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Experimental Therapeutics |
2Department of Immunology [A. S.], Mayo School of Medicine [J. L. H.], and Division of Oncology Research [L. M. K.], Mayo Clinic, Rochester, Minnesota 55905, and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 [C. C. H., P. Y., D. M. O., R. T. A.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The molecular pharmacology underlying the cellular effects of rapamycin is now understood in considerable detail. The principal rapamycin "receptor" is a widely expressed intracellular protein termed FKBP4 -12. In mammalian cells, the interaction of rapamycin with FKBP12 generates a pharmacologically active complex that binds with high affinity to the mTOR [Ref. 4 ; also named FRAP (5) , RAFT1 (6) , or RAPT1 (7) by others]. This rapamycin target protein is a member of a recently described family of protein kinases, termed PIKKs. The PIKK family members share a COOH-terminal catalytic domain that bears significant sequence homology to the lipid kinase domains of PI3Ks (8) . Other members of the PIKK family include TOR1p and TOR2p, the budding yeast orthologues of mTOR. The finding that rapamycin interacts with FPR1p, the budding yeast orthologue of FKBP12, to arrest yeast cell growth in G1 phase (9) suggests that the TOR signaling pathway has been at least partially conserved during eukaryotic evolution.
The specificity of rapamycin as an inhibitor of mTOR function facilitated the identification of the downstream signaling events governed by mTOR in mitogen-stimulated cells. To date, the rapamycin-sensitive signaling activities ascribed to mTOR impinge primarily on the translational machinery. Rapamycin treatment triggers the rapid dephosphorylation and inactivation of p70S6K in mitogen-stimulated cells (10, 11, 12, 13, 14) . The overall effect of p70S6K activation is to stimulate ribosome biogenesis, and, in turn, to increase the capacity of the translational machinery, which allows cells to meet the increased demand for protein synthesis imposed by cell cycle progression (15, 16, 17) . Although p70S6K activation involves a complex series of phosphorylation events catalyzed by multiple protein kinases (18, 19, 20, 21) , the prompt reversal of p70S6K activation by rapamycin (11 , 14) suggests that this protein kinase requires continuous signaling through mTOR to both achieve and maintain the activated state. The exact nature of the input supplied by mTOR remains unclear; however, recent findings suggest that mTOR phosphorylates and suppresses the activity of a type 2A protein phosphatase bound directly to p70S6K (22) . Hence, rapamycin treatment may inactivate p70S6K by removing a mTOR-dependent inhibitory constraint on the activity of a p70S6K-targeted type 2A protein phosphatase PP2A.
A second downstream protein targeted by mTOR is the translational repressor, PHAS-I, also termed 4E-BP1. PHAS-I represses translation initiation through association with eIF-4E, the mRNA cap-binding subunit of the eIF-4F complex. The binding of PHAS-I to eIF-4E blocks assembly of the eIF-4F complex at the 5'-cap structure of the mRNA template, thereby decreasing the efficiency of translation initiation (23) . Stimulation of cells with insulin or growth factors causes the phosphorylation of PHAS-I at five sites, which leads to the release of eIF-4E, and, in turn, an increase in eIF-4F-dependent translation initiation (24, 25, 26, 27, 28, 29) . The phosphorylation of PHAS-I induced by hormonal stimuli is strongly inhibited by rapamycin (27 , 30 , 31) . Earlier results suggested that mTOR is directly responsible for the phosphorylation of PHAS-I in intact cells (32 , 33) , although additional proline-directed kinases appear to be required for full phosphorylation of PHAS-I in insulin-stimulated cells (34) .
The signaling pathway that couples growth factor receptor occupancy to mTOR activation is only partially understood. However, accumulating evidence places mTOR downstream of both PI3K and the PI3K-regulated protein kinase, AKT (also termed PKB), in growth factor-stimulated cells. This model is based in part on genetic and pharmacological evidence that links activation of PI3K and/or AKT to the two intracellular events known to be governed by mTOR, the activation of p70S6K and the phosphorylation of PHAS-I (35) . The notion that mTOR participates in signaling downstream from PI3K may be particularly relevant to the antitumor activity of rapamycin. Recent studies have identified a negative regulator of PI3K-mediated signaling, PTEN, as a tumor suppressor gene product (36) . The tumor suppressor function of PTEN is attributed to its activity as a phosphoinositide 3-phosphatase, which effectively terminates PI3K-mediated signaling via dephosphorylation of the second messengers, phosphatidylinositol-3,4,5-trisphosphate and phosphatidylinositol-3,4-bisphosphate. Thus, loss of PTEN function leads to hyperactivation of the PI3K signaling cascade, which promotes abnormal cell growth, survival, and migration.
The importance of PI3K signaling during tumorigenesis is underscored by observations that mutations in the PTEN gene occurs frequently in a variety of human cancers, including prostate cancer and glioblastoma (37) . If mTOR also resides downstream of PI3K and/or AKT, then mTOR activity should also be deregulated in PTEN-deficient tumor cells, and consequently, PTEN status might be an important predictor of cancer cell sensitivity to the mTOR inhibitor, rapamycin. Given these speculative arguments, it becomes increasingly important to define the interactions among PI3K, AKT, and mTOR as the rapamycin analogue, CCI-779, moves into clinical trials in patients with different types of cancer.
At the inception of this study, the most direct evidence for epistatic relationships among PI3K, AKT, and mTOR stems from results obtained with a polyclonal antibody, termed mTAb1, which recognizes a COOH-terminal peptide sequence in mTOR (residues 2433–2450; Ref. 38 ). The authors noted that cellular stimulation with insulin, or expression of mutationally activated AKT, caused a decrease in the immunoreactivity of mTOR in anti-mTAb1 immunoblot analyses (39) . The loss of mTAb1 binding activity was reversed by treatment of the immunoprecipitated mTOR with a protein phosphatase prior to immunoblot analysis. Collectively, these results suggested that insulin or AKT stimulation caused the phosphorylation of mTOR at a site(s) that resulted in a decrease in the recognition of this protein by the mTAb1 antibody.
The goal of the present study was to further understand the role of the PI3K-AKT signaling pathway in the regulation of mTOR function by extracellular stimuli. We demonstrate that stimulation of myeloid progenitor cells with IL-3 triggers a rapid increase in the protein kinase activity of mTOR. The IL-3-dependent increase in mTOR activity is blocked by low concentrations of the PI3K inhibitor, wortmannin. Furthermore, we provide in vitro and in vivo evidence that AKT phosphorylates mTOR at a site(s) located in a region that represses the catalytic activity of the mTOR kinase domain. Deletion of this repressor domain generates a mTOR mutant bearing a constitutively elevated level of protein kinase activity. These findings outline a direct linkage between the PI3K-AKT pathway and mTOR and suggest that deregulated signaling through mTOR may contribute to the transformed phenotype of PTEN-deficient cancer cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ile mutation and are usually
designated with the suffix "SI" (e.g., AmTOR-SI).
PCR-based mutagenesis was used to construct single and double mTOR
point mutants in which Thr2446 and
Ser2448 were changed to alanine (A) residues. The
internal deletion mutant, AmTOR
, which lacks amino acids 2430–2450,
was prepared by the PCR-based SOEing technique (40)
. The
expression vectors for HA-tagged wild-type AKT (cAKT), catalytically
inactive AKT (AKT-kd) and the constitutively active myristylated form
of AKT (myrAKT), were kind gifts from P. N. Tsichlis (Fox-Chase Cancer
Center, Philadelphia, PA). The cDNA encoding
p70S6K (kindly provided by Dr. Naohiro Terada,
National Jewish Medical and Research Center, Denver, CO) was appended
with nucleotides encoding an NH2-terminal FLAG
epitope tag and was cloned into pcDNA3 using EcoRI and
XbaI restriction sites. All PCR products were subcloned and
then sequenced to ensure the fidelity of the amplification step. Recombinant murine IL-3 was purchased from R&D Systems, Inc. (Minneapolis, MN). Recombinant human insulin (Recombulin) and G418 (Geneticin) were obtained from Life Technologies, Inc. (Gaithersburg, MD), and FuGene transfection reagent was purchased from Boehringer Mannheim (Indianapolis, IN). Wortmannin (Sigma) was dissolved in DMSO (Me2SO) to yield a 1.2 mM stock solution. The wortmannin stock solution was aliquoted and stored at -70°C. Rapamycin (Sigma) was prepared as a 10 µM stock solution in ethanol and aliquoted and stored as described above.
The 
-AU1 and 12CA5 (-HA) mAbs were purchased from Babco
(Richmond, CA), and the -mTOR monoclonal antibody, 26E3, was a
generous gift from Dr. Peter Houghton (St. Jude Children’s Research
Hospital, Memphis, TN). Peptides corresponding to amino acid residues
2433–2450 in mTOR were synthesized (Research Genetics, Huntsville, AL)
with or without phosphate at either or both of the underlined residues
in the sequence CDTNAKGNKRSRTRTDSYS. Polyclonal
antibodies directed against the nonphosphorylated peptide were raised
by immunizing rabbits with the peptide coupled to keyhole limpet
hemocyanin. The antiserum (designated -mTOR 367) was
affinity-purified over a peptide-coupled Sulfolink bead column
according to the manufacturer’s procedure (Pierce, Rockford, IL).
Phosphospecific antibodies were prepared in a similar fashion, except
that a keyhole limpet hemocyanin-coupled, dually phosphorylated peptide
(containing phosphate at both Thr2446 and
Ser2448 served as the immunogen. The resulting
antiserum was first passed over a column consisting of
nonphosphorylated peptide immobilized on Sulfolink beads, and the
flow-through fraction was then passed through a second column
containing the immobilized, dually phosphorylated peptide. The bound
antibodies (designated -mTORp2) were eluted at low pH and were
stored in PBS containing 0.05% azide.
Cell Culture and Transfections.
The murine IL-3-dependent myelomonocytic progenitor cell line, FDC-P1,
was cultured in standard growth medium [RPMI 1640 supplemented with
10% (v/v) FBS (Hyclone, Logan, UT), 2 mM
L-glutamine, 50 µM 2-mercaptoethanol, 10
mM HEPES (pH 7.2), and 10% (v/v) WEHI-3 cell-conditioned
medium as a source of IL-3]. Stably transfected FDC-P1 cells
expressing AmTORwt were prepared by suspending
1 x 107 exponentially growing
cells in 350 µl of standard growth medium at 4°C. The cells were
mixed with a total of 45 µg of plasmid DNA suspended in the same
medium. Mock transfectants received 45 µg of pcDNA3 only, whereas
mTOR transfectants were electroporated with 25 µg of mTOR-encoding
plasmid plus 20 µg of pcDNA3 as filler. Prior to electroporation, the
cell-DNA mixtures were incubated for 10 min at room temperature. The
cells were electroporated with a BTX model T820 square-wave
electroporator (San Diego, CA) at a setting of 350 V (10-ms pulse
duration). The electroporated cells were mixed gently and then allowed
to stand at room temperature for an additional 10 min. The cells were
then diluted into 20 ml of standard growth medium and cultured for
24 h, at which time the cells were transferred into fresh growth
medium containing 800 µg of G418/ml. Stable clones that expressed
AmTORwt and AmTORkd were
isolated by limiting dilution, and expression levels of the transfected
proteins were assessed by immunoblotting with the AU1 mAb.
For experiments, exponentially growing FDC-P1 cells (2 x 107 cells/sample were washed twice in PBS. The cells were resuspended in 20 ml of starvation medium [RPMI 1640 containing 100 µg/ml BSA, 2 mM L-glutamine, and 50 µM 2-mercaptoethanol, buffered to pH 7.2 with 10 mM HEPES]. After 4–6 h in culture, the factor-deprived cells were treated for 30 min with the indicated pharmacological inhibitors and then were restimulated with either 30 ng/ml IL-3 or 20% FBS.
HEK 293 and 293T cells were maintained in monolayer cultures in DMEM (Life Technologies, Inc.) supplemented with 10% FBS or 5% FBS, respectively. Prior to transfection, 6 x 105 cells were seeded into a 60-mm tissue culture dish. The cells were cultured for 24 h under standard conditions and then were transfected with a total of 5 µg of plasmid DNA mixed with 8 µl of FuGene transfection reagent/dish. The standard amounts of plasmid DNAs used for each transfection were: AKT, 0.25 µg; mTOR, 3 µg; and p70S6K, 2 µg. When necessary, the total amount of plasmid DNA was brought to 5 µg by addition of the empty pcDNA3 expression vector. The transfected cells were cultured for 16 h, washed one time in PBS, and arrested for 24 h in serum-free DMEM. The serum-deprived cells were pretreated for 30 min with wortmannin or rapamycin and then stimulated with insulin for the indicated times. The procedures for transfection of HEK 293T cells were similar, except that the cells were deprived of serum for 2 h prior to drug treatment.
DU 145 and PC-3 prostate cancer cells were maintained in monolayer cultures in RPMI 1640 supplemented with 10% FBS. Prior to assay, 2 x 105 cells were seeded in 60-mm tissue culture dishes. After 24 h, the cells were transferred into serum-free RPMI 1640 and were cultured for an additional 20 h. The cells were washed in PBS and lysed in LB buffer [25 mM Tris-HCl, pH 7.4, 50 mM NaCl, 10% (w/v) glycerol, 1% Triton X-100, 50 mM ß-glycerophosphate, 20 nM microcystin- LR, 100 µg/ml PMSF, and protease inhibitor cocktail (5 µg/ml aprotinin, 5 µg/ml pepstatin, and 10 µg/ml leupeptin)]. The lysates were cleared of insoluble material, and the cleared extracts were assayed for total protein to equalize sample loading prior to SDS-PAGE.
Immunoprecipitations.
Mock-transfected or AmTOR-transfected FDC-P1 cells
(2 x 107 cells/sample) were
growth factor deprived and restimulated as described above. The cells
were washed in PBS and lysed by sonication in 1 ml of buffer L [50
mM Tris-HCl, 50 mM ß-glycerophosphate, 100
mM NaCl (pH 7.4), containing 10% glycerol, 0.2% Tween 20,
1 mM DTT, 1 mM
Na3VO4, 1 mM
MgCl2, 50 nM microcystin-LR, 1
mM PMSF, and protease inhibitor cocktail]. The lysates
were cleared of insoluble material by centrifugation, and the extracts
were immunoprecipitated with 1 µl of -AU1 mAb for 2 h at
4°C. The immunoprecipitates were washed three times in buffer W [50
mM Tris-HCl, 50 mM ß-glycerophosphate, 100
mM NaCl (pH 7.4), containing 10% glycerol, 0.2% Tween 20,
and 1 mM DTT] and twice in buffer K [10 mM
HEPES, 50 mM NaCl, 50 mM ß-glycerophosphate
(pH 7.4), 50 nM microcystin-LR, and the protease inhibitor
cocktail].
Immunoblot Analyses.
For immunoblot analyses with -mTOR 367 or -mTORp2 antibodies,
recombinant AmTOR was immunoprecipitated with the tag-specific -AU1
mAb from transfected FDC-P1, HEK 293, or HEK 293T cells. The
immunoprecipitated proteins were separated by SDS-PAGE through 6%
polyacrylamide gels. After transfer to Immobilon-P, the membranes were
blocked and probed with 5 µg per ml affinity-purified antibodies in
Tris-buffered saline-0.2% Tween 20 (TBST) containing 2% (w/v) BSA
(for -mTORp2 antibodies) or 5% milk (for -mTOR 367 antibodies).
Immunoreactive proteins were detected with horseradish
peroxidase-conjugated to protein A and the Renaissance reagent (New
England Nuclear, Boston, MA). The blots were then stripped and reprobed
with the -AU1 mAb in TBST-milk solution. The phosphorylation state
of endogenous mTOR in DU 145 or PC-3 prostate cancer cells was analyzed
by immunoblotting with -mTORp2 as described above, followed by
reblotting of the same membrane with the -mTOR mAb, 26E3, in
TBST-milk solution.
Kinase Assays.
The protein kinase activity of immunoprecipitated mTOR was assayed with
recombinant PHAS-I as the substrate (Stratagene, La Jolla, CA; Ref.
32
). The samples were separated by SDS-PAGE, and
radiolabeled PHAS-I was detected by autoradiography. Incorporation of
32P into PHAS-I was quantitated with a Molecular
Dynamics Storm 840 Phosphorimager (Sunnyvale, CA) and ImageQuant
software.
Phosphorylation of mTOR by AKT in vitro was performed by
transfection of AmTORwt and HA-tagged myrAKT,
c-AKT, or catalytically inactive AKT into separate populations of HEK
293 cells. The cells were plated in 60-mm culture dishes and were
transfected as described above. After 16 h, the transfected cells
were transferred into serum-free DMEM and cultured for 24 h.
Cellular extracts were prepared by removal of the culture medium,
followed by addition of 400 µl of buffer P per dish [50
mM Tris-HCl, 100 mM NaCl,
50 mM ß-glycerophosphate (pH 7.4), containing
10% (w/v) glycerol, 1% Triton X-100, 1 mM DTT,
50 nM microcystin, 1 mM
PMSF, and protease inhibitor cocktail]. The detached cells were
disrupted by sonication, and cleared extracts from the HA-tagged
AKT-expressing cells and the AmTORwt-expressing
cells were mixed at a total protein ratio of 1:9. The epitope-tagged
mTOR and AKT proteins were coimmunoprecipitated with 1 µl of AU1 mAb
and 5 µg of 12CA5 mAb bound to protein A-Sepharose beads that had
been precoupled to rabbit antimouse immunoglobulin antibodies. The
immunoprecipitates were washed three times in buffer N [25
mM HEPES (pH 7.6), 0.5 M
NaCl, 10% glycerol, 1 mM
Na3VO4, and 0.2% Tween
20] and two times in kinase buffer F [50 mM
Tris (pH 7.5), 10 mM MgCl2,
50 mM
Na3VO4, and 1
mM DTT]. The coimmunoprecipitated proteins were
incubated for 50 min at 30°C in 20 µl of kinase buffer F
supplemented with 10 µM ATP and 20 µCi of
[
-32P]ATP (specific activity, 4500 Ci/mmol).
The reaction products were separated by SDS-PAGE and transferred to an
Immobilon-P membrane. The incorporation of 32P
into wild-type or mutated forms of AmTOR was detected by
autoradiography and quantitated by phosphorimager analysis as described
above.
To measure the activity of transiently transfected FLAG-p70S6K, serum-deprived HEK 293 cells were prepared as described above. The cells were stimulated with 1 µM insulin and then lysed in TNEE buffer [50 mM Tris-HCl, 150 mM NaCl, 2.5 mM EDTA, 2 mM EGTA, 25 mM ß-glycerophosphate, 25 mM NaF (pH 7.5), containing, 0.5% Triton X-100, 100 µM sodium orthovanadate, 2 mM DTT, and protease inhibitor cocktail]. The epitope-tagged p70S6K was immunoprecipitated from cellular extracts with anti-FLAG M2 affinity resin (Sigma Chemical Co., St. Louis, MO), and protein kinase activity was determined with a p70S6K assay kit (Upstate Biotechnology, Inc., Lake Placid, NY).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the initial studies, AmTORwt-expressing cells
were deprived of serum and IL-3 for 6 h and then were restimulated
for 10 min with IL-3 prior to the preparation of cellular extracts. The
extracts were immunoprecipitated with -AU1 mAb, and mTOR kinase
activities were determined with PHAS-I as the substrate. Parallel
samples were prepared from identically treated cells that expressed the
AmTORkd mutant. Stimulation of
AmTORwt-expressing FDC-P1 cells with IL-3
significantly increased the in vitro kinase activity of the
immunoprecipitated AmTORwt but did not change the
amount of AmTORwt in these immunoprecipitates
(Fig. 1A
, left panel). The activation of mTOR by IL-3 was
maximal at 5–10 min after cytokine stimulation and then dropped to a
lower, but still elevated, plateau level of activity that was sustained
for at least 4 h after cytokine addition (Fig. 1B
and
data not shown). In contrast, AU1 immunoprecipitates from either
mock-transfected or AmTORkd-expressing cells
contained low levels of background protein kinase activity that was not
substantially increased by cellular stimulation with IL-3.
Interestingly, serum-starved FDC-P1 cells also displayed a clear
increase in AmTORwt activity after a 10-min
exposure to fresh serum (Fig. 1A
, right panel).
Thus, ligation of receptors for IL-3, as well as undefined serum
components (possibly insulin-like growth factors), initiates a
signaling pathway leading to mTOR activation in FDC-P1 cells.
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100
nM (42)
. As shown in Fig. 2
, the activation of AmTORwt by IL-3 was virtually
abrogated by pretreatment of the FDC-P1 cells with 10 nM
wortmannin. Thus, the sensitivity of IL-3-dependent mTOR activation to
wortmannin strongly suggests that this response is dependent on the
activation of PI3K.
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-mTOR 367) specific for
the same region of mTOR (amino acid residues 2433–2450), we observed a
similar time-dependent decrease in the immunoreactivity of AU1-tagged
mTOR isolated from IL-3-stimulated FDC-P1 cells (Fig. 3
, upper panel). Interestingly, the time course of the
alteration in anti-mTOR antibody reactivity corresponded closely to the
changes in mTOR kinase activity induced by IL-3 (Fig. 1B)
.
The IL-3-dependent decrease in mTOR immunoreactivity was abrogated by
pretreatment of the cells with 100 nM wortmannin,
suggesting that this alteration was mediated through the activation of
PI3K. As will be described below, parallel immunoblot analyses with a
phospho-mTOR-specific antibody (-mTORp2) indicated that the decrease
in -mTOR 367 reactivity induced by IL-3 stimulation is attributable
to the phosphorylation of at least one amino acid
(Ser2448) located within the -mTOR 367 epitope
(Fig. 3
, middle panel; see below for description).
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-mTOR 367
antibodies revealed that this region contained two consensus
phosphorylation sites (Thr2446 and
Ser2448) for AKT (Fig. 4)
. To determine whether mTOR was an in vitro substrate for
AKT, we expressed AmTORwt, HA-tagged wild-type
AKT (cAKT), activated AKT (myrAKT), or a catalytically inactive AKT
(AKTkd) in different populations of HEK 293 cells. Cellular extracts
were then mixed, and AmTORwt was
coimmunoprecipitated with each form of AKT. The mixed immune complexes
were then subjected to in vitro kinase assays. As shown in
Fig. 5A
, a relatively low level of phosphorylation was observed in
immunoprecipitates containing AmTORwt only. This
background phosphorylation reflects the in vitro autokinase
activity of mTOR (43
, 44)
. The incorporation of
radiolabeled phosphate into AmTORwt was strongly
increased by coimmunoprecipitation with the activated form of AKT
(myrAKT) and, to a lesser extent, with wild-type AKT. The increase in
mTOR phosphorylation was dependent on the protein kinase activity of
AKT, because coimmunoprecipitation of mTOR with a catalytically
inactive form of AKT failed to enhance the phosphorylation of mTOR in
the immune complex kinase assay. Identical results were obtained when
the different AKT proteins were coimmunoprecipitated with a
catalytically inactive version of AmTOR, indicating that mTOR kinase
activity was not required for the in vitro phosphorylation
of the mTOR polypeptide by AKT (results not shown).
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-mTOR 367 epitope were modified by AKT in
vitro, we coimmunoprecipitated myrAKT with mutated mTOR
polypeptides containing single or double Ala substitutions at
Thr2446 and Ser2448. The
results presented in Fig. 5B
demonstrate that Ala
substitutions at both Thr2446 and
Ser2448 (the "AA" mutant) almost completely
eliminated the in vitro phosphorylation of AmTOR by myrAKT.
Single alanine substitutions at Thr2446 ("TA"
mutant) or Ser2448 ("SA" mutant) each showed
an approximate 50% reduction in phosphorylation by myrAKT (data not
shown). Hence, both of the identified sites within the -mTOR 367
binding site were targeted for modification by myrAKT in
vitro. Deletion of the entire -mTOR 367 target sequence
(residues 2430–2450) decreased the phosphorylation of the resulting
"mTOR-" mutant to a level similar to that obtained with the AA
double mutant. Thus, the in vitro phosphorylation of mTOR by
myrAKT occurs largely, if not entirely, at two closely spaced residues
(Thr2446 and Ser2448)
located within the peptide sequence recognized by -mTOR 367 and
mTAb1 (39)
antibodies.
Phosphorylation of mTOR at Ser2448 in Intact Cells.
The observation that AKT phosphorylates the COOH terminal region of
mTOR prompted efforts to determine whether these residues underwent
reversible modification in growth factor- or hormone-stimulated cells.
To examine the phosphorylation states of Thr2446
and Ser2448 in intact cells, we generated
phospho-mTOR-specific (-mTORp2) antibodies (see "Materials and
Methods" for details). These antibodies specifically recognized
phosphopeptides corresponding to amino acids 2433–2450 of mTOR and
containing phosphate at either Thr2446 or
Ser2448, as well as the doubly phosphorylated
peptide.5
To determine whether physiological stimuli trigger the phosphorylation
of these sites, we first returned to the protein blot shown in Fig. 3
.
When this blot was stripped and reprobed with -mTORp2 antibodies, we
observed that IL-3 stimulation caused a prompt increase in the
reactivity of mTOR with these phosphospecific antibodies (Fig. 3
,
middle panel). Notably, the time-dependent increase
in -mTORp2 immunoreactivity mirrored precisely the decrease in
-mTOR 367 binding provoked by IL-3 stimulation (Fig. 3
, upper
panel). Pretreatment of these cells with 100
nM wortmannin blocked the increase in -mTORp2
binding stimulated by IL-3, indicating that this alteration was
dependent on the activation of PI3K.
Transient transfection studies in HEK 293 cells revealed that
insulin stimulation also provoked a rapid increase in the
-mTORp2 reactivity of the rapamycin-resistant AmTOR-SI mutant
(Fig. 6A)
. AmTOR-SI contains a single amino acid substitution [Ser
(2035)Ile] that renders the hormone-dependent signaling functions
of this protein kinase resistant to rapamycin in intact cells (see
below for additional explanation). It should be noted that this
upstream "SI" mutation did not influence the binding of -mTORp2
to the ectopically expressed mTOR, because identical results were
obtained in HEK 293 cells transfected with the AmTOR-WT-encoding
plasmid (results not shown). To determine whether
Thr2446 or Ser2448, or both
sites, were targeted for phosphorylation in vivo, cells were
transfected with AmTOR-SI expression constructs containing single or
double mutations at Ser2448 and
Thr2446 (designated SA, TA, and AA,
respectively). Stimulation of serum-starved HEK 293 cells for 10 min
with insulin caused a wortmannin-sensitive increase in the reactivity
of AmTOR-SI (designated WT in the figure) with -mTORp2 antibodies.
Virtually identical results were obtained when the TA mutant was
immunoblotted with -mTORp2 antibodies, indicating that
Thr2446 was not a major phosphorylation site in
insulin-stimulated cells. In contrast, both the SA and AA mutants
failed to react with -mTORp2 antibodies, either before or after
insulin stimulation. To determine whether the kinase activity of mTOR
itself was required for the phosphorylation of
Ser2448, we transfected HEK 293 cells with the
catalytically inactive AmTOR-SI/kd double mutant. As shown in the final
lane of Fig. 6A
, insulin treatment also triggers the
phosphorylation of AmTOR-SI/kd, which indicates that this modification
is not explained by the previously reported autokinase activity of mTOR
(44)
.
|
. The -mTORp2 reactivities of both
the WT and TA forms of AmTOR-SI were markedly increased in
myrAKT-expressing cells. Once again, mutation of
Ser2448 to Ala in the SA and AA mutants caused a
near-total loss of the myrAKT-dependent increase in -mTORp2 binding.
Although we consistently observed a slight increase in -mTORp2
reactivity after coexpression of the SA mutant with myrAKT, the
physiological relevance of this residual Thr2446
phosphorylation remains unclear.
Impact of AKT-mediated Phosphorylation on mTOR Signaling Functions.
To examine the role of Ser2448 phosphorylation in
mTOR signaling functions, we focused on the two known downstream
targets in this pathway, p70S6K and the
translational repressor, PHAS-I. Because qualitatively similar results
were obtained with both of these downstream reporters for mTOR
activity, only the data obtained with p70S6K will
be presented in this report. The studies were performed by transfection
of HEK 293 cells with the various AmTOR-SI constructs, together with a
FLAG-tagged p70S6K expression plasmid. As
explained above, the Ser (2035) to Ile mutation (SI) renders the
recombinant AmTOR-SI resistant to inhibition by the FKBP12·rapamycin
complex. Thus, when the transfected cells are treated with rapamycin,
endogenous mTOR activity is strongly suppressed, and insulin-mediated
p70S6K activation is contingent on the function
of the transiently expressed AmTOR-SI protein (32)
. In the
control sample, transiently transfected wild-type (WT) AmTOR failed to
support insulin-stimulated activation of p70S6K
in the presence of rapamycin (Fig. 7A)
. In contrast, cells transfected with the
rapamycin-resistant mTOR-SI mutant displayed a robust increase in
p70S6K activity in response to insulin.
Surprisingly, AmTOR-SI constructs containing mutations at either
Ser2448 (SI/SA) or Thr2446
(SI/TA) or both sites (SI/AA) were as effective as the intact mTOR-SI
in supporting insulin-stimulated p70S6K
activation. Similar results were obtained when cotransfected myrAKT,
rather than exogenous insulin, served as the stimulus for
p70S6K activation or PHAS-I phosphorylation (data
not shown).
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. The results clearly
indicated that, even under conditions in which AmTOR-SI expression was
limiting for p70S6K activation, loss of the
Thr2446 and/or Ser2448
phosphoacceptor sites had no effect on the coupling of insulin receptor
occupancy to p70S6K activation. Once again,
similar results were obtained when the titration study was repeated
with myrAKT as the stimulus for p70S6K activation
(results not shown).
Amino Acids 2430–2450 of mTOR Define a "Repressor" Domain.
Although mutations of the AKT phosphorylation sites in mTOR failed to
interfere with either p70S6K activation or PHAS-I
phosphorylation in HEK 293 cells, we remained intrigued by the
observation that antibody binding to amino acids 2433–2450 of mTOR
significantly increased the protein kinase activity of mTOR in
vitro (38)
. To further investigate the biochemical
basis of this phenomenon, we deleted amino acids 2430–2450 from both
the AmTOR and AmTOR-SI constructs to create the corresponding ""
mutants. AmTOR- was expressed in HEK 293 cells, and the PHAS-I
kinase activity of this protein was determined under linear in
vitro reaction conditions (Fig. 8A)
. The AmTOR- mutant consistently displayed an elevated
level of protein kinase activity relative to the full-length
AmTORwt protein. When corrected for the
background activity present in -AU1 immunoprecipitates from
mock-transfected cells (first lane in Fig. 8A
),
the protein kinase activity of AmTOR- was 3.5–10-fold higher than
that of wild-type AmTOR over five independent trials.
|
mutant also displayed enhanced
signaling function in vivo, we compared the abilities of
AmTOR-SI and AmTOR-SI/ to support p70S6K
activation in rapamycin-treated HEK 293 cells. Over a broad range of
expression levels, the AmTOR-SI/ deletion mutant consistently drove
p70S6K activity in serum-starved HEK 293 cells to
at least a 3-fold higher level than was obtained in cells expressing
full-length AmTOR-SI (Fig. 8B)
. The increase in
FLAG-p70S6K activity induced by AmTOR-SI/ was
paralleled by the appearance of a more slowly migrating form of
FLAG-p70S6K, indicating that expression of this
activated form of mTOR was sufficient to increase the phosphorylation
of p70S6K in serum-starved cells (Fig. 8B
, inset). Despite the increase in basal
p70S6K activity provoked by AmTOR-SI/, insulin
stimulation was still required for maximal phosphorylation and
activation of p70S6K. Qualitatively similar
results were obtained when PHAS-I was substituted for
p70S6K as the reporter for signaling through
mTOR. The fact that expression of AmTOR-SI/ does not fully replace
the requirement for insulin is consistent with models suggesting that
both mTOR-dependent and -independent signaling pathways contribute to
p70S6K activation and PHAS-I phosphorylation in
hormone-stimulated cells (18, 19, 20, 21
, 34) .
Phosphorylation of mTOR in Prostate Cancer Cell Lines.
The finding that AKT is an upstream protein kinase for mTOR prompted
speculation that cancer cells bearing mutations leading to autonomous
activation of the PI3K-AKT pathway might show constitutive
phosphorylation of mTOR at Ser2448. The human
prostate cancer cell line, PC-3, exhibits constitutive activation of
the PI3K-AKT pathway because of loss of function mutations in the tumor
suppressor gene, PTEN, together with overexpression of the
AKT3 isoform (45)
. A second prostate cancer cell line, DU
145, retains functional PTEN but displays a 20–40-fold increase in
AKT3 protein expression and enzyme activity (45)
. The
growth of these cell types, as measured in clonogenic assays, is
strongly inhibited by treatment with 20 nM
rapamycin.6
In the present studies, we examined the phosphorylation of mTOR at
Ser2448 under serum starvation conditions and
after restimulation with serum. In HEK 293 cells, phosphorylation of
endogenous mTOR at Ser2448 was strongly dependent
on cellular stimulation with insulin or serum (Fig. 9)
. In contrast, Ser2448 phosphorylation in DU 145
and PC-3 cells was substantially resistant to serum starvation. Thus,
prostate cancer cell lines that contain overexpressed and/or
persistently activated AKT also maintain the phosphorylation of
Ser2448 in mTOR in the absence of serum-derived
growth factors.
|
|
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the initial experiments, we examined the effect of IL-3 stimulation on mTOR kinase activity in FDC-P1 myeloid progenitor cells that stably expressed either AmTORwt or AmTORkd. Like the parental cell line, the transfected cells require IL-3 and serum for survival and proliferation, and the cell growth rate is reduced by 50–60% in the presence of rapamycin.7 Restimulation of AmTORwt-expressing cells with either IL-3 or serum triggered a rapid increase in the protein kinase activity of AmTORwt, as measured in immune complex kinase assays with PHAS-I as the substrate. The time course of IL-3-dependent mTOR activation was nearly identical to that reported with insulin-stimulated 3T3-L1 adipocytes (39) or serum-stimulated HEK 293 cells (33) . An essential role for PI3K in this pathway was clearly suggested by the sensitivity of IL-3-dependent mTOR kinase activation to a low concentration (10 nM) of the PI3K inhibitor, wortmannin. The potency of wortmannin as an inhibitor of IL-3-dependent mTOR activation is considerably greater than that reported for the mTOR kinase itself (44) , which supports the conclusion that a wortmannin-sensitive PI3K activity mediates the activation of mTOR in response to IL-3 receptor occupancy.
The first evidence supporting a direct role for a PI3K-dependent kinase
in the regulation of mTOR function was reported by Scott et
al. (39)
, who showed that AKT activation triggered a
modification of mTOR that resulted in a decrease in the reactivity of
mTAb1 antibodies with the full-length protein. We confirmed this result
with our own -mTOR 367 antibodies, which bind to the same region of
mTOR (residues 2433–2450) as that recognized by mTAb1. The peptide
sequence recognized by the mTAb1 and -mTOR 367 antibodies is notable
in several respects. Among the members of the TOR protein family, the
mTAb1/-mTOR 367 target sequence is unique to the mammalian TOR
protein. The absence of this stretch of amino acids from the yeast
orthologues, TOR1p and TOR2p, suggested that these residues may have
been inserted during metazoan evolution to receive a regulatory,
hormonal input not present in single yeast cells. Furthermore, we
surmised that the -mTOR 367 epitope, although located downstream of
the catalytic domain, may allosterically regulate the protein kinase
activity of mTOR, because the binding of mTAb1 or -mTOR 367
antibodies stimulated mTOR kinase activity
(38)
.4 Finally, the presence of two
consensus phosphorylation sites for AKT in this region suggested that
amino acid residues 2433–2450 of mTOR conferred responsiveness to an
afferent regulatory signal(s) triggered by the activation of PI3K.
Our studies with the phosphospecific -mTORp2 antibodies demonstrate
that Ser2448 is rapidly phosphorylated in both
IL-3- and insulin-stimulated cells. In contrast to
Ser2448, the second candidate AKT phosphorylation
site, Thr2446, was detectably modified only in
HEK 293T cells that overexpressed activated myrAKT. These results
strongly suggest that cytokine-dependent phosphorylation of mTOR is
carried out, at least in part, by AKT, and that
Ser2448 is the major site of modification,
whereas Thr2446 is at best a minor
phosphorylation site in insulin-stimulated cells. It should be noted,
however, that our conclusion concerning the phosphorylation site
preference of AKT is based on studies with mTOR mutants containing Ala
substitutions at either Thr2446 or
Ser2448. At this point, we cannot rule out the
possibility that mTOR phosphorylation during hormone or growth factor
stimulation is processive, with Ser2448
phosphorylation setting the stage for Thr2446
phosphorylation by AKT or another protein kinase. While this manuscript
was in preparation, Nave et al. (46)
also
identified Ser2448 in mTOR as a phosphorylation
site for AKT in insulin-stimulated HEK 293 cells; however, this study
did not address the functional significance of
Ser2448 phosphorylation in any biological assays.
Nonetheless, two independent studies now suggest that hormonally
activated AKT regulates mTOR function through phosphorylation of the
COOH-terminal region at Ser2448. The exact nature
of this AKT-regulated function remains a centrally important question.
Our results indicate that mutations of Ser2448
and/or Thr2446 to nonphosphorylatable alanine
residues had no detectable effect on the abilities of the resulting
AmTOR-SI mutants to support insulin- or myr-AKT-stimulated
p70S6K activation and PHAS-I phosphorylation in
rapamycin-treated HEK 293 cells. Although we cannot completely rule out
artifacts attributable to overexpression of the recombinant AmTOR-SI
proteins in HEK 293 cells, titration studies with plasmids encoding the
AmTOR-SI phosphorylation site mutants failed to uncover any
quantitative defect attributable to the lack of
Ser2448 and/or Thr2446
phosphorylation.
A more provocative explanation for the present findings is that
phosphorylation of mTOR by AKT is required for the transmission of
signals other than those leading to modulation of
p70S6K or PHAS-I activities. A relevant example
stems from recent findings that mTOR kinase activity is involved in
regulation of the activities of novel PKC isoforms (PKC-
and
PKC-
; Ref. 47
). These studies identified a hydrophobic
sequence located in the PKC- and PKC- COOH-terminal regions, the
phosphorylation of which is rapamycin sensitive and mTOR dependent in
serum-stimulated cells. Interestingly, the activation of PKC- and
PKC- requires at least one additional phosphorylation event, which
appears to be executed by PDK1. An intriguing possibility is that PI3K
also governs the mTOR-dependent limb of the PKC activation pathway by
triggering the phosphorylation of mTOR at
Ser2448. Given the reported contributions of
these PKC isotypes to mitogenic signaling (48
, 49)
, it
will be important to determine whether this AKT-mediated
phosphorylation event contributes to the regulation of PKC- and
PKC- activities by polypeptide growth factors.
Indirect evidence supporting a regulatory function for
Ser2448 phosphorylation stems from the discovery
that deletion of the region (amino acids 2430–2450) surrounding these
sites generates a hyperactivated version of mTOR. The AmTOR-
deletion mutant displayed a 3.5–10-fold elevation in protein kinase
activity in vitro relative to the full-length
AmTORwt protein. In intact cells, expression of
AmTOR-SI/ significantly increased the basal levels of
p70S6K activity and PHAS-I phosphorylation
(results not shown) in serum-starved and rapamycin-treated HEK 293
cells and enhanced both responses when the starved cells were
stimulated with insulin. On the basis of the biochemical and phenotypic
consequences of deleting amino acids 2430–2450 from mTOR, we propose
that this region serves as a "repressor domain," the function of
which is normally modulated by growth factors and other stimuli that
affect mTOR-dependent signaling. The present findings suggest that
Ser2448 phosphorylation is not sufficient to
relieve the inhibitory activity of the repressor domain on mTOR kinase
activity. However, the presence of six serine or threonine residues in
the repressor domain hints that this region might channel inputs from
multiple upstream kinases to the mTOR catalytic domain. The generation
of mTOR constructs bearing additional point mutations and internal
deletions within the repressor domain will help to unravel its roles in
the regulation of mTOR kinase activity and function in intact cells.
Rapamycin displays potent cytostatic activities against certain tumors and may augment tumor cell killing by certain anticancer agents (50) . Early observations demonstrated that different tumor cell lines displayed highly variable sensitivities to rapamycin. The phenotypic parameters that govern tumor cell sensitivity versus resistance to rapamycin are poorly understood; however, the ability to predict tumor responsiveness to rapamycin therapy becomes more critical as the rapamycin analogue, CCI-779, moves into Phase II clinical trials as an anticancer agent. The present report offers preliminary evidence to support the notion that the status of the PI3K-AKT signaling pathway in cancer cells is an important determinant of the cellular response to rapamycin. The prostate cancer cell lines, DU 145 and PC-3, exhibit hyperactivation of the PI3K-AKT pathway, either through loss of PTEN (PC-3) and/or increased protein expression and enzymatic activity of AKT (DU 145). In each case, we found that the phosphorylation of mTOR at the AKT phosphorylation site (Ser2448) was abnormally persistent under serum starvation conditions. These findings raise the possibility that tumor cells bearing activating mutations in the PI3K pathway are "hard-wired" through mTOR (and hence sensitive to rapamycin) for the stimulation of G1-phase progression. The finding that serum-independent S-phase entry in rodent fibroblasts transformed with activated AKT is exquisitely sensitive to rapamycin supports this model (51) . Clearly, additional experiments are required to establish the relationship between deregulated PI3K-AKT activity and rapamycin sensitivity in human cancer cells. If this hypothesis proves correct, then screening of tumor biopsy specimens for PTEN loss, AKT activation, and mTOR phosphorylation may provide a rational basis for identification of those cancer patients most likely to benefit from therapy with mTOR inhibitors, such as rapamycin or CCI-779.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported by USPHS Grants CA 76193
and CA 52995 (to R. T. A.) from the National Cancer Institute, by a
Collaborative Research Program in Cancer Research Grant (to R. T. A.)
from Glaxo-Wellcome, and by the Mayo Foundation. C. C. H. is the
recipient of postdoctoral fellowship PF-99-100–01-TBE from the
American Cancer Society. R. T. A. is a Glaxo-Wellcome Professor of
Molecular Cancer Biology. 
2 These authors contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at Department of Pharmacology and Cancer Biology, Room C333B
LSRC, Box 3813, Duke University Medical Center, Durham, NC 27710.
Phone: (919) 613-8650; Fax: (919) 684-8461 E-mail: abrah008{at}mc.duke.edu
4 The abbreviations used are: FKBP, FK506-binding
protein; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide
3'-kinase; PIKK, PI3K-related kinases; p70S6K, ribosomal
p70 S6 kinase; eIF, eukaryotic initiation factor; FBS, fetal bovine
serum; IL, interleukin; HEK, human embryonic kidney; HA, hemagglutinin;
mAb, monoclonal antibody; PMSF, phenylmethylsulfonyl fluoride; PDK,
phosphoinositide-dependent kinase; PKC, protein kinase C.
5 C. C. Hudson and R. T. Abraham, unpublished
results.
6 C. C. Hudson, P. Yin, and R. T. Abraham,
unpublished data.
7 A. Sekulic and R. T. Abraham, unpublished
results.
Received 1/10/00. Accepted 4/27/00.
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M. Jankiewicz, B. Groner, and S. Desrivieres Mammalian Target of Rapamycin Regulates the Growth of Mammary Epithelial Cells through the Inhibitor of Deoxyribonucleic Acid Binding Id1 and Their Functional Differentiation through Id2 Mol. Endocrinol., October 1, 2006; 20(10): 2369 - 2381. [Abstract] [Full Text] [PDF]
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M. Hidalgo, J. C. Buckner, C. Erlichman, M. S. Pollack, J. P. Boni, G. Dukart, B. Marshall, L. Speicher, L. Moore, and E. K. Rowinsky A Phase I and Pharmacokinetic Study of Temsirolimus (CCI-779) Administered Intravenously Daily for 5 Days Every 2 Weeks to Patients with Advanced Cancer. Clin. Cancer Res., October 1, 2006; 12(19): 5755 - 5763. [Abstract] [Full Text] [PDF]
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A. Suryawan, J. Escobar, J. W. Frank, H. V. Nguyen, and T. A. Davis Developmental regulation of the activation of signaling components leading to translation initiation in skeletal muscle of neonatal pigs Am J Physiol Endocrinol Metab, October 1, 2006; 291(4): E849 - E859. [Abstract] [Full Text] [PDF]
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 T. C. Vary and C. J. Lynch Meal Feeding Stimulates Phosphorylation of Multiple Effector Proteins Regulating Protein Synthetic Processes in Rat Hearts J. Nutr., September 1, 2006; 136(9): 2284 - 2290. [Abstract] [Full Text] [PDF]
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L. Wang, C. J. Rhodes, and J. C. Lawrence Jr. Activation of Mammalian Target of Rapamycin (mTOR) by Insulin Is Associated with Stimulation of 4EBP1 Binding to Dimeric mTOR Complex 1 J. Biol. Chem., August 25, 2006; 281(34): 24293 - 24303. [Abstract] [Full Text] [PDF]
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P. K. Dash, S. A. Orsi, and A. N. Moore Spatial Memory Formation and Memory-Enhancing Effect of Glucose Involves Activation of the Tuberous Sclerosis Complex-Mammalian Target of Rapamycin Pathway J. Neurosci., August 2, 2006; 26(31): 8048 - 8056. [Abstract] [Full Text] [PDF]
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A. Charest, E. W. Wilker, M. E. McLaughlin, K. Lane, R. Gowda, S. Coven, K. McMahon, S. Kovach, Y. Feng, M. B. Yaffe, et al. ROS Fusion Tyrosine Kinase Activates a SH2 Domain-Containing Phosphatase-2/Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Signaling Axis to Form Glioblastoma in Mice. Cancer Res., August 1, 2006; 66(15): 7473 - 7481. [Abstract] [Full Text] [PDF]
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F. Vega, L. J. Medeiros, V. Leventaki, C. Atwell, J. H. Cho-Vega, L. Tian, F.-X. Claret, and G. Z. Rassidakis Activation of Mammalian Target of Rapamycin Signaling Pathway Contributes to Tumor Cell Survival in Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma. Cancer Res., July 1, 2006; 66(13): 6589 - 6597. [Abstract] [Full Text] [PDF]
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 D. M. Thomson and S. E. Gordon Impaired overload-induced muscle growth is associated with diminished translational signalling in aged rat fast-twitch skeletal muscle J. Physiol., July 1, 2006; 574(1): 291 - 305. [Abstract] [Full Text] [PDF]
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M. I. Lewis, S. C. Bodine, N. Kamangar, X. Xu, X. Da, and M. Fournier Effect of severe short-term malnutrition on diaphragm muscle signal transduction pathways influencing protein turnover J Appl Physiol, June 1, 2006; 100(6): 1799 - 1806. [Abstract] [Full Text] [PDF]
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E. Connolly, S. Braunstein, S. Formenti, and R. J. Schneider Hypoxia Inhibits Protein Synthesis through a 4E-BP1 and Elongation Factor 2 Kinase Pathway Controlled by mTOR and Uncoupled in Breast Cancer Cells Mol. Cell. Biol., May 15, 2006; 26(10): 3955 - 3965. [Abstract] [Full Text] [PDF]
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 H. Ito, H. Aoki, F. Kuhnel, Y. Kondo, S. Kubicka, T. Wirth, E. Iwado, A. Iwamaru, K. Fujiwara, K. R. Hess, et al. Autophagic cell death of malignant glioma cells induced by a conditionally replicating adenovirus. J Natl Cancer Inst, May 3, 2006; 98(9): 625 - 636. [Abstract] [Full Text] [PDF]
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I. Yamaoka, M. Doi, M. Nakayama, A. Ozeki, S. Mochizuki, K. Sugahara, and F. Yoshizawa Intravenous administration of amino acids during anesthesia stimulates muscle protein synthesis and heat accumulation in the body Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E882 - E888. [Abstract] [Full Text] [PDF]
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T. C. Vary and C. J. Lynch Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation Am J Physiol Endocrinol Metab, April 1, 2006; 290(4): E631 - E642. [Abstract] [Full Text] [PDF]
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K. Funai, J. D. Parkington, S. Carambula, and R. A. Fielding Age-associated decrease in contraction-induced activation of downstream targets of Akt/mTor signaling in skeletal muscle Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2006; 290(4): R1080 - R1086. [Abstract] [Full Text] [PDF]
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K. S. Klos, S. L. Wyszomierski, M. Sun, M. Tan, X. Zhou, P. Li, W. Yang, G. Yin, W. N. Hittelman, and D. Yu ErbB2 Increases Vascular Endothelial Growth Factor Protein Synthesis via Activation of Mammalian Target of Rapamycin/p70S6K Leading to Increased Angiogenesis and Spontaneous Metastasis of Human Breast Cancer Cells Cancer Res., February 15, 2006; 66(4): 2028 - 2037. [Abstract] [Full Text] [PDF]
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F. Kaper, N. Dornhoefer, and A. J. Giaccia Mutations in the PI3K/PTEN/TSC2 Pathway Contribute to Mammalian Target of Rapamycin Activity and Increased Translation under Hypoxic Conditions Cancer Res., February 1, 2006; 66(3): 1561 - 1569. [Abstract] [Full Text] [PDF]
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 R. E. Brown, P. L. Zhang, M. Lun, S. Zhu, P. K. Pellitteri, A. Law, G. C. Wood, and T. L. Kennedy Morphoproteomic and Pharmacoproteomic Rationale for mTOR Effectors as Therapeutic Targets in Head and Neck Squamous Cell Carcinoma Ann. Clin. Lab. Sci., January 1, 2006; 36(3): 273 - 282. [Abstract] [Full Text] [PDF]
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 G. Ohji, S. Hidayat, A. Nakashima, C. Tokunaga, N. Oshiro, K.-i. Yoshino, K. Yokono, U. Kikkawa, and K. Yonezawa Suppression of the mTOR-Raptor Signaling Pathway by the Inhibitor of Heat Shock Protein 90 Geldanamycin J. Biochem., January 1, 2006; 139(1): 129 - 135. [Abstract] [Full Text] [PDF]
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B. J. Krawiec, R. A. Frost, T. C. Vary, L. S. Jefferson, and C. H. Lang Hindlimb casting decreases muscle mass in part by proteasome-dependent proteolysis but independent of protein synthesis Am J Physiol Endocrinol Metab, December 1, 2005; 289(6): E969 - E980. [Abstract] [Full Text] [PDF]
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P. Amornphimoltham, V. Patel, A. Sodhi, N. G. Nikitakis, J. J. Sauk, E. A. Sausville, A. A. Molinolo, and J. S. Gutkind Mammalian Target of Rapamycin, a Molecular Target in Squamous Cell Carcinomas of the Head and Neck Cancer Res., November 1, 2005; 65(21): 9953 - 9961. [Abstract] [Full Text] [PDF]
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 D. J. VanderWeele and C. M. Rudin Mammalian Target of Rapamycin Promotes Vincristine Resistance through Multiple Mechanisms Independent of Maintained Glycolytic Rate Mol. Cancer Res., November 1, 2005; 3(11): 635 - 644. [Abstract] [Full Text] [PDF]
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 N. Hedhli, M. Pelat, and C. Depre Protein turnover in cardiac cell growth and survival Cardiovasc Res, November 1, 2005; 68(2): 186 - 196. [Abstract] [Full Text] [PDF]
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A. Hahn-Windgassen, V. Nogueira, C.-C. Chen, J. E. Skeen, N. Sonenberg, and N. Hay Akt Activates the Mammalian Target of Rapamycin by Regulating Cellular ATP Level and AMPK Activity J. Biol. Chem., September 16, 2005; 280(37): 32081 - 32089. [Abstract] [Full Text] [PDF]
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J. Cortes and H. Kantarjian New Targeted Approaches in Chronic Myeloid Leukemia J. Clin. Oncol., September 10, 2005; 23(26): 6316 - 6324. [Abstract] [Full Text] [PDF]
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A. Creer, P. Gallagher, D. Slivka, B. Jemiolo, W. Fink, and S. Trappe Influence of muscle glycogen availability on ERK1/2 and Akt signaling after resistance exercise in human skeletal muscle J Appl Physiol, September 1, 2005; 99(3): 950 - 956. [Abstract] [Full Text] [PDF]
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P. Wlodarski, M. Kasprzycka, X. Liu, M. Marzec, E. S. Robertson, A. Slupianek, and M. A. Wasik Activation of Mammalian Target of Rapamycin in Transformed B Lymphocytes Is Nutrient Dependent but Independent of Akt, Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase, Insulin Growth Factor-I, and Serum Cancer Res., September 1, 2005; 65(17): 7800 - 7808. [Abstract] [Full Text] [PDF]
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M. K. Holz and J. Blenis Identification of S6 Kinase 1 as a Novel Mammalian Target of Rapamycin (mTOR)-phosphorylating Kinase J. Biol. Chem., July 15, 2005; 280(28): 26089 - 26093. [Abstract] [Full Text] [PDF]
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G. G. Chiang and R. T. Abraham Phosphorylation of Mammalian Target of Rapamycin (mTOR) at Ser-2448 Is Mediated by p70S6 Kinase J. Biol. Chem., July 8, 2005; 280(27): 25485 - 25490. [Abstract] [Full Text] [PDF]
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Z.-q. Yuan, D. Kim, S. Kaneko, M. Sussman, G. M. Bokoch, G. D. Kruh, S. V. Nicosia, J. R. Testa, and J. Q. Cheng ArgBP2{gamma} Interacts with Akt and p21-activated Kinase-1 and Promotes Cell Survival J. Biol. Chem., June 3, 2005; 280(22): 21483 - 21490. [Abstract] [Full Text] [PDF]
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 Y. Luo, A. R. Shoemaker, X. Liu, K. W. Woods, S. A. Thomas, R. de Jong, E. K. Han, T. Li, V. S. Stoll, J. A. Powlas, et al. Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo Mol. Cancer Ther., June 1, 2005; 4(6): 977 - 986. [Abstract] [Full Text] [PDF]
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L.-Y. C. Wing, H.-M. Chen, P.-C. Chuang, M.-H. Wu, and S.-J. Tsai The Mammalian Target of Rapamycin-p70 Ribosomal S6 Kinase but Not Phosphatidylinositol 3-Kinase-Akt Signaling Is Responsible for Fibroblast Growth Factor-9-induced Cell Proliferation J. Biol. Chem., May 20, 2005; 280(20): 19937 - 19947. [Abstract] [Full Text] [PDF]
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H. Takeuchi, Y. Kondo, K. Fujiwara, T. Kanzawa, H. Aoki, G. B. Mills, and S. Kondo Synergistic Augmentation of Rapamycin-Induced Autophagy in Malignant Glioma Cells by Phosphatidylinositol 3-Kinase/Protein Kinase B Inhibitors Cancer Res., April 15, 2005; 65(8): 3336 - 3346. [Abstract] [Full Text] [PDF]
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F. Lefranc, J. Brotchi, and R. Kiss Possible Future Issues in the Treatment of Glioblastomas: Special Emphasis on Cell Migration and the Resistance of Migrating Glioblastoma Cells to Apoptosis J. Clin. Oncol., April 1, 2005; 23(10): 2411 - 2422. [Abstract] [Full Text] [PDF]
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Y. Shi, A. Sharma, H. Wu, A. Lichtenstein, and J. Gera Cyclin D1 and c-myc Internal Ribosome Entry Site (IRES)-dependent Translation Is Regulated by AKT Activity and Enhanced by Rapamycin through a p38 MAPK- and ERK-dependent Pathway J. Biol. Chem., March 25, 2005; 280(12): 10964 - 10973. [Abstract] [Full Text] [PDF]
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X. Wan, A. Mendoza, C. Khanna, and L. J. Helman Rapamycin Inhibits Ezrin-Mediated Metastatic Behavior in a Murine Model of Osteosarcoma Cancer Res., March 15, 2005; 65(6): 2406 - 2411. [Abstract] [Full Text] [PDF]
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 K. Inoki, H. Ouyang, Y. Li, and K.-L. Guan Signaling by Target of Rapamycin Proteins in Cell Growth Control Microbiol. Mol. Biol. Rev., March 1, 2005; 69(1): 79 - 100. [Abstract] [Full Text] [PDF]
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M. Tan, P. Li, K. S. Klos, J. Lu, K.-H. Lan, Y. Nagata, D. Fang, T. Jing, and D. Yu ErbB2 Promotes Src Synthesis and Stability: Novel Mechanisms of Src Activation That Confer Breast Cancer Metastasis Cancer Res., March 1, 2005; 65(5): 1858 - 1867. [Abstract] [Full Text] [PDF]
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L. Khamzina, A. Veilleux, S. Bergeron, and A. Marette Increased Activation of the Mammalian Target of Rapamycin Pathway in Liver and Skeletal Muscle of Obese Rats: Possible Involvement in Obesity-Linked Insulin Resistance Endocrinology, March 1, 2005; 146(3): 1473 - 1481. [Abstract] [Full Text] [PDF]
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E. V. Gerasimovskaya, D. A. Tucker, and K. R. Stenmark Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation J Appl Physiol, February 1, 2005; 98(2): 722 - 731. [Abstract] [Full Text] [PDF]
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E. V. Gerasimovskaya, D. A. Tucker, M. Weiser-Evans, J. M. Wenzlau, D. J. Klemm, M. Banks, and K. R. Stenmark Extracellular ATP-induced Proliferation of Adventitial Fibroblasts Requires Phosphoinositide 3-Kinase, Akt, Mammalian Target of Rapamycin, and p70 S6 Kinase Signaling Pathways J. Biol. Chem., January 21, 2005; 280(3): 1838 - 1848. [Abstract] [Full Text] [PDF]
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I. Briaud, L. M. Dickson, M. K. Lingohr, J. F. McCuaig, J. C. Lawrence, and C. J. Rhodes Insulin Receptor Substrate-2 Proteasomal Degradation Mediated by a Mammalian Target of Rapamycin (mTOR)-induced Negative Feedback Down-regulates Protein Kinase B-mediated Signaling Pathway in {beta}-Cells J. Biol. Chem., January 21, 2005; 280(3): 2282 - 2293. [Abstract] [Full Text] [PDF]
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X. Cao, F. Kambe, L. C. Moeller, S. Refetoff, and H. Seo Thyroid Hormone Induces Rapid Activation of Akt/Protein Kinase B-Mammalian Target of Rapamycin-p70S6K Cascade through Phosphatidylinositol 3-Kinase in Human Fibroblasts Mol. Endocrinol., January 1, 2005; 19(1): 102 - 112. [Abstract] [Full Text] [PDF]
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D. J. VanderWeele, R. Zhou, and C. M. Rudin Akt up-regulation increases resistance to microtubule-directed chemotherapeutic agents through mammalian target of rapamycin Mol. Cancer Ther., December 1, 2004; 3(12): 1605 - 1613. [Abstract] [Full Text] [PDF]
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M. Koziczak and N. E. Hynes Cooperation between Fibroblast Growth Factor Receptor-4 and ErbB2 in Regulation of Cyclin D1 Translation J. Biol. Chem., November 26, 2004; 279(48): 50004 - 50011. [Abstract] [Full Text] [PDF]
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J. E. Kim and J. Chen Regulation of Peroxisome Proliferator-Activated Receptor-{gamma} Activity by Mammalian Target of Rapamycin and Amino Acids in Adipogenesis Diabetes, November 1, 2004; 53(11): 2748 - 2756. [Abstract] [Full Text] [PDF]
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J. E. Thompson and C. B. Thompson Putting the Rap on Akt J. Clin. Oncol., October 15, 2004; 22(20): 4217 - 4226. [Abstract] [Full Text] [PDF]
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X. Zhou, M. Tan, V. Stone Hawthorne, K. S. Klos, K.-H. Lan, Y. Yang, W. Yang, T. L. Smith, D. Shi, and D. Yu Activation of the Akt/Mammalian Target of Rapamycin/4E-BP1 Pathway by ErbB2 Overexpression Predicts Tumor Progression in Breast Cancers Clin. Cancer Res., October 15, 2004; 10(20): 6779 - 6788. [Abstract] [Full Text] [PDF]
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W. H. Mondesire, W. Jian, H. Zhang, J. Ensor, M.-C. Hung, G. B. Mills, and F. Meric-Bernstam Targeting Mammalian Target of Rapamycin Synergistically Enhances Chemotherapy-Induced Cytotoxicity in Breast Cancer Cells Clin. Cancer Res., October 15, 2004; 10(20): 7031 - 7042. [Abstract] [Full Text] [PDF]
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J. Dong and D. Pan Tsc2 is not a critical target of Akt during normal Drosophila development Genes & Dev., October 15, 2004; 18(20): 2479 - 2484. [Abstract] [Full Text] [PDF]
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T. Yamazaki, T. Akada, O. Niizeki, T. Suzuki, H. Miyashita, and Y. Sato Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) binds and catalyzes PDK1, allowing VEGF-stimulated activation of S6K for endothelial cell proliferation and angiogenesis Blood, October 15, 2004; 104(8): 2345 - 2352. [Abstract] [Full Text] [PDF]
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I. Mothe-Satney, N. Gautier, C. Hinault, J. C. Lawrence Jr., and E. Van Obberghen In Rat Hepatocytes Glucagon Increases Mammalian Target of Rapamycin Phosphorylation on Serine 2448 but Antagonizes the Phosphorylation of Its Downstream Targets Induced by Insulin and Amino Acids J. Biol. Chem., October 8, 2004; 279(41): 42628 - 42637. [Abstract] [Full Text] [PDF]
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L. A. deGraffenried, L. Fulcher, W. E. Friedrichs, V. Grunwald, R. B. Ray, and M. Hidalgo Reduced PTEN expression in breast cancer cells confers susceptibility to inhibitors of the PI3 kinase/Akt pathway Ann. Onc., October 1, 2004; 15(10): 1510 - 1516. [Abstract] [Full Text] [PDF]
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A. D. Sinor and L. Lillien Akt-1 Expression Level Regulates CNS Precursors J. Neurosci., September 29, 2004; 24(39): 8531 - 8541. [Abstract] [Full Text] [PDF]
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 M. Campbell, W. E. Allen, C. Sawyer, B. Vanhaesebroeck, and E. R. Trimble Glucose-Potentiated Chemotaxis in Human Vascular Smooth Muscle Is Dependent on Cross-Talk Between the PI3K and MAPK Signaling Pathways Circ. Res., August 20, 2004; 95(4): 380 - 388. [Abstract] [Full Text] [PDF]
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N. Hay and N. Sonenberg Upstream and downstream of mTOR Genes & Dev., August 15, 2004; 18(16): 1926 - 1945. [Abstract] [Full Text] [PDF]
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L. Hou and E. Klann Activation of the Phosphoinositide 3-Kinase-Akt-Mammalian Target of Rapamycin Signaling Pathway Is Required for Metabotropic Glutamate Receptor-Dependent Long-Term Depression J. Neurosci., July 14, 2004; 24(28): 6352 - 6361. [Abstract] [Full Text] [PDF]
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J. D. Parkington, N. K. LeBrasseur, A. P. Siebert, and R. A. Fielding Contraction-mediated mTOR, p70S6k, and ERK1/2 phosphorylation in aged skeletal muscle J Appl Physiol, July 1, 2004; 97(1): 243 - 248. [Abstract] [Full Text] [PDF]
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