin in regular cell
culture conditions (37). Additionally, the increase in deacylated tRNAs
is thought to be minimized by the drugs when amino acid supply is
limited (38). Exponentially growing Jurkat cells were transferred to
amino acid-depleted medium in the presence or absence of cycloheximide
(1 µM) or puromycin (100 µg/ml) (Fig.
7). A decrease in amino acid
concentration to 1/4 did not change p70s6k activity
significantly, and a decrease to
reduced p70s6k
activity by about one-half in 1 h. Both cycloheximide and
puromycin enhanced p70s6k activity when cells were
transferred to the medium containing 1/4 and
concentrations of amino acids. Inhibition of p70s6k
activity by decrease in amino acid concentration to
was
completely blocked by the drugs. These data support the idea that
increase in deacylated tRNA or decrease in aminoacylated tRNA is a
factor suppressing p70s6k.

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Fig. 7.
Effects of cycloheximide or puromycin on
amino acid depletion-induced p70s6k suppression.
Exponentially growing Jurkat cells were transferred to RPMI 1640 medium
containing 1/4× or × concentrations of amino acids + 10% dialyzed FCS, in the presence of cycloheximide (1 µM) or puromycin (100 µg/ml), or in the absence of the
drugs (control) for 1 h. Cells were harvested, and the
activity of p70s6k was measured as described above. The
p70s6k activity of cells transferred to the medium
containing a 1× concentration of amino acids was 13,894 cpm. The data
are shown by percentage of activity compared to this value. Results
show one representative experiment out of two.
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Study Using a Temperature-sensitive Mutant of tRNA
Synthetase--
There have been several reports describing
temperature-sensitive mutants of mammalian cells that contain mutations
in the tRNA synthetases. Here, we utilized tsBN250 cells that were
derived from a golden hamster kidney BHK21 cell line (39). tsBN250 has a point mutation at the histidyl-tRNA synthetase gene, which likely alters normal protein folding (39). The activity of the mutated tRNA
synthetase is very low or undetectable and cannot support cell growth
unless excess amounts of His are supplemented at nonpermissible temperature (39 °C) (39). Using these lines, we examined the correlation of tRNA synthetase activity in the cells with
p70s6k activity. BHK21 and tsBN250 cells were maintained at
permissible 33 °C. Cells were transferred to 39 °C incubator and
cell numbers and p70s6k activity were monitored. As shown
in Fig. 8A, growth of tsBN250 cells was arrested in nonpermissible temperature, and it was partially recovered by addition of high concentrations of His, but not of Lys, to
the culture medium. Transfer to nonpermissible temperature decreased
p70s6k in tsBN250 cells within 30 min but not in parental
BHK21 cells (Fig. 8B). There was a rebound of the kinase
activity in tSBN250 cells within 2-6 h after transferring cells to
39 °C as seen in a His deprivation experiment (Fig. 6C).
The inhibition of p70s6k in tsBN250 cells was partially
recovered by addition of high concentrations of His, the corresponding
amino acid for the mutated tRNA synthetase, but not by Lys (Fig.
8C). In contrast to p70s6k, activity of
p90rsk in tsBN250 cells was not altered by the changing
temperature or by addition of extra amino acids. These data suggest
that inhibition of tRNAHis aminoacylation led to the
suppression of p70s6k activity.

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Fig. 8.
Study using a temperature-sensitive mutant of
tRNA synthetase. A golden hamster kidney BHK21 cell line and its
temperature-sensitive mutant tsBN250 cells, which have a point mutation
at the histidyl-tRNA synthetase gene, were maintained at 33 °C.
A, cell growth. BHK21 and tsBN250 cells were transferred to
a 39 °C incubator with or without supplementation of high
concentrations of His (2 mM) or Lys (10 mM).
Cell numbers were monitored at the indicated times (day). B,
p70s6k activity. BHK21 and tsBN250 cells were transferred
to a 39 °C incubator. Cells were harvested at the indicated times,
and the activity of p70s6k was measured as described above.
The kinase activities of cells before transferring to 39 °C were
12,241 cpm (BHK21) and 29,448 cpm (tsBN250). The data are shown by
percentage of activity compared to this value. Results are
representative of one of three similar experiments. C,
p70s6k and p90rsk activity. tsBN250 cells were
transferred to a 39 °C incubator for 30 min with or without
supplementation of high concentrations of His (2 mM) or Lys
(10 mM). The activity of p70s6k and
p90rsk was measured as described above. Results show one
representative experiment out of four.
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DISCUSSION |
Amino acid deprivation inactivated and amino acid supplementation
activated p70s6k, indicating that the kinase activity is
dependent on amino acid concentration in cell culture media. The
activities of other serine/threonine kinases were less affected by
amino acid concentration, and moreover, none of these kinases
demonstrated the pattern for amino acid dependence that was seen in
p70s6k. Amino acid concentration affected phosphorylation
of not only p70s6k but also 4E-BP1, which is another
translation regulatory molecule controlled by mTOR. Additionally,
p70s6k activation and 4E-BP1 phosphorylation induced by
amino acid addition was inhibited by rapamycin, a specific inhibitor of
mTOR. In contrast, a concentration of wortmannin (100 nM)
that almost completely inhibits PI3K activity had only a minor effect
on the amino acid-induced p70s6k. mTOR activity is
sensitive to only higher concentrations of wortmannin
(IC50, ~200 nM), and a partial inhibition of
p70s6k by wortmannin (100 nM) may be explained
by a partial inhibition of mTOR by the drug. Moreover, amino acid
supplementation induced p70s6k in the presence of rapamycin
in cells constitutively expressing a rapamycin-resistant mutant of mTOR
(S2035I-mTOR). These data indicate that amino
acid-dependent regulation of p70s6k requires
mTOR but not PI3K. Our data here support the conclusion by Hara
et al. (27) that amino acids regulate p70s6k
through mTOR itself or an mTOR-controlled downstream element, such as a
protein phosphatase. Burnett et al. (19) have recently reported that mTOR has the ability to directly phosphorylate
p70s6k at Thr389, which is an essential
phosphorylation site for both the activity of p70s6k, and
the sensitivity to rapamycin. Furthermore, in yeast, both addition of
rapamycin or loss of TOR function (a yeast homolog of mTOR) causes a
starvation response in Saccharomyces cerevisiae, suggesting
that TOR is a factor to control mRNA translation upon nutrient
starvation (40). Additionally, Noda and Ohsumi (41) have recently
reported that TOR controls autophagy (a proteolytic response), which is
an event also regulated by amino acid concentration. Taken together,
mTOR (or TOR in yeast) itself may be regulated by amino acid
concentration. In this scenario, an amino acid-sensitive site(s) in
mTOR should not reside within its FKBP12/rapamycin binding domain,
because amino acids regulated S2035I-mTOR, which does not interact with
the FKBP12/rapamycin complex. Because mTOR has a long N-terminal
regulatory region (>2,000 amino acids), multiple regulatory mechanisms
on mTOR activity would not be unexpected. In the case where the
mTOR-controlled downstream element is affected by amino acids, amino
acids should regulate this molecule independent of the mTOR-regulatory
site because amino acid deprivation decreased S2035I-mTOR-dependent p70s6k activity.
The next question we attempted to answer is how amino acids affect the
mTOR/p70s6k pathway. The results obtained using amino acid
alcohols, inhibitors of mRNA translation elongation, and
temperature-sensitive mutants of tRNA synthetase indicated involvement
of tRNA aminoacylation in amino acid-dependent control of
p70s6k. All of the inhibitors of neutral amino acid
transporters partially suppressed amino acid-induced p70s6k
activity, which does not conflict with the idea that intracellular amino acid concentration regulates the pathway through tRNA
aminoacylation. Indeed, mechanisms by which unicellular organisms
recognize and respond to amino acid starvation is all through the
aminoacylation status of tRNA. One aspect of the response to amino acid
starvation of Escherichia coli is the accumulation of a
regulatory nucleotide, guanosine 3'-diphosphate, 5'-diphosphate
(ppGpp), which inhibits several reactions, including transcription of
rRNA ("stringent" control). The synthesis of ppGpp is induced by
deacylated tRNA (42). Additionally, "attenuation" in amino acid
biosynthetic operons is controlled by availability of aminoacyl tRNA
(43). The best characterized mechanism by which eukaryotes cope with amino acid starvation is the amino acid-dependent control
of yeast GCN2 in S. cerevisiae (44, 45). This
serine/threonine kinase has a 530-amino acid sequence related to
histidyl-tRNA synthetase at its carboxyl terminus (46). Studies using
an RNA binding assay have shown that deacylated tRNA can bind to this
domain of GCN2 (47). Upon amino acid starvation, surplus deacylated tRNAs are thought to bind to GCN2 and activate GCN2 protein kinase activity (48). Although the carboxyl terminus of GCN2 has homology to
histidyl-tRNA synthetase, it seems that deacylated forms of other tRNAs
in addition to histidyl-tRNA can activate GCN2 as well. It seems that
there has been sufficient genetic drift in the histidyl-tRNA synthetase
domain of GCN2 that it now lacks the ability to discriminate between
the binding of different tRNAs (45). mTOR or p70s6k does
not have any sequence homology with tRNA synthetases, implying that
tRNA may not interact with mTOR or p70s6k directly. Indeed,
addition of a yeast tRNA mixture to immunoprecipitated mTOR and
p70s6k does not suppress activity of the kinases to
phosphorylate 4E-BP1 and S6, respectively, in vitro (data
not shown). If amino acid-dependent control of
p70s6k is through mTOR, tRNA may interact with an
unidentified upstream regulator of mTOR. Withdrawal of most individual
amino acids inhibited p70s6k in Chinese hamster ovary
cells, although with differing potency (27). In our system, a variety
of L-amino acid alcohols was effective to inhibit
p70s6k. These data suggest that most deacylated tRNAs would
be sufficient to suppress p70s6k with differing potency, as
they were demonstrated to activate GCN2 in yeast. One candidate for an
upstream regulator of mTOR would be a mammalian homologue of GCN2,
although it has not been cloned or identified yet to date. Histidinol
was among the most effective amino acid alcohols in the suppression of
p70s6k, which may support the idea that the upstream
regulatory molecule of mTOR contains a domain homologous to
histydyl-tRNA synthetase, as does yeast GCN2.
A rebound of p70s6k activity was detected during
deprivation of His or by addition of lower doses of histidinol. Similar
results were obtained by deprivation of other individual amino acids or by addition of other amino acid alcohols, although to various extents
(data not shown). Additionally, a rebound of p70s6k
activity was also observed when tsBN250 cells were transferred to
nonpermissible temperature. These effects might be explained by
proteolytic responses induced by amino acid deprivation. Because TOR
was demonstrated to be involved in autophagic responses in yeast (41),
inhibition of mTOR by amino acid deprivation might induce a proteolytic
response in mammalian cells, which in turn increases intracellular
amino acid concentrations and subsequently reactivates the
mTOR/p70s6k pathway.
In contrast to intensive studies on signaling mechanisms regulated by
growth factors, our understanding of intracellular signal transduction
controlled by nutrients such as amino acids is very limited, especially
in mammalian cells. p70s6k (and potentially its upstream
kinase mTOR) has been shown to be a protein kinase that is tightly
controlled by amino acid concentration. Moreover, the study reported
here demonstrates for the first time the involvement of tRNA
aminoacylation for the regulation. Our data give us a basis for study
directions to reveal an entire signaling mechanism for amino
acid-dependent control of mRNA translation (and
potentially protein degradation as well) in mammalian cells.