[CANCER RESEARCH 60, 3504 –3513, July 1, 2000]
`
`A Direct Linkage between the Phosphoinositide 3-Kinase-AKT Signaling Pathway
`and the Mammalian Target of Rapamycin in Mitogen-stimulated and
`Transformed Cells1
`Aleksandar Sekulic´,2 Christine C. Hudson,2 James L. Homme, Peng Yin, Diane M. Otterness, Larry M. Karnitz, and
`Robert T. Abraham3
`Department 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.]
`
`ABSTRACT
`
`The microbially derived antiproliferative agent rapamycin inhibits cell
`growth by interfering with the signaling functions of the mammalian
`target of rapamycin (mTOR). In this study, we demonstrate that inter-
`leukin-3 stimulation induces a wortmannin-sensitive increase in mTOR
`kinase activity in a myeloid progenitor cell line. The involvement of
`phosphoinositide 3*-kinase (PI3K) in the regulation of mTOR activity was
`further suggested by findings that mTOR was phosphorylated in vitro and
`in vivo by the PI3K-regulated protein kinase, AKT/PKB. Although AKT
`phosphorylated mTOR at two COOH-terminal sites (Thr2446 and Ser2448)
`in vitro, Ser2448 was the major phosphorylation site in insulin-stimulated
`or -activated AKT-expressing human embryonic kidney cells. Transient
`transfection assays with mTOR mutants bearing Ala substitutions at
`Ser2448 and/or Thr2446 indicated that AKT-dependent mTOR phosphoryl-
`ation was not essential for either PHAS-I phosphorylation or p70S6K
`activation in HEK cells. However, a deletion of amino acids 2430 –2450 in
`mTOR, which includes the potential AKT phosphorylation sites, signifi-
`cantly increased both the basal protein kinase activity and in vivo signal-
`ing functions of mTOR. These results demonstrate that mTOR is a direct
`target of the PI3K-AKT signaling pathway in mitogen-stimulated cells,
`and that the identified AKT phosphorylation sites are nested within a
`“repressor domain” that negatively regulates the catalytic activity of
`mTOR. Furthermore, the activation status of the PI3K-AKT pathway in
`cancer cells may be an important determinant of cellular sensitivity to the
`cytostatic effect of rapamycin.
`
`INTRODUCTION
`
`Rapamycin is a potent immunosuppressive drug and investigational
`anticancer agent, the major mechanism of action of which involves the
`inhibition of lymphoid or tumor cell proliferation, through interfer-
`ence with an event(s) required for G1-to-S phase progression in
`cycling cells. The block to G1 phase progression imposed by rapa-
`mycin occurs prior to the “restriction point,” based on the observa-
`tions that rapamycin inhibits the phosphorylation of the retinoblas-
`toma protein and that rapamycin-treated cells are not fully committed
`to enter S-phase of the cell cycle after release from drug-induced G1
`arrest (1–3). The sensitivity of certain tumor cell lines to the cytostatic
`effects of rapamycin has prompted considerable interest in the possi-
`bility that this drug might be a useful cancer chemotherapeutic agent.
`Indeed, a rapamycin analogue (CCI-779; Wyeth-Ayerst) is now in
`
`Received 1/10/00; accepted 4/27/00.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`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 Amer-
`ican 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@
`mc.duke.edu.
`
`Phase I clinical trials in cancer patients in the United States and
`Europe.
`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 or-
`thologue 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 gov-
`erned 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 –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–
`17). Although p70S6K activation involves a complex series of phos-
`phorylation events catalyzed by multiple protein kinases (18 –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 find-
`ings 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 transla-
`tional 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 59-cap structure
`of the mRNA template, thereby decreasing the efficiency of transla-
`tion initiation (23). Stimulation of cells with insulin or growth factors
`
`4 The abbreviations used are: FKBP, FK506-binding protein; mTOR, mammalian
`target of rapamycin; PI3K, phosphoinositide 39-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, mono-
`clonal antibody; PMSF, phenylmethylsulfonyl fluoride; PDK, phosphoinositide-depen-
`dent kinase; PKC, protein kinase C.
`3504
`
`.
`
`Roxane Labs., Inc.
`Exhibit 1021
`Page 001
`
`

`
`mTOR REGULATION BY THE PI3K-AKT PATHWAY
`
`MATERIALS AND METHODS
`
`suggest that deregulated signaling through mTOR may contribute to
`the transformed phenotype of PTEN-deficient cancer cells.
`
`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 –29). The phosphorylation of PHAS-I in-
`duced by hormonal stimuli is strongly inhibited by rapamycin (27, 30,
`31). Earlier results suggested that mTOR is directly responsible for the
`Plasmids, Reagents, and Antibodies. The expression vectors encoding
`phosphorylation of PHAS-I in intact cells (32, 33), although addi-
`AU1-tagged wild-type mTOR (AmTORwt) and catalytically inactive (“kinase-
`tional proline-directed kinases appear to be required for full phospho-
`dead”) mTOR (AmTORkd) were described previously (32). The rapamycin-
`rylation of PHAS-I in insulin-stimulated cells (34).
`resistant versions of mTOR contain an additional Ser (2035)3 Ile mutation
`The signaling pathway that couples growth factor receptor occu-
`and are usually designated with the suffix “SI” (e.g., AmTOR-SI). PCR-based
`pancy to mTOR activation is only partially understood. However,
`mutagenesis was used to construct single and double mTOR point mutants in
`accumulating evidence places mTOR downstream of both PI3K and
`which Thr2446 and Ser2448 were changed to alanine (A) residues. The internal
`the PI3K-regulated protein kinase, AKT (also termed PKB), in growth
`deletion mutant, AmTORD, which lacks amino acids 2430 –2450, was pre-
`factor-stimulated cells. This model is based in part on genetic and
`pared by the PCR-based SOEing technique (40). The expression vectors for
`HA-tagged wild-type AKT (cAKT), catalytically inactive AKT (AKT-kd) and
`pharmacological evidence that links activation of PI3K and/or AKT to
`the constitutively active myristylated form of AKT (myrAKT), were kind gifts
`the two intracellular events known to be governed by mTOR, the
`from P. N. Tsichlis (Fox-Chase Cancer Center, Philadelphia, PA). The cDNA
`activation of p70S6K and the phosphorylation of PHAS-I (35). The
`encoding p70S6K (kindly provided by Dr. Naohiro Terada, National Jewish
`notion that mTOR participates in signaling downstream from PI3K
`Medical and Research Center, Denver, CO) was appended with nucleotides
`may be particularly relevant to the antitumor activity of rapamycin.
`encoding an NH2-terminal FLAG epitope tag and was cloned into pcDNA3
`Recent studies have identified a negative regulator of PI3K-mediated
`using EcoRI and XbaI restriction sites. All PCR products were subcloned and
`signaling, PTEN, as a tumor suppressor gene product (36). The tumor
`then sequenced to ensure the fidelity of the amplification step.
`suppressor function of PTEN is attributed to its activity as a phos-
`Recombinant murine IL-3 was purchased from R&D Systems, Inc. (Min-
`phoinositide 3-phosphatase, which effectively terminates PI3K-
`neapolis, MN). Recombinant human insulin (Recombulin) and G418 (Geneti-
`mediated signaling via dephosphorylation of the second messengers,
`cin) were obtained from Life Technologies, Inc. (Gaithersburg, MD), and
`FuGene transfection reagent was purchased from Boehringer Mannheim (In-
`phosphatidylinositol-3,4,5-trisphosphate
`and phosphatidylinositol-
`dianapolis, IN). Wortmannin (Sigma) was dissolved in DMSO (Me2SO) to
`3,4-bisphosphate. Thus, loss of PTEN function leads to hyperactiva-
`yield a 1.2 mM stock solution. The wortmannin stock solution was aliquoted
`tion of the PI3K signaling cascade, which promotes abnormal cell
`and stored at 270°C. Rapamycin (Sigma) was prepared as a 10 mM stock
`growth, survival, and migration.
`solution in ethanol and aliquoted and stored as described above.
`The importance of PI3K signaling during tumorigenesis is under-
`The a-AU1 and 12CA5 (a-HA) mAbs were purchased from Babco (Rich-
`scored by observations that mutations in the PTEN gene occurs
`mond, CA), and the a-mTOR monoclonal antibody, 26E3, was a generous gift
`frequently in a variety of human cancers, including prostate cancer
`from Dr. Peter Houghton (St. Jude Children’s Research Hospital, Memphis,
`and glioblastoma (37). If mTOR also resides downstream of PI3K
`TN). Peptides corresponding to amino acid residues 2433–2450 in mTOR were
`and/or AKT, then mTOR activity should also be deregulated in
`synthesized (Research Genetics, Huntsville, AL) with or without phosphate at
`PTEN-deficient tumor cells, and consequently, PTEN status might be
`either or both of the underlined residues in the sequence CDTNAKGNKRSR-
`TRTDSYS. Polyclonal antibodies directed against the nonphosphorylated pep-
`an important predictor of cancer cell sensitivity to the mTOR inhib-
`tide were raised by immunizing rabbits with the peptide coupled to keyhole
`itor, rapamycin. Given these speculative arguments, it becomes in-
`limpet hemocyanin. The antiserum (designated a-mTOR 367) was affinity-
`creasingly important to define the interactions among PI3K, AKT, and
`purified over a peptide-coupled Sulfolink bead column according to the man-
`mTOR as the rapamycin analogue, CCI-779, moves into clinical trials
`ufacturer’s procedure (Pierce, Rockford, IL). Phosphospecific antibodies were
`in patients with different types of cancer.
`prepared in a similar fashion, except that a keyhole limpet hemocyanin-
`At the inception of this study, the most direct evidence for epistatic
`coupled, dually phosphorylated peptide (containing phosphate at both Thr2446
`relationships among PI3K, AKT, and mTOR stems from results
`and Ser2448 served as the immunogen. The resulting antiserum was first passed
`obtained with a polyclonal antibody, termed mTAb1, which recog-
`over a column consisting of nonphosphorylated peptide immobilized on Sul-
`nizes a COOH-terminal peptide sequence in mTOR (residues 2433–
`folink beads, and the flow-through fraction was then passed through a second
`2450; Ref. 38). The authors noted that cellular stimulation with
`column containing the immobilized, dually phosphorylated peptide. The bound
`antibodies (designated a-mTORp2) were eluted at low pH and were stored in
`insulin, or expression of mutationally activated AKT, caused a de-
`PBS containing 0.05% azide.
`crease in the immunoreactivity of mTOR in anti-mTAb1 immunoblot
`Cell Culture and Transfections. The murine IL-3-dependent myelomono-
`analyses (39). The loss of mTAb1 binding activity was reversed by
`cytic progenitor cell line, FDC-P1, was cultured in standard growth medium
`treatment of the immunoprecipitated mTOR with a protein phospha-
`[RPMI 1640 supplemented with 10% (v/v) FBS (Hyclone, Logan, UT), 2 mM
`tase prior to immunoblot analysis. Collectively, these results sug-
`L-glutamine, 50 mM 2-mercaptoethanol, 10 mM HEPES (pH 7.2), and 10%
`gested that insulin or AKT stimulation caused the phosphorylation of
`(v/v) WEHI-3 cell-conditioned medium as a source of IL-3]. Stably transfected
`mTOR at a site(s) that resulted in a decrease in the recognition of this
`FDC-P1 cells expressing AmTORwt were prepared by suspending 1 3 107
`protein by the mTAb1 antibody.
`exponentially growing cells in 350 ml of standard growth medium at 4°C. The
`The goal of the present study was to further understand the role of
`cells were mixed with a total of 45 mg of plasmid DNA suspended in the same
`the PI3K-AKT signaling pathway in the regulation of mTOR function
`medium. Mock transfectants received 45 mg of pcDNA3 only, whereas mTOR
`transfectants were electroporated with 25 mg of mTOR-encoding plasmid plus
`by extracellular stimuli. We demonstrate that stimulation of myeloid
`20 mg of pcDNA3 as filler. Prior to electroporation, the cell-DNA mixtures
`progenitor cells with IL-3 triggers a rapid increase in the protein
`were incubated for 10 min at room temperature. The cells were electroporated
`kinase activity of mTOR. The IL-3-dependent increase in mTOR
`with a BTX model T820 square-wave electroporator (San Diego, CA) at a
`activity is blocked by low concentrations of the PI3K inhibitor,
`setting of 350 V (10-ms pulse duration). The electroporated cells were mixed
`wortmannin. Furthermore, we provide in vitro and in vivo evidence
`gently and then allowed to stand at room temperature for an additional 10 min.
`that AKT phosphorylates mTOR at a site(s) located in a region that
`The cells were then diluted into 20 ml of standard growth medium and cultured
`represses the catalytic activity of the mTOR kinase domain. Deletion
`for 24 h, at which time the cells were transferred into fresh growth medium
`of this repressor domain generates a mTOR mutant bearing a consti-
`containing 800 mg of G418/ml. Stable clones that expressed AmTORwt and
`tutively elevated level of protein kinase activity. These findings out-
`AmTORkd were isolated by limiting dilution, and expression levels of the
`line a direct linkage between the PI3K-AKT pathway and mTOR and
`transfected proteins were assessed by immunoblotting with the AU1 mAb.
`3505
`
`Roxane Labs., Inc.
`Exhibit 1021
`Page 002
`
`

`
`mTOR REGULATION BY THE PI3K-AKT PATHWAY
`
`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 ml of buffer P per dish [50 mM Tris-HCl, 100 mM
`NaCl, 50 mM b-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 ml of AU1 mAb
`and 5 mg of 12CA5 mAb bound to protein A-Sepharose beads that had been
`precoupled to rabbit antimouse immunoglobulin antibodies. The immunopre-
`cipitates 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 ml of kinase buffer F supplemented with 10 mM ATP and 20 mCi
`of [g-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 mM insulin and then lysed in TNEE buffer [50 mM Tris-HCl,
`150 mM NaCl, 2.5 mM EDTA, 2 mM EGTA, 25 mM b-glycerophosphate, 25
`mM NaF (pH 7.5), containing, 0.5% Triton X-100, 100 mM sodium orthovana-
`date, 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).
`
`For experiments, exponentially growing FDC-P1 cells (2 3 107 cells/
`sample were washed twice in PBS. The cells were resuspended in 20 ml of
`starvation medium [RPMI 1640 containing 100 mg/ml BSA, 2 mM L-gluta-
`mine, and 50 mM 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, respec-
`tively. Prior to transfection, 6 3 105 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 mg of plasmid DNA mixed with 8 ml of
`FuGene transfection reagent/dish. The standard amounts of plasmid DNAs
`used for each transfection were: AKT, 0.25 mg; mTOR, 3 mg; and p70S6K, 2
`mg. When necessary, the total amount of plasmid DNA was brought to 5 mg
`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 3 105
`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 b-glyc-
`erophosphate, 20 nM microcystin- LR, 100 mg/ml PMSF, and protease inhib-
`itor cocktail (5 mg/ml aprotinin, 5 mg/ml pepstatin, and 10 mg/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 3 107 cells/sample) were growth factor deprived and restimulated as
`described above. The cells were washed in PBS and lysed by sonication in 1
`Stimulation of mTOR Catalytic Activity by Serum or IL-3. Our
`ml of buffer L [50 mM Tris-HCl, 50 mM b-glycerophosphate, 100 mM NaCl
`initial objective was to determine whether mTOR activity was regu-
`(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
`lated in a PI3K-dependent fashion by IL-3, a cytokine that promotes
`inhibitor cocktail]. The lysates were cleared of insoluble material by centrif-
`the proliferation and survival of myeloid lineage progenitor cells. To
`ugation, and the extracts were immunoprecipitated with 1 ml of a-AU1 mAb
`facilitate the analyses of mTOR kinase activity, we stably expressed
`for 2 h at4°C. The immunoprecipitates were washed three times in buffer W
`AU1-tagged wild-type or catalytically inactive (“kinase-dead”) ver-
`[50 mM Tris-HCl, 50 mM b-glycerophosphate, 100 mM NaCl (pH 7.4), con-
`sions of mTOR (AmTORwt and AmTORkd, respectively) in IL-3-
`taining 10% glycerol, 0.2% Tween 20, and 1 mM DTT] and twice in buffer K
`dependent FDC-P1 cells. Importantly, the stable cell lines selected for
`[10 mM HEPES, 50 mM NaCl, 50 mM b-glycerophosphate (pH 7.4), 50 nM
`these studies were not overexpressing the recombinant protein, as
`microcystin-LR, and the protease inhibitor cocktail].
`indicated by immunoblot analyses of the transfected clones for total
`Immunoblot Analyses. For immunoblot analyses with a-mTOR 367 or
`mTOR protein levels with antibodies that recognize both the endog-
`a-mTORp2 antibodies, recombinant AmTOR was immunoprecipitated with
`the tag-specific a-AU1 mAb from transfected FDC-P1, HEK 293, or HEK
`enous and transfected proteins (results not shown). The tagged Am-
`293T cells. The immunoprecipitated proteins were separated by SDS-PAGE
`TOR proteins therefore serve as a “tracer” subpopulation, the behavior
`through 6% polyacrylamide gels. After transfer to Immobilon-P, the mem-
`of which in response to physiological stimuli should reflect that of the
`branes were blocked and probed with 5 mg per ml affinity-purified antibodies
`endogenous mTOR.
`in Tris-buffered saline-0.2% Tween 20 (TBST) containing 2% (w/v) BSA (for
`In the initial studies, AmTORwt-expressing cells were deprived of
`a-mTORp2 antibodies) or 5% milk (for a-mTOR 367 antibodies). Immuno-
`serum and IL-3 for 6 h and then were restimulated for 10 min with
`reactive proteins were detected with horseradish peroxidase-conjugated to
`IL-3 prior to the preparation of cellular extracts. The extracts were
`protein A and the Renaissance reagent (New England Nuclear, Boston, MA).
`immunoprecipitated with a-AU1 mAb, and mTOR kinase activities
`The blots were then stripped and reprobed with the a-AU1 mAb in TBST-milk
`were determined with PHAS-I as the substrate. Parallel samples were
`solution. The phosphorylation state of endogenous mTOR in DU 145 or PC-3
`prepared from identically treated cells that expressed the AmTORkd
`prostate cancer cells was analyzed by immunoblotting with a-mTORp2 as
`mutant. Stimulation of AmTORwt-expressing FDC-P1 cells with IL-3
`described above, followed by reblotting of the same membrane with the
`a-mTOR mAb, 26E3, in TBST-milk solution.
`significantly increased the in vitro kinase activity of the immunopre-
`Kinase Assays. The protein kinase activity of immunoprecipitated mTOR
`cipitated AmTORwt but did not change the amount of AmTORwt in
`was assayed with recombinant PHAS-I as the substrate (Stratagene, La Jolla,
`these immunoprecipitates (Fig. 1A, left panel). The activation of
`CA; Ref. 32). The samples were separated by SDS-PAGE, and radiolabeled
`mTOR by IL-3 was maximal at 5–10 min after cytokine stimulation
`PHAS-I was detected by autoradiography. Incorporation of 32P into PHAS-I
`and then dropped to a lower, but still elevated, plateau level of activity
`was quantitated with a Molecular Dynamics Storm 840 Phosphorimager
`that was sustained for at least 4 h after cytokine addition (Fig. 1B and
`(Sunnyvale, CA) and ImageQuant software.
`data not shown). In contrast, AU1 immunoprecipitates from either
`Phosphorylation of mTOR by AKT in vitro was performed by transfection
`mock-transfected or AmTORkd-expressing cells contained low levels
`of AmTORwt and HA-tagged myrAKT, c-AKT, or catalytically inactive AKT
`of background protein kinase activity that was not substantially in-
`into separate populations of HEK 293 cells. The cells were plated in 60-mm
`creased by cellular stimulation with IL-3. Interestingly, serum-starved
`culture dishes and were transfected as described above. After 16 h, the
`3506
`
`RESULTS
`
`Roxane Labs., Inc.
`Exhibit 1021
`Page 003
`
`

`
`mTOR REGULATION BY THE PI3K-AKT PATHWAY
`
`noreactivity 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 abro-
`gated by pretreatment of the cells with 100 nM wortmannin, suggest-
`ing that this alteration was mediated through the activation of PI3K.
`As will be described below, parallel immunoblot analyses with a
`phospho-mTOR-specific antibody (a-mTORp2) indicated that
`the
`decrease in a-mTOR 367 reactivity induced by IL-3 stimulation is
`attributable to the phosphorylation of at least one amino acid (Ser2448)
`located within the a-mTOR 367 epitope (Fig. 3, middle panel; see
`below for description).
`Examination of the peptide sequence recognized by a-mTOR 367
`antibodies revealed that this region contained two consensus phos-
`phorylation 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
`
`Fig. 1. Stimulation of mTOR kinase activity by IL-3 or serum. FDC-P1 cells were
`stably transfected with wild-type AU1-tagged mTOR or a catalytically inactive version of
`AmTOR. Transfected clones were cultured for 6 h inmedium without serum and IL-3. A,
`left panel, factor-deprived FDC-P1 cells were stimulated for 10 min with IL-3 or medium
`only (2). The protein kinase activities of wild-type (wt) AmTOR or the “kinase-dead”
`(kd) AmTOR were determined in immune complex kinase assays with [g-32P]ATP and
`recombinant PHAS-I as the substrate (lower panels). Incorporation of 32Pi into PHAS-I
`was quantitated with a Molecular Dynamics Storm 840 phosphorimaging system and
`reported as cpm. The amounts of immunoprecipitated mTOR were assessed by immuno-
`blotting with AU1 mAb (upper panels). Right panel, AmTORwt-expressing FDC-P1 cells
`were restimulated for 10 min with IL-3 or serum, and AmTORwt kinase activities were
`measured as above. B, mTORwt-expressing FDC-P1 cells were restimulated for the
`indicated times with IL-3. The protein kinase activities in AmTORwt immunoprecipitates
`were determined as described in A. Incorporation of 32Pi into PHAS-I was normalized to
`the basal level of phosphorylation in the immunoprecipitate prepared from unstimulated
`cells.
`
`Fig. 2. Inhibition of IL-3 dependent mTOR activation by cellular treatment with
`wortmannin. AmTORwt-transfected FDC-P1 cells were deprived of growth factors as
`described in Fig. 1. AmTORwt-expressing cells were treated for 30 min with the indicated
`concentrations of wortmannin (Wm). After stimulation of the cells for 10 min with IL-3,
`AmTORwt was immunoprecipitated, and immune complex kinase assays were performed
`as described in the Fig. 1 legend. The amount of AmTORwt in each immunoprecipitate
`was determined by a-AU1 immunoblotting (upper panel).
`
`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.
`Role of PI3K in IL-3-dependent mTOR Activation. Earlier stud-
`ies implicated the PI3K pathway in the activation of mTOR-depend-
`ent signaling events in HEK 293 cells and 3T3-L1 preadipocytes (35,
`39). Stimulation of the IL-3 receptor also triggers a rapid increase in
`PI3K activity (41), which suggested that PI3K might be responsible
`for the activation of mTOR in IL-3-stimulated FDC-P1 cells. If the
`activation of mTOR by IL-3 is dependent on PI3K, then this response
`should be inhibited by pretreatment of the cells with wortmannin at
`drug concentrations #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.
`Direct Phosphorylation of mTOR by the PI3K-regulated Ki-
`Fig. 3. IL-3-dependent alterations in the reactivity of AmTORwt with R domain-
`nase, AKT. An earlier report provided evidence that activation of the
`directed a-mTOR antibodies. AmTORwt-expressing FDC-P1 cells were deprived of
`growth factors and then stimulated for the indicated times with medium only (2) or with
`PI3K-AKT pathway led to the phosphorylation of the mTAb1 anti-
`IL-3. Wortmannin (100 nM) was added to the indicated sample 30 min prior to IL-3
`body epitope located in the COOH-terminal region of mTOR (39).
`stimulation. The cells were lysed, and AmTORwt was immunoprecipitated from cleared
`extracts with a-AU1 mAb. The control lane (Co) represents an a-AU1 immunoprecipitate
`Using independently derived polyclonal antibodies (a-mTOR 367)
`from mock-transfected (empty plasmid only) FDC-P1 cells. The immunoprecipitates were
`specific for the same region of mTOR (amino acid residues 2433–
`resolved by SDS-PAGE and sequentially immunoblotted with a-mTOR 367 antibodies,
`2450), we observed a similar time-dependent decrease in the immu-
`phosphospecific a-mTORp2 antibodies (see Figs. 4 and 5 for details), and a-AU1 mAb.
`3507
`
`Roxane Labs., Inc.
`Exhibit 1021
`Page 004
`
`

`
`mTOR REGULATION BY THE PI3K-AKT PATHWAY
`
`Fig. 4. Schematic diagram of mTOR functional domains and mutants used in this
`study. The NH2-terminal AU1 epitope tag is followed by an extended NH2-terminal
`region of unknown function. The FKBP12zrapamycin binding (FRB) domain is labeled, as
`is the Ser (2035)3 Ile mutation used to generate the rapamycin-resistant AmTOR-SI
`mutants. The FRB domain is followed by the catalytic region (CAT). Finally, the putative
`“repressor” (R) domain (residues 2430 –2450) is shown together with the newly identified
`AKT phosphorylation sites at Thr2446 and Ser2448.
`
`observed that IL-3 stimulation caused a prompt increase in the reac-
`tivity of mTOR with these phosphospecific antibodies (Fig. 3, middle
`panel). Notably, the time-dependent increase in a-mTORp2 immuno-
`reactivity mirrored precisely the decrease in a-mTOR 367 binding
`provoked by IL-3 stimulation (Fig. 3, upper panel). Pretreatment of
`these cells with 100 nM wortmannin blocked the increase in
`a-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
`a-mTORp2 reactivity of the rapamycin-resistant AmTOR-SI mutant
`(Fig. 6A). AmTOR-SI contains a single amino acid substitution [Ser
`(2035)3 Ile] that renders the hor