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Department of Medicine, University of Virginia Health System, 450 Ray C Hunt Dr, Charlottesville, Virginia 22903, USA
1 Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202-5121, USA
2 Department of Surgery, Tokyo Dental College, 5-11-13 Sugano, Ichikawa, Chiba 272-8513, Japan
3 Department of Molecular and Cellular Oncology, University of Texas M D Anderson Cancer Center, 1515 Holcombe Blvd, Houston, Texas 77030-4009, USA
(Requests for offprints should be addressed to R J Santen; Email: rjs5y{at}virginia.edu)
This paper was presented at the 1st Tenovus/AstraZeneca Workshop, Cardiff (2005). AstraZeneca has supported the publication of these proceedings.
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Abstract |
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(ER) and the MAP kinase, phosphoinositol 3 kinase (PI3-K) and mammalian target of rapamycin (mTOR) growth factor pathways. ER is four- to tenfold up-regulated and co-opts a classical growth factor pathway using Shc, Grb-2 and Sos. This induces rapid non-genomic effects which are enhanced in LTED cells. The molecules involved in the non-genomic signaling process have been identified. Estradiol binds to cell membrane-associated ER, which physically associates with the adaptor protein Shc, and induces its phosphorylation. In turn, Shc binds Grb-2 and Sos, which result in the rapid activation of MAP kinase. These non-genomic effects of estradiol produce biologic effects as evidenced by Elk-1 activation and by morphologic changes in cell membranes. Additional effects include activation of the PI3-K and mTOR pathways through estradiol-induced binding of ER to the IGF-I and epidermal growth factor receptors. A major question is how ER locates in the plasma membrane since it does not contain an inherent membrane localization signal. We have provided evidence that the IGF-I receptor serves as an anchor for ER in the plasma membrane. Estradiol causes phosphorylation of the adaptor protein, Shc and the IGF-I receptor itself. Shc, after binding to ER, serves as the ‘bus’ which carries ER to Shc-binding sites on the activated IGF-I receptors. Use of small inhibitor (si) RNA methodology to knockdown Shc allows the conclusion that Shc is needed for ER to localize in the plasma membrane. In order to abrogate growth factor-induced hypersensitivity, we have utilized a drug, farnesylthiosalicylic acid, which blocks the binding of GTP-Ras to its membrane acceptor protein, galectin 1, and reduces the activation of MAP kinase. We have also shown that this drug is a potent inhibitor of mTOR as an additional mechanism of inhibition of cell proliferation. The concept of ‘adaptive hypersensitivity’ and the mechanisms responsible for this phenomenon have important clinical implications. The efficacy of aromatase inhibitors in patients relapsing on tamoxifen could be explained by this mechanism and inhibitors of growth factor pathways should reverse the hypersensitivity phenomenon and result in prolongation of the efficacy of hormonal therapy for breast cancer.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Farnesylthiosalicylic acid (FTS) was a gift from Drs Yoel Kloog (Tel-Aviv University, Tel-Aviv, Israel) and Wayne Bardin (Thyreos, New York, NY, USA). Estradiol was from Steraloids (Wilton, NH, USA). Fulvestrant was kindly provided by Dr Alan Wakeling (AstraZeneca Pharmaceuticals, Cheshire, UK). Sources of antibodies for Western analysis were as follows: phospho-MAP kinase (MAPK) monoclonal antibodies (Sigma), total MAPK (Zymed Laboratories, Inc., South San Francisco, CA, USA), Ser473-phospho-Akt, total Akt, Thr389-phospho-p70 S6K, total p70 S6K, Ser65-phospho-PHAS-I and total PHAS-I (Cell Signaling Technology, Beverly, MA, USA) and Thr229-phospho-p70 S6K (R&D Systems, Inc., Minneapolis, MN, USA). Secondary antibodies conjugated with horse-radish peroxidase were purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Cell culture medium, improved modified Eagles’ medium (IMEM) was from Biosource International, Inc. (Camrillo, CA, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagles’ medium/F12, glutamine and trypsin were from Invitrogen (Carlsbad, CA, USA). Most commonly used chemicals were obtained from Sigma.
Cell culture
Wild-type MCF-7 cells (kindly provided by Dr R Bruggemeier, Ohio State University, Columbus, OH, USA) were grown in IMEM containing 5% FBS. The estrogen hypersensitive MCF-7 subline was generated from MCF-7 cells by long-term culture under estrogen-deprived conditions and are called long-term estradiol-deprived (LTED) cells (Masamura et al. 1995). These cells represent a model of breast cancer treated with a hormonal therapy in which the cells have adapted in response to this treatment. LTED cells were routinely grown in phenol red-free IMEM containing 5% charcoal–dextran-stripped FBS.
Growth assay
Cells were plated in six-well plates at a density of 60 000 cells/well in their culture media. Two days later the cells were treated with various agents for 5 days with medium change on day 3. The final concentration of vehicle (ethanol or dimethyl sulphoxide) was 0.1%. At the end of treatment, cells were rinsed twice with saline. Nuclei were prepared by sequential addition of 1 ml HEPES–MgCl2 solution (0.01 M HEPES and 1.5 mM MgCl2) and 0.1 ml ZAP solution (0.13 M ethylhexadecyldimethylammonium bromide in 3% glacial acetic acid (v/v)) and counted using a Coulter counter.
Immunoblotting
Cells grown in 60 mm dishes were washed with ice-cold phosphate-buffered saline. To each dish was added 0.5 ml lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1% Triton X 100, 1 mM ß-glycerophosphate, 1 µg/ml leupeptin and aprotinin and 1 mM phenylmethylsulphonyl fluoride). After 5 min the lysates were pulse-sonicated and centrifuged at 14 000 r.p.m. for 10 min. Cell lysates were stored at –80 °C until analysis. Samples (50 µg total protein) were subjected to SDS-PAGE using 10% polyacrylamide gels before proteins were transferred to nitrocellulose membranes. The membranes were probed with primary antibodies in 5% bovine serum albumin dissolved in Tris-buffered saline with 0.05% Tween 20. Secondary antibodies conjugated to horse-radish peroxidase (1 : 2000) were then applied. After reacting with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA), targeted protein bands were visualized by exposing the membrane to X-ray film. Bands of specific proteins were scanned and relative optical densities of bands were determined using a Molecular Dynamics scanner and ImageQuant program (Yue et al. 2002).
Mammalian target of rapamycin (mTOR) studies
The methods used for immunopreciptiation, transfections with cDNA constructs, overexpression of AU1-mTOR, HA-raptor and HA-mLST8, the immune complex assays of mTOR activity and PAGE analysis are all described in detail in McMahon et al. (2005).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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) (Yue et al. 2002). We reasoned that the development of hypersensitivity could involve modulation of the genomic effects of estradiol acting on transcription, non-genomic actions involving plasma membrane-related receptors, cross-talk between growth factor- and steroid hormone-stimulated pathways or interactions among these various effects (Masamura et al. 1995, Jeng et al. 1998, 2000, Shim et al. 2000, Yue et al. 2002, 2003, 2005).
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(ER) increased four- to tenfold during long-term estradiol deprivation (Jeng et al. 2000). Accordingly, to directly examine whether enhanced sensitivity to estradiol in LTED cells occurred at the level of ER-mediated transcription, we quantitated the effects of estradiol on transcription in LTED and in wild-type MCF-7 cells. As transcriptional readouts, we measured the effect of estradiol on progesterone receptor (PgR) and pS2 protein concentrations and on ERE-CAT reporter activity (Jeng et al. 1998, Yue et al. 2005) (Fig. 1B, C and D
). We observed no shift to the left in estradiol dose–response curves (the end point utilized to detect hypersensitivity) for any of these responses (i.e. PgR, pS2 and CAT activity) when comparing LTED with wild-type MCF-7 cells. On the other hand, basal levels (i.e. no estrogen added) of transcription of three ER/ERE-related reporter genes were greater in LTED than in wild-type MCF-7 cells (Fig. 1E, F and G) (Jeng et al. 1998).
To interpret these data, we used the classic definition for hypersensitivity, namely a significant shift to the left in the dose causing 50% of maximal stimulation. Accordingly, these data suggested that hypersensitivity of LTED cells to the proliferative effects of estradiol did not occur primarily at the level of ER-mediated gene transcription (Fig. 1A–D) but may be influenced by the higher rates of maximal transcription (Fig. 1E, F and G).
We next considered that adaptation might involve dynamic interactions between pathways utilizing steroid hormones and those involving MAPK and phosphoinositol 3 kinase (PI3-K) for growth factor signaling (Masamura et al. 1995, Jeng et al. 1998, 2000, Shim et al. 2000, Yue et al. 2002, 2003, 2005) (Fig. 2A). Our initial approach demonstrated that basal levels of MAPK were elevated in LTED cells in vitro (Fig. 2B, top) and in xenografts (data not shown) and were inhibited by the pure antiestrogen fulvestrant (Jeng et al. 2000, Yue et al. 2003).
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(TGF) (data not shown). Administration of TGF caused a 2 log shift to the left in the ability of estradiol to stimulate the growth of wild-type MCF-7 cells. To demonstrate that this effect related specifically to MAPK and not to a non-MAPK-mediated effect of TGF, we co-administered PD98059. Under these circumstances, the 2 log left shift in estradiol dose–response returned back to the baseline dose–response curve (Yue et al. 2002). As further evidence of the role of MAPK, we administered U-0126 to LTED cells and examined its effect on level of sensitivity to estradiol. This agent partially shifted dose–response curves to the right by approximately one-half log (data not shown).
Whilst an important component, MAPK did not appear to be solely responsible for hypersensitivity to estradiol. Blockade of this enzyme did not completely abrogate hypersensitivity. Accordingly, we examined the PI3K pathway to determine if it was also up-regulated in LTED cells (Fig. 2B, second, third and fourth panels) (Yue et al. 2005). We determined that LTED cells exhibited an enhanced activation of Akt (Fig. 2B, second panel), P70S6 kinase (Fig. 2B, third panel) and 4E binding protein 1 (4E-BP1)/PHAS-I (Fig. 2B, fourth panel) (Yue et al. 2005). Dual inhibition of PI3K with Ly 294002 (a specific PI3K inhibitor) and MAPK with U-0126 shifted the level of sensitivity to estradiol more dramatically: more than 2 logs to the right (Fig. 2C) (Yue et al. 2002).
One possible mechanism to explain the activation of MAPK would be through non-genomic effects of estrogen acting through ER located in or near the cell membrane (Migliaccio et al. 1996, Kelly et al. 1999, Valverde et al. 1999). We have postulated that membrane-associated ER might utilize a classical growth factor pathway to transduce its effects in LTED cells. The adaptor protein Shc represents a key modulator of tyrosine kinase-activated peptide hormone receptors (Song et al. 2002). Upon receptor activation and autophosphorylation, Shc binds rapidly to specific phosphotyrosine residues of receptors through its phosphotyrosine binding (PTB) or Src homology (SH)-2 domain and becomes phosphorylated itself on tyrosine residues of the collagen homology (CH) domain (Pelicci et al. 1995). The phosphorylated tyrosine residues on the CH domain provide the docking sites for the binding of the SH2 domain of Grb2 and hence recruit Sos, a guanine nucleotide exchange protein. Formation of this adapter complex allows Ras activation via Sos, leading to the activation of the MAPK pathway (Song et al. 2002).
We have postulated that estrogen deprivation might trigger activation of a non-genomic, estrogen-regulated MAPK pathway which utilizes Shc (Dikic et al. 1995, Boney et al. 2000, Song et al. 2002). We employed MAPK activation as an endpoint with which to demonstrate rapid non-genomic effects of estradiol (Fig. 3). The addition of estradiol stimulated MAPK phosphorylation in LTED cells within minutes. The increased MAPK phosphorylation by estradiol was time and dose dependent, being greatly stimulated at 15 min and remaining elevated for at least 30 min. Maximal stimulation of MAPK phosphorylation was at 10–10 M estradiol.
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We reasoned that the adapter protein Shc may directly or indirectly associate with ER in LTED cells and thereby mediate estradiol-induced activation of MAPK. We considered this likely in light of recent evidence regarding ER membrane localization (Collins & Webb 1999, Watson et al. 1999a,b). To test this hypothesis, we immunoprecipitated Shc from non-stimulated and estradiol-stimulated LTED cells and then probed immunoblots with anti-ER antibodies. Our data showed that the ER/Shc complex pre-existed before estradiol treatment and estradiol time dependently increased this association (Song et al. 2002). In parallel with Shc phosphorylation, we observed a maximally induced association between ER and Shc at 3 min (data not shown). MAPK pathway activation by Shc requires Shc association with the adapter protein Grb2 and then further association with Sos. By immunoprecipitation of Grb2 and detection of both Shc and Sos, we demonstrated that the Shc–Grb2–Sos complex constitutively existed at relatively low levels in LTED cells, but was greatly increased by treatment of cells with 10–10M estradiol for 3 min (Song et al. 2002).
To provide evidence that the ER–Shc–MAPK pathway exerts biologic effects, we evaluated the role of MAPK on the activation of Elk-1. When activated, Elk-1 serves as a downstream mediator of cell proliferation. The phosphorylation of Elk-1 by MAPK can up-regulate its transcriptional activity through phosphorylation. By co-transfection of LTED cells with both GAL4-Elk and its reporter gene GAL4-luc (Roberson et al. 1995, Duan et al. 2001) we were able to show that estradiol dose dependently increased Elk-1 activation at 6 h as shown by luciferase assay (Song et al. 2002).
We also wished to demonstrate the biologic effects on cell morphology. To examine the effects of estradiols on reorganization of the actin cytoskeleton, we visualized the distribution of F-actin by phalloidin staining and also redistribution of the ER localization in LTED and MCF-7 cells (data not shown) (Song et al. 2002). Untreated MCF-7 cells expressed low actin polymerization and a few focal adhesion points. After estradiol stimulation, in contrast, the cytoskeleton underwent remodeling associated with formation of cellular ruffles, lamellipodias and leading edges, alterations of cell shape and loss of mature focal adhesion points. A subcellular redistribution of ER to these dynamic membranes upon estradiol stimulation represented another important feature. The ER antagonist ICI 182,780 at 10–9 M blocked estradiol-induced ruffle formation as well as redistribution of ER to the membrane with little effect by itself. These studies therefore further demonstrated the rapid action of estradiol with respect to dynamic membrane alterations in LTED cells.
A key unanswered question was how the ER could localize in the plasma membrane when it does not contain membrane localization motifs. We postulated that the IGF-I receptor (IGF-IR) and Shc might be involved in this process (Fig. 4A) (Song et al. 2004). A series of studies by other investigators suggested that ER and the IGF-IR might interact (Song et al. 2004). We tested the model that estradiol caused binding of Shc to ER but also caused phosphorylation of the IGF-IR. In this way, Shc would serve as the ‘bus’ which would carry ER to the plasma membrane where it would bind to the Shc acceptor site. To assess this possibility, we immunoprecipitated IGF-IRs before and after addition of estradiol. This caused Shc to bind to the IFG-IR and caused the IGF-IR to become phosphorylated (Fig. 4B). In order to prove a causal effect for this role of Shc, we utilized a small inhibitor (si) RNA methodology to knockdown Shc and showed that this prevented ER from binding to the IGF-IR (Song et al. 2004). As further evidence, we conducted confocal microscopy experiments to show that knockdown of Shc prevented ER from localizing in the plasma membrane (Fig. 5).
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plays a role in cell proliferation and in activation of MAPK. It appeared likely then that LTED cells might exhibit enhanced functionality of the membrane ER system. As evidence of this, we examined the ability of estradiol to cause the phosphorylation of Shc in wild-type and MCF-7 cells and also to cause association of Shc with the membrane ER. We demonstrated a marked enhancement of both of these processes in LTED as opposed to wild-type cells. At the present time, it is not clear what is responsible for enhancement of the non-genomic ER- mediated process. If adaptive hypersensitivity results from the up-regulation of growth factor pathways, an inhibitor of MAPK and downstream PI-3 kinase pathways could be important in abolishing hypersensitivity and in inhibiting cell proliferation. We have studied the effects of an MAPK inhibitor, FTS, which has been shown to block proliferation of LTED cells. This agent interferes with the binding of GTP-Ras to its acceptor site in the plasma membrane, a protein called galectin 1 (Haklai et al. 1998). While examining its downstream effects, we have shown that this agent is also a potent inhibitor of mTOR (McMahon et al. 2005). We postulated that an agent which blocks not only the MAPK pathway but also downstream actions of the PI3K pathway might be ideal to inhibit hypersensitivity. Accordingly, we have studied the effects of FTS on mTOR intensively.
mTOR is a Ser/Thr protein kinase involved in the control of cell growth and proliferation (Harris & Lawrence 2003). One of the best characterized substrates of mTOR is PHAS-I (also called 4E-BP1) (Brunn et al. 1997, Lawrence & Brunn 2001). PHAS-I/4E-BP1 binds to elongation factor E (eIF4E) and represses cap-dependent translation by preventing eIF4E from binding to eIF4G (Brunn et al. 1997, Lawrence & Brunn 2001). When phosphorylated by mTOR, PHAS-I/4E-BP1 dissociates from eIF4E, allowing eIF4E to engage eIF4G, thus increasing the formation of the eIF4F complex needed for the proper positioning of the 40S ribosomal subunit and for efficient scanning of the 5'-untranslated region (Harris & Lawrence 2003). In cells, mTOR is found in mTORC1, a complex also containing raptor, a newly discovered protein of 150 000 Da. It has been proposed that raptor functions in TORC1 as a substrate-binding subunit which presents PHAS-I/4E-BP1 to mTOR for phosphorylation (Lawrence & Brunn 2001, Harris & Lawrence 2003). Our results suggest that FTS inhibits phosphorylation of the mTOR effectors, PHAS-I/4E-BP1 and S6K1, in response to estrogen stimulation of breast cancer cells (McMahon et al. 2005).
To investigate the effects of FTS on mTOR function, we utilized 293T cells and monitored changes in the phosphorylation of PHAS-I/4E-BP1, a well-characterized target of mTOR (McMahon et al. 2005). Incubating cells with increasing concentrations of FTS decreased the phosphorylation of PHAS-I/4E-BP1, as evidenced by a decrease in the electrophoretic mobility. To determine whether FTS also promoted dephosphorylation of Thr36 and Thr45, the preferred sites for phosphorylation by mTOR (Harris & Lawrence 2003), an immunoblot was prepared with PThr36/45 antibodies. Increasing FTS markedly decreased the reactivity of PHAS-I/4E-BP1 with the phosphospecific antibodies (Fig. 6) (McMahon et al. 2005).
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). AU1-mTOR and HA-tagged forms of raptor and mLST8 were overexpressed in 293T cells, which were then incubated with increasing concentrations of FTS before AU1-mTOR was immunoprecipitated with anti-AU1 antibodies. Immunoblots were prepared with anti-HA antibodies to assess the relative amounts of HA-raptor and HA-mLST8 that co-immunoprecipitated with AU1-mTOR. Both HA-tagged proteins were readily detectable in immune complexes from cells incubated in the absence of FTS, indicating that mTOR, raptor and mLST8 form a complex in 293T cells. FTS did not change the amount of AU1-mTOR that immunoprecipitated; however, increasing concentrations of FTS produced a progressive decrease in the amount of HA-raptor that co-immunoprecipitated. The half-maximal effect on raptor dissociation from mTOR was observed at approximately 30 µM FTS (Fig. 6). Results obtained with overexpressed proteins are not necessarily representative of responses of endogenous proteins. Experiments were therefore conducted to investigate the effect of FTS on the endogenous TORC1 in non-transfected cells. Similar results were found, indicating that the FTS blocks the association of raptor from mTOR (McMahon et al. 2005).
Incubating cells with FTS produced a stable decrease in mTOR activity that persisted even when mTOR was immunoprecipitated. The dose–response curves for FTS-mediated inhibition of AU1-mTOR activity (Fig. 6) and dissociation of AU1-mTOR and HA-raptor were very similar, with half-maximal effects occurring between 20 and 30 µM. These results indicated that FTS inhibits mTOR in cells by promoting dissociation of raptor from mTORC1.
These studies provide direct evidence that FTS inhibits mTOR activity. The finding that the inhibition of mTOR activity by increasing concentrations of FTS correlated closely with the dissociation of the mTOR–raptor complex, both in cells and in vitro (Fig. 6), supports the conclusion that FTS acts by promoting dissociation of raptor from mTORC1.
Since FTS blocks both MAPK and mTOR, it was reasonable to conclude that it could block cell proliferation. For that reason, we conducted extensive studies to demonstrate that FTS blocks the growth of LTED cells. As shown in Fig. 7, FTS blocks the growth on LTED cells both in vitro and in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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present in cell extracts and an increase in basal level of transcription of several estradiol-stimulated genes. The lack of shift to the left in the dose–response curves of these transcriptional endpoints suggested that hypersensitivity is not mediated primarily at the transcriptional level (Fig. 1). On the other hand, rapid, non-genomic effects of estradiol such as the phosphorylation of Shc and binding of Shc to ER are easily demonstrable and appear to be enhanced in the LTED cells. Taken together, these observations suggested that adaptive hypersensitivity is associated with an increased utilization of non-genomic, plasma membrane-mediated pathways. This resulted in an increased level of activation of the MAPK as well as the PI3K and mTOR pathways. All of these signals converge on downstream effectors which are directly involved in cell cycle functionality and which probably exert synergistic effects at that level. As a reflection of this synergy, E2F1, an integrator of cell cycle stimulatory and inhibitory events, is hypersensitive to the effects of estradiol in LTED cells (Yue et al. 2002). Thus, our working hypothesis at present is that hypersensitivity reflects upstream non-genomic ER events as well as downstream synergistic interactions of several pathways converging at the level of the cell cycle. It is clear that primary endocrine therapies can exert pressure on breast cancer cells that causes them to adapt as a reflection of their inherent plasticity. Based upon this concept, we postulate that certain patients may become resistant to tamoxifen as a result of developing hypersensitivity to the estrogenic properties of tamoxifen. Up-regulation of growth factor pathways involving erb-B-2, IGF-IR and the epidermal growth factor (EGF) receptor are associated with this process (McMahon et al. 2005). The estrogen agonistic properties of tamoxifen under these circumstances might explain the superiority of clinical responses in patients receiving aromatase inhibitors as opposed to tamoxifen. It is possible to counteract the effects of the adaptive processes leading to growth factor up-regulation. If breast cancer cells are exceedingly sensitive to small amounts of estradiol or to the estrogenic properties of tamoxifen, one therefore needs highly potent aromatase inhibitors to block estrogen synthesis or pure antiestrogens such as fulvestrant. Blockade of the downstream effects of the IGF-IR, EFG receptor and erb-B-2 pathways would also be beneficial and allow continuing responsiveness to aromatase inhibitors or tamoxifen.
Disruption of each of several key steps could reduce the level of sensitivity to estradiol and block cell growth. Figure 8 illustrates the potential sites for disruption of adaptive hypersensitivity. An agent that blocks the nodal points through which several growth factor pathways must pass might be a more suitable therapy than combination of several growth factor-blocking agents. Our preliminary data suggest that FTS blocks two nodal points, the functionality of Ras and the activity of mTOR. FTS also effectively inhibits the proliferation of MCF-7 breast cancer cells in culture. Since this agent blocks MAPK as well as mTOR, it may be ideal for the prevention of adaptive hypersensitivity and prolongation of the effects of hormonal therapy in breast cancer. We are currently conducting further studies in xenograft models to demonstrate its efficacy. We envision the possibility that women with breast cancer will receive a combination of aromatase inhibitors plus FTS. In this way, the beneficial effects of the aromatase inhibitor may be prolonged and relapses due to growth factor over-expression might be prevented or retarded.
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References |
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J M W Gee, A Howell, W J Gullick, C C Benz, R L Sutherland, R J Santen, L-A Martin, F Ciardiello, W R Miller, M Dowsett, et al. Consensus Statement Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S1 - S7. [Abstract] [Full Text] [PDF]
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32P]ATP and recombinant (HIS6) PHAS-I then subjected to SDS-PAGE. (A) Phosphorimage of a dried gel was obtained to detect 32P-PHAS-I (4E-BP1) and an immunoblot was prepared with PThr36/45 antibodies. Other samples of the immune complexes were subjected to SDS-PAGE and immunoblots were prepared with antibodies to the HA epitope or to mTOR (
