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. 2008 Feb;10(2):99–107. doi: 10.1593/neo.07905

Regulation of mTORC1 Signaling by Src Kinase Activity Is Akt1-Independent in RSV-Transformed Cells1

Martina Vojtěchová *, Jolana Turečková *, Dana Kučerová *, Eva Šloncová *, Jiří Vachtenheim †, Zdena Tuháčková *
PMCID: PMC2244684  PMID: 18283331

Abstract

Increased activity of the Src tyrosine protein kinase that has been observed in a large number of human malignancies appears to be a promising target for drug therapy. In the present study, a critical role of the Src activity in the deregulation of mTOR signaling pathway in Rous sarcoma virus (RSV)-transformed hamster fibroblasts, H19 cells, was shown using these cells treated with the Src-specific inhibitor, SU6656, and clones of fibroblasts expressing either the active Src or the dominant-negative Src kinase-dead mutant. Disruption of the Src kinase activity results in substantial reduction of the phosphorylation and activity of the Akt/protein kinase B (PKB), phosphorylation of tuberin (TSC2), mammalian target of rapamycin (mTOR), S6K1, ribosomal protein S6, and eukaryotic initiation factor 4E-binding protein 4E-BP1. The ectopic, active Akt1 that was expressed in Src-deficient cells significantly enhanced phosphorylation of TSC2 in these cells, but it failed to activate the inhibited components of the mTOR pathway that are downstream of TSC2. The data indicate that the Src kinase activity is essential for the activity of mTOR-dependent signaling pathway and suggest that mTOR targets may be controlled by Src independently of Akt1/TSC2 cascade in cells expressing hyperactive Src protein. These observations might have an implication in drug resistance to mTOR inhibitor-based cancer therapy in certain cell types.

Introduction

Growth-regulating signals originating from within and outside the cell are integrated through serine/threonine protein kinase mammalian target of rapamycin (mTOR) that has a key role in many aspects of cellular physiology. The mTOR signaling network has been implicated in the regulation of many cell functions including transcription, ribosome biogenesis, mRNA turnover, autophagy, cytoskeletal organization, and translation of specific subsets of mRNAs. There are several links between mTOR signaling and human diseases such as cancer (reviewed in the study of Wullschleger et al. [1]).

mTOR exists in two heteromeric complexes, mTORC1 and mTORC2. mTORC1 is the only known target of the specific inhibitor of mTOR, drug rapamycin. It consists of three proteins, mTOR, mLST8, and regulatory-associated protein of TOR, and the mTORC1-dependent pathway signals to the translational control of protein synthesis. The mTORC2 complex has been shown to contain mTOR, mLST8, and rapamycin-insensitive companion of mTOR and to control actin cytoskeleton dynamics [2]. Recently, protein PRAS40 that associates with 14-3-3 proteins has been identified as another target for regulation by mTORC1, but not by mTORC2 [3,4].

mTORC1 regulates cap-dependent translation especially of mRNAs with structured 5′-UTRs. It is facilitated by the phosphorylation and inactivation of 4E-BPs that are the suppressors of the cap-binding protein eIF4E, which plays a central role in cap-dependent translation by interacting with the 5′-cap structure of the mRNA. Many of these mRNAs encode proteins that control cell cycle progression and proliferation. mTORC1-regulated phosphorylation of ribosomal protein S6 by p70 S6 kinase (S6K1) increases selective translation of mRNAs encoding critical protein components of the proteosynthetic apparatus and cell size. Deregulation of the selective mRNA translation may play an important role in carcinogenesis, has direct consequences on cancer initiation, and can contribute to cell transformation (reviewed in the study of Holland et al. [5]).

In cancer cells, some of the proteins shown upstream or downstream of mTOR may be deregulated. An important negative regulator of mTOR is the suppressor protein tuberous sclerosis complex (TSC1/2) comprising hamartin (TSC1) and tuberin (TSC2). TSC2 is a 198-kDa protein, which contains the GTPase activator domain that promotes hydrolysis of GTP bound to the small GTPase Rheb activating mTOR [6]. Phosphorylation of TSC2 inactivates the GTPase activator domain function of TSC2, disrupts the TSC1/2 complex and stimulates activity of Rheb and mTOR [7]. The TSC1/2 complex may receive inputs from diverse signaling pathways; many growth-regulated proteins interact with the TSC1/2 complex and thus regulate mTOR. However, regulation of mTOR in a TSC1/2-independent fashion has also been reported [8].

TSC2 acts downstream of phosphatidylinositol 3-kinase (PI-3K) and protein kinase B (Akt/PKB), being directly phosphorylated at Ser939 and Thr1462 and thereby regulated by Akt/PKB [9]. TSC2 may also be phosphorylated by AMP-activated protein kinase [10] and by RSK (p90 S6 kinase), the downstream effector of the Ras/mitogen-activated protein kinase signaling pathway [11].

The serine/threonine kinase Akt/PKB, originally identified as an oncogene, plays a key role in cell survival, growth, proliferation, angiogenesis, metabolism, and migration. The three Akt isoforms (Akt1, Akt2, and Akt3) that may have distinct physiological functions (reviewed in the study of Manning and Cantley [12]), comprise an amino-terminal pleckstrin homology (PH) domain and a kinase domain. A multistep mechanism of the activation of Akt/PKB has been proposed, including interaction of the PH domain within Akt/PKB with phosphatidylinositol triphosphates produced by PI3K and subsequent translocation of Akt/PKB to the plasma membrane. Following membrane location, Akt/PKB can interact with the tyrosine kinase Src [13]. The interaction between the SH3 domain of Src and a proline-rich motif (PXXP) in the C-terminus of Akt/PKB may allow Src kinase to phosphorylate Akt/PKB at Tyr315 and Tyr326 [13,14] and probably also at Tyr474 [15]. Tyrosine phosphorylation was shown to be required for Akt/PKB phosphorylation at Thr308 in the activation loop by phosphoinositide-dependent kinase 1 (PDK1) and subsequent full activation by the phosphorylation at Ser473 within the carboxy-terminal tail catalyzed by other kinases, not yet clearly identified. The mTORC2 complex has been recently shown to mediate phosphorylation of Akt/PKB at Ser473 [16]. The involvement of the mTORC2 complex was also observed in the feedback phosphorylation of Akt/PKB at Ser473 when treated with the Akt/PKB inhibitor A-443654 [17].

Despite the great progress in the analyses of mTOR signaling, some aspects of its function are still unclear and many new questions have arisen, especially regarding the involvement of mTOR in the regulatory positive and negative feedback mechanisms. Treatment with rapamycin inhibits proliferation and may lead to apoptosis, and thus rapamycin and its analogs are evaluated in clinical trials as important anticancer agents. Although rapamycin is a selective inhibitor of mTOR and its downstream signaling, the inhibition of mTOR by rapamycin was shown in a subset of human cancer cell lines also to induce Akt/PKB phosphorylation at Ser473 mediated through receptor tyrosine kinase activation [18] or PI-3K-dependent phosphorylation of Akt/PKB and eIF4E [19]. These events seem to attenuate growth-inhibitory effects of rapamycin and serve as a negative feedback mechanism that could induce drug resistance.

Conversely, prolonged rapamycin treatment inhibits Akt/PKB signaling through suppression of mTORC2 assembly [20] and tumors expressing activated Akt/PKB that are resistant to conventional chemotherapy agents could be sensitized to chemotherapy-induced death by rapamycin [21]. Currently, it seems rather difficult to define a role of mTOR, particularly in the positive and negative feedback regulatory mechanisms.

We have previously shown that a model cell line (Rous sarcoma virus (RSV)-transformed hamster fibroblasts, H19 cells) expressing hyperactive Src protein displayed enhanced protein synthesis and upregulated PI-3K/Akt and mTOR-dependent signaling [22]. Significant changes in these pathways suggested that Akt/PKB was not an upstream effector of the mTORC1 targets, S6K1 and 4E-BP1, in RSV-transformed cells. In the present study, we examined a role of the active, ectopic Akt1 expressed in hamster fibroblasts in which the activity of the Src kinase was suppressed. Using the pharmacological inhibitor of the Src kinase activity, SU6656, and a dominantnegative kinase-dead double Y416F-K295N mutant of Src [23] to disrupt the Src enzymatic activity, we observed that inhibition of the protein kinase activity of Src blocked the mTORC1 signaling and that this block could not be overridden by the constitutively active form of Akt1. It may imply that mTORC1 downstream signaling is controlled by the Src kinase activity independently of Akt/PKB in RSV-transformed cells.

Materials and Methods

All general reagents were from Sigma (St. Louis, MO) unless otherwise stated. Protein G-Sepharose and [γ-32P]ATP were obtained from GE Healthcare Bio-Sciences (Uppsala, Sweden). Anti-phospho- Akt(Tyr326), anti-phospho-Akt(Thr308), anti-phospho-Akt(Ser473), anti-phospho-mTOR(Ser2448), anti-phospho-S6(Ser235/236), anti-phospho-tuberin(Thr1462), anti-S6K1, anti-S6, anti-4EBP1, anti-tuberin, and anti-Akt antibodies were purchased from Cell Signaling Technology (Danvers, MA), and anti-haemaglutinin (HA)-tag antibody was from Santa Cruz Biotechnologies (Santa Cruz, CA).The anti-Src antibody LA074 and the anti-tubulin antibodywere as in the study of Vojtěchová et al. [23]. The inhibitor SU6656 was obtained from Biomol International (Exeter, UK). Recombinant protein Akt and the active recombinant glutathione S-transferase (GST)-Src fusion protein were from Upstate Biotechnologies (Charlottesville, VA).

Cell Culture and Preparation of Cell Extracts

RSV-transformed hamster fibroblasts H19, nontransformed hamster fibroblasts NIL-2, NIL-A cells expressing the active wild-type v-Src, and NIL-B cells expressing the kinase-dead double mutant v-Src were grown in the presence of 5% fetal calf serum as described [23]. Cells were brought to quiescence by an overnight incubation with 2% fetal calf serum and then treated with the inhibitor SU6656 or rapamycin added to a final concentration of 5 µM and 20 nM (DMSO solution diluted in a medium, 1:106), respectively, or DMSO carrier alone for 30 minutes. The cells were lysed in an ice-cold extraction buffer [40 mM β-glycerophosphate, 50 mM HEPES pH 7.4, 5 mM EGTA, 1 mM Na3VO4, 1 mM benzamidine, 3 mMNa4P2O7, 0.5 mM PMSF, 1% Triton X-100, and the proteinase inhibitors pepstatin, antipain, and leupeptin (0.4 µg/ml each)] unless otherwise indicated. Cell debris was pelleted by centrifuging at 20,000g for 10 minutes, and supernatants were assayed for protein concentration.

Immunoprecipitation of Proteins, Western Blot Analysis, and Purification of S6 Protein

Proteins were immunoprecipitated from the cell extracts on Protein G-Sepharose beads, and the resulting immunoprecipitates were used either for SDS-PAGE followed by immunoblot analysis or for the in vitro kinase assays as described previously [22]. The S6 ribosomal protein was purified from the nontransformed NIL-2 cells by SPSepharose column chromatography and HPLC on a C18 column [24].

Soft Agar Assay

The assay was carried out in 60-mm Petri dishes. Approximately 5 x 103 cells were seeded in the upper layer containing 3 ml of modified Eagle's medium, 0.3% agar, 5% FCS supplemented with DMSO or inhibitors in a final concentration of 5 ’M SU6656 or 20 nM rapamycin. The cell media were refreshed every 3 to 4 days. The colonies were stained with p-indonitrotetrazolium violet (#18377; Sigma), counted, and photographed (magnification, x 20) after 12 days.

Protein Kinase Assays

Src activity. The reaction was carried out in 25 µl of a mixture containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl2 or 10 mM MnCl2, 50 µM Na3VO4, 5 mM DTT, 50 µM ATP, 1 µCi [γ-32P] ATP, 1 µg of denatured enolase, and Src protein immunoprecipitated from analyzed cells, and was incubated for 25 minutes at 30°C. The samples were subjected to SDS-PAGE/immunoblot analysis and autoradiography.

S6K1 activity. The assay was carried out in 25 µl of a mixture containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 40 µM Na3VO4, 5 mM DTT, 0.25 µl of PKA inhibitor substrate, 50 µM ATP, 6 µCi [γ-32P]ATP, and 2 µg of purified S6 protein. The samples were subjected to SDS-PAGE/immunoblot analysis and autoradiography.

Akt/PKB activity. The kinase activities in either anti-Akt or anti-HA-tag immunoprecipitates were assayed with RPRAATF peptide as described previously [22]. To analyze ectopic iAkt and endogenous Akt/PKB separately, the iAkt was precipitated with the anti-HA antibody, and the endogenous Akt/PKB was then immunoprecipitated from the resulting supernatants with anti-Akt antibody.

Construction and Use of an Inducible Akt Adenovirus

We used a recombinant adenovirus expressing the estrogen-dependent, constitutively active form of human Akt/PKB. This construct was derived from the human Akt-1 by replacing the PH domain with the N-terminal myristoylation signal and fusing the mutant murine anti-estrogen receptor modified for tamoxifen to its C-terminus. On infection of the cells, the Akt1 protein is expressed, but its full enzymatic activity requires induction by 4-hydroxytamoxifen (HT) [25].

Infections of the Cells with an Inducible Akt Adenovirus

The cells were coinfected with equal amounts of two recombinant adenoviruses, one encoding a tetracycline-inhibited transactivator, and the other a tetracycline-inhibited transactivator-regulated gene expressing HT-inducible HA-tagged membrane-targeted Akt1 as described [26]. After infection, cells were grown until their confluence and then incubated overnight in F10/DMEM with 1% FCS in the absence or presence of 1 µM HT. The next day, cells were treated either with a vehicle (DMSO) or with 5 µM SU6656 or 20 nM rapamycin for 30 minutes and extracted as described above.

Src Kinase Mutants

The active v-Src kinase and the Src kinase-dead double Y416F-K295N mutant were prepared as reported [23]. The active, wild-type H19 v-src gene or the kinase-dead double Y416F-K295N v-src mutant were stably transfected in the embryonic hamster fibroblasts NIL-2. The cells were cloned and two cell lines were established, namely, the NIL-A cells expressing the active v-Src protein and the NIL-B cells expressing the Src kinase-dead protein.

Results

The Inhibitor of Src Tyrosine Kinase Activity, SU6656, Suppresses Akt/PKB and mTOR Signaling in H19 Cells

It has been recently reported that tyrosine kinase inhibitors PP1 and PP2 inhibited protein translation through implication of tyrosine phosphorylation in the cap-dependent translation [27]. The inhibition of serine/threonine phosphorylation and the activity of S6K1, Akt/PKB, and ERK1/2 by inhibitors PP1 and PP2 was also reported [27,28], and it was suggested that Src kinase activity may be involved in the regulation of the PI-3K/mTOR signaling pathway. This pathway was shown to be highly upregulated in RSV-transformed hamster fibroblasts (H19 cells) expressing hyperactive Src protein [22,29]. To analyze the involvement of the Src kinase activity in the regulation of the mTOR-dependent signaling in H19 cells in more detail, we treated these cells with a specific inhibitor of the Src family kinase, SU6656 [30].

The inhibitory efficiency of SU6656 is demonstrated by a reduced phosphorylation of Src at its catalytic Tyr416 in RSV-transformed fibroblasts treated with SU6656 (Figure 1A) and by a decreased activity of Src immunoprecipitated from the inhibited H19 cells in the in vitro kinase assay (Figure 1B). Akt/PKB was also upregulated in cells transformed by the v-src oncogene [22], and Src has been suggested to act as a putative priming kinase for Akt/PKB [14]. In agreement with this hypothesis, the decreased activity of Src correlated well with a decreased phosphorylation of Akt/PKB at Tyr326 and also at Ser473 and Thr308 residues, when treating the H19 cells with SU6656 (Figure 1C). Phosphorylation of recombinant Akt/PKB by the active GST-Src fusion protein showed that Akt/PKB is indeed directly phosphorylated by Src in vitro (Figure 1D).

Figure 1.

Figure 1

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Inhibition of Src activity by SU6656 results in the reduced phosphorylation of Akt/PKB in H19 cells. (A) Phosphorylation of Src at Tyr416 residue. Crude extracts of H19 cells that were treated with either SU6656 (5 µM) or the vehicle (DMSO) were analyzed by SDS-PAGE followed by immunoblot analysis using anti-phospho-Src(Tyr416) and anti-Src antibodies. (B) In vitro kinase activity of Src. Auto-radiography of SDS-PAGE of the reaction mixtures containing Src immunoprecipitated from H19 cells treated with 5 µM SU6656 or with the vehicle (DMSO) that were incubated in the presence of [γ-32P]ATP and enolase (1 µg) as described in the Materials and Methods section. (C) Effect of the Src-specific inhibitor SU6656 on the phosphorylation of Akt at Tyr326, Ser473, and Thr308 residues. Crude extracts of H19 cells treated with SU6656 (5 µM) or with the vehicle (DMSO) were analyzed by SDS-PAGE followed by immunoblot analysis using anti-phospho-Akt(Tyr326), anti-phospho-Akt(Thr308), anti-phospho-Akt(Ser473), and anti-Akt antibodies. (D) In vitro phosphorylation of Akt/PKB catalyzed by Src. Autoradiography of SDS-PAGE of the reaction mixtures containing recombinant protein Akt/PKB with (1) or without active recombinant GST-Src fusion protein (2) that were incubated in the presence of [γ-32P]ATP as described in the Materials and Methods section.

Treatment of H19 cells with SU6656 also resulted in a decreased phosphorylation level of mTOR at Ser2448, presumably phosphorylated by Akt/PKB or by S6K1 in feedback mechanism [31,32], and in reduced phosphorylation of the downstream targets of mTOR, S6K1 and 4E-BP1, as manifested by higher electrophoretic mobility of these proteins on SDS-PA gels (Figure 2A). Decreased phosphorylation of S6K1 is accompanied by a substantial reduction of the S6K1 activity as judged by the dephosphorylation degree of its physiological substrate, ribosomal protein S6 in vivo (Figure 2A), and the decreased in vitro phosphorylation of the purified ribosomal S6 protein catalyzed by S6K1 that was immunoprecipitated from H19 cells treated with SU6656 (Figure 2B). These data imply that the deregulation of mTORC1 signaling may critically depend on the Src tyrosine kinase activity in RSV-transformed cells.

Figure 2.

Figure 2

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Src kinase inhibitor SU6656 suppresses mTOR signaling. (A) Crude extracts of H19 cells nontreated (DMSO) or treated with SU6656 (5 µM) were subjected to immunoblot analysis using anti-phospho-mTOR(Ser2448), anti-S6K1, anti-phospho-S6(Ser235/236), anti-S6, anti-4E-BP1, and anti-tubulin antibodies. The phosphorylation status of S6K1 and 4E-BP1 was determined by means of their migration on SDS-PAGE. The least phosphorylated form of S6K1 or 4E-BP1 is represented by the most rapidly migrating protein band α [29]. Under these conditions, the total amount of S6 protein was not affected. Tubulin was used as a loading control. (B) In vitro phosphorylation of S6 protein purified from normal fibroblasts catalyzed by S6K1. Autoradiography of SDS-PAGE of the reaction mixtures containing S6K1 immunoprecipitated from H19 cells treated with SU6656 (5 µM) or with the vehicle (DMSO) that were incubated in the presence of [γ-32P]ATP and purified S6 protein (1 µg) as described in the Materials and Methods section.

Suppressed Phosphorylation of TSC2 But Not of mTORC1 Downstream Targets Was Restored By the Active, Ectopic Akt Expressed in Src-Inhibited H19 Cells

Although Akt/PKB was shown to be an upstream regulator of mTOR signaling in many cell types, we have found previously [22] (and unpublished results) that, when treating the RSV-transformed cells with the selective inhibitors of PI-3K wortmannin and LY294002, the phosphorylation and activity of Akt/PKB decreased, but it was not accompanied by the inhibition of mTOR and its targets S6K1 and 4E-BP1. It might indicate that Akt/PKB is not involved in mTORC1 downstream signaling in H19 cells. Therefore, we examined the possibility that suppression of mTOR signaling by the inhibition of the Src activity could be overcome by ectopic, constitutively active Akt.

A recombinant adenovirus encoding HT-inducible HA-tagged membrane-targeted Akt1 (iAkt) was used as described in the Materials and Methods section. This ectopic, myristoylated PH domain-deleted Akt1 is PI-3K-independent, does not require phosphatidylinositol triphosphates, PI-3K products, for its activity, and was not inhibited by LY294002 [26]. In the infected cells, the iAkt protein is expressed, but its full enzymatic activity requires presence of HT [25] as demonstrated in Figure 3A. In the presence of HT, ectopic iAkt was phosphorylated at Ser473 and Thr308 (Figure 3A, lanes 3 and 4), and unexpectedly, unlike in endogenous Akt/PKB, this phosphorylation was not changed when treating the cells with the Src inhibitor SU6656 (Figure 3A, lanes 4 and 5). Phosphorylation of Ser473 and Thr308 residues of endogenous Akt/PKB significantly decreased in both uninfected and infected cells H19 when treating with SU6656 (Figure 3A, lanes 1 and 2 and lanes 4 and 5, respectively), suggesting that whereas the endogenous Akt/PKB is a target of the Src kinase activity, the ectopic iAkt is not regulated by Src in H19 cells.

Figure 3.

Figure 3

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Effect of ectopic, active iAkt on the phosphorylation of endogenous Akt/PKB and tuberin reduced by SU6656 in H19 cells. (A) Crude extracts of H19 cells uninfected or infected with ectopic, constitutively active iAkt as indicated were treated with SU6656 (5 µM) or with the vehicle (DMSO) and analyzed by SDS-PAGE followed by immunoblot analysis using anti-phospho-Akt(Tyr326), anti-phospho-Akt(Ser473), anti-phospho-Akt(Thr308), and anti-Akt antibodies. (B) In vitro kinase activity of Akt was assayed as described [22] using the RPRAATF peptide (150 µg) as a substrate in the reaction mixtures containing iAkt expressed and immunoprecipitated from the infected H19 cells by anti-HA antibody and the endogenous Akt/PKB immunoprecipitated from H19 cells either treated with the vehicle (DMSO) or with SU6656 (5 µM) as indicated. Data are means ± SD of triplicate determination of three separate experiments. Mixtures containing no peptide substrate were subtracted from the results. (C) Crude extracts of H19 cells uninfected or infected with ectopic iAkt as indicated were treated with SU6656 (5 µM) or vehicle (DMSO), and analyzed by SDS-PAGE followed by immunoblot analysis using anti-phospho-tuberin(Thr1462) and anti-tuberin antibodies.

In agreement with this observation, the ectopic iAkt was not phosphorylated at Tyr326 in H19 cells either in the absence or presence of HT (Figure 3A, lanes 3–5). The lack of tyrosine phosphorylation in iAkt was confirmed by immunoblot analysis using a general anti-phosphotyrosine antibody (not shown). It indicates that this mutant form of Akt can be phosphorylated at Ser473 and Thr308, irrespective of its tyrosine phosphorylation status. Moreover, this ectopic iAkt displayed a very high enzymatic activity as measured by in vitro phosphorylation of the substrate peptide RPRAATF catalyzed by the ectopic iAkt immunoprecipitated by anti-HA-tag antibody from the analyzed cells (Figure 3B).

Dephosphorylation of TSC2 at Thr1462, which is a phosphorylation site for Akt/PKB [9], is in agreement with the fact that Akt/PKB was inhibited in H19 cells treated with SU6656. Because TSC2 is the upstream negative regulator of mTOR, suppression of its phosphorylation and thus activation could be a reason of attenuation of the mTOR pathway activity mediated by Src inhibition (Figure 3C, lanes 1 and 2). We tested whether expression of the active iAkt could restore inhibited phosphorylation of TSC2 and the mTOR signaling pathway. As shown by immunoblot analysis, an increased level of phosphotuberin appeared in H19 cells infected with constitutively active iAkt compared to the cells containing only endogenous Akt/PKB (Figure 3C, lanes 3–5). Conversely, the expression of iAkt did not overcome the inhibition of components of the mTOR signaling pathway by SU6656 that are downstream to tuberin. As demonstrated in Figure 4 (lanes 1 and 2 and lanes 5 and 6, respectively), infection of H19 cells with iAkt had no effect on the decreased phosphorylation of S6K1, ribosomal protein S6 and 4E-BP1 induced by SU6656 treatment in H19 cells (Figure 4, lanes 2 and 6).

Figure 4.

Figure 4

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Effect of the ectopic, active iAkt on the phosphorylation of S6K1, ribosomal protein S6, and 4E-BP1 inhibited by SU6656 or rapamycin treatment in H19 cells. Crude extracts of H19 cells uninfected or infected with ectopic iAkt were treated with the vehicle (DMSO) or with either SU6656 (5 µM) or rapamycin (20 nM) and analyzed by SDS-PAGE followed by immunoblot analysis using anti-S6K1, anti-phospho-S6(Ser235/236), anti-S6, and anti-4E-BP1 antibodies.

To compare the efficiency of SU6656-mediated suppression of mTOR signaling with the inhibition of mTOR signaling pathway by the mTOR-specific inhibitor rapamycin, we examined the effect of rapamycin in H19 cells uninfected or infected with the ectopic iAkt. Although the inhibition of mTOR by rapamycin may induce activation of endogenous Akt/PKB in a regulatory negative feedback [17–20], no effect of rapamycin treatment on the phosphorylation of endogenous or ectopic Akt was observed in the H19 cells (not shown).

SU6656 showed to be rather less effective than rapamycin. On treatment with SU6656, the phosphorylation of S6K1, ribosomal protein S6, and 4E-BP1 decreased, but not to that induced by rapamycin (Figure 4, lanes 1–3). The activity of S6K1 was also less inhibited by SU6656 than by rapamycin, as indicated by the degree of dephosphorylation of the ribosomal S6 protein in treated H19 cells (Figure 4, lanes 2 and 3). Infection of H19 cells with ectopic, active iAkt failed to overcome the inhibitory effects of rapamycin as well (Figure 4, lanes 3 and 7).

Thus, in H19 cells, the overexpression of the ectopic, active iAkt does not restore the phosphorylation of S6K1, ribosomal protein S6, and 4E-BP1 that was suppressed when treating the cells with rapamycin or SU6656. Nevertheless, iAkt enhanced the phosphorylation of TSC2 suppressed on Src inhibition, which may imply that the Src activity affects the mTOR downstream targets independently of TSC2.

Dominant Negative Src Inhibits Both Akt/PKB and mTOR Signaling

In addition to the pharmacological inhibition of the Src kinase activity by SU6656, we analyzed the cells in which the Src kinase activity was suppressed by mutations in the kinase domain of the active Src protein, as described in the Materials and Methods section. Expression of the kinase-dead double Src mutant Y416F-K295N in NIL-B cells decreased Tyr416 phosphorylation of the total Src protein present in these cells (Figure 5A, upper panel), in agreement with a dominant negative effect of this Src mutant on the endogenous c-Src in hamster fibroblasts shown previously [23]. The presence of inactive Src in NIL-B cells abrogated the phosphorylation of endogenous Akt/PKB at Tyr326, confirming that this residue is indeed the Src target (Figure 5A).

Figure 5.

Figure 5

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Dominant-negative Src inhibits phosphorylation and activity of endogenous Akt/PKB and S6K1. Crude extracts of cells NIL-A and NIL-B either uninfected or infected with ectopic iAkt as described in the Materials and Methods section were analyzed by SDS-PAGE followed by immunoblot analysis using: (A) anti-phospho-Src(Tyr416), anti-Akt, anti-phospho-Akt(Tyr326), anti-phospho-Akt(Ser473), and anti-phospho-Akt(Thr308) antibodies; (B) anti-tuberin, anti-phospho-tuberin(Thr1462), anti-S6K1, anti-phospho-S6K1(Thr389), anti-phospho-S6 (Ser235/236), anti-S6 antibodies.

Infection of NIL-A cells (expressing wild-type v-Src) and NIL-B cells (expressing kinase-dead Src) with the ectopic, active iAkt showed that iAkt was not phosphorylated at Tyr326 in these cells, similarly as in H19 cells. No decrease in the phosphorylation of Ser473 and Thr308 residues was observed in the ectopic iAkt in NIL-B cells, whereas phosphorylation of these residues in the endogenous Akt/PKB was significantly reduced in NIL-B cells compared to NIL-A cells (Figure 5A). This correlates well with the decreased phosphorylation of endogenous Akt/PKB at Ser473 and Thr308 observed in H19 cells when treated with SU6656 as shown in Figure 1C.

Phosphorylation of TSC2 was reduced in the Src-deficient NIL-B cells and increased in both NIL-A and NIL-B cells that were infected with the active iAkt compared to the uninfected cells containing only endogenous Akt/PKB (Figure 5B). Conversely, the expression of constitutively active iAkt did not increase reduced phosphorylation of S6K1 in NIL-B cells as measured by the electrophoretic mobility of S6K1 on SDS-PA gels and by the phosphorylation of S6K1 at Thr389. In addition, decreased phosphorylation of ribosomal protein S6 in the Src-deficient NIL-B cells was not overcome by the expression of constitutively active iAkt (Figure 5B), similarly as observed in H19 cells when treated with SU6656 (Figure 4).

The data obtained by the pharmacological inhibition of the Src kinase activity by SU6656 in RSV-transformed hamster fibroblasts appeared to correlate well with the data observed in the analyses of hamster fibroblasts in which the total Src activity was suppressed by the expression of the inactive Src protein. The results confirmed that the Src tyrosine kinase activity plays an important role in mTORC1 signaling that is TSC2-independent in v-src-transformed cells.

Rapamycin and SU6656 Blocked the Soft Agar Colony Formation of RSV-Transformed Cells

Because the Src-specific inhibitor SU6656, similarly as the mTOR-specific inhibitor rapamycin, effectively downregulated mTORC1-dependent signaling to translational control of gene expression in H19 cells (Figure 4), we performed the soft agar colony formation assay to examine their capability to influence the phenotype of RSV-transformed hamster fibroblasts. As shown in Figure 6, both SU6656 and rapamycin blocked the oncogenic transformation of H19 cells. Rapamycin was less efficient than SU6656, and the number of colonies did not change when both inhibitors were applied simultaneously. Anchorage-independent growth induced by the v-src oncogene thus correlates with the activation of mTORC1-dependent signaling in H19 cells, suggesting that mTOR is an important mediator of the oncogenic signals issued by the v-src oncogene.

Figure 6.

Figure 6

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Rapamycin and SU6656 inhibit colony formation of RSV-transformed cells in soft agar. The assay was carried out in 60-mm Petri dishes as described in the Materials and Methods section. The number of viable colonies was checked after 12 days of incubation by staining with p-indonitrotetrazolium violet. Plotted results are the mean of two independent experiments.

Taken together, the results obtained with the Src-specific inhibitor SU6656 and the dominant negative mutant form of the Src protein showed that the inhibition of the Src activity may suppress the activity of mTORC1-dependent signaling pathway. Furthermore, the data indicate that the suppressive mechanism is not dependent on endogenous Akt/PKB, which is also inhibited by the disruption of the Src activity.

Discussion

The results presented in this study suggest a direct control of Akt/PKB and mTORC1 signaling by the Src kinase activity in RSV-transformed fibroblasts (H19 cells). The phosphorylation and activity of Akt/PKB and the phosphorylation of tuberin (TSC2), mTOR, S6K1, S6 protein, and 4E-BP1 decreased when the Src kinase activity was disrupted both by treatment with the Src inhibitor SU6656 or by the expression of Src kinase-dead double Y416F-K295N mutant that has a dominant negative effect on the endogenous c-Src [23]. The inhibition of downstream components of the mTOR-dependent signaling pathway, S6K1 and 4E-BP1, was not overcome by the expression of ectopic iAkt in H19 cells. These data suggesting that Akt/PKB does not control the mTORC1 targets in these cells are thus in agreement with our previous results demonstrating the inhibition of Akt/PKB but not inhibition of S6K1 and 4E-BP1 when treating the H19 cells with the PI-3K inhibitors wortmannin and LY294002 [22] (and unpublished results).

However, decreased phosphorylation of tuberin, which is the Akt/PKB substrate upstream of mTOR, was restored by the active, ectopic iAkt in Src-deficient cells, as shown in this study. Increased phosphorylation of TSC2 by iAkt was therefore insufficient to overcome the suppression of mTORC1 signaling induced by the inhibition of the Src activity.

It has been already reported that regulation of the mTOR pathway may involve mechanisms that operate independently of TSC2 in certain types of mammalian cells [8]. Regulation of mTOR signaling by the Src activity may be such additional input implicated in the control of mTOR-dependent pathway in RSV-transformed hamster fibroblasts. Interestingly, Src has been recently shown to bind and directly phosphorylate the mTOR target, S6K1 [33].

Preclinical and clinical data strongly support the potent effect of tyrosine kinase inhibitors as successful anticancer drugs. Similarly, as the inhibitor of the Src tyrosine kinase activity, SU6656, induced inhibition of the serine/threonine phosphorylation of signaling proteins of Akt/TSC2 and mTOR pathways demonstrated here, SU6656 treatment of NIH3T3 cells expressing the ETV6-NTRK3 oncoprotein attenuated the activation of Ras-ERK1/2 and PI-3K/Akt cascades [34]. Tyrosine kinase inhibitor Tyrphostin (AG1024), which inhibits cell proliferation and delays tumor growth in vivo, also inhibits phosphorylation of Akt/PKB besides tyrosine phosphorylation of the Bcr-Abl oncoprotein [35]. The inhibition of the serine/threonine phosphorylation and the activity of eIF4E, S6K1, Akt/PKB, and ERK1/2 by tyrosine kinase inhibitors has also been reported [27,28].

Inhibition of the mTORC1 signaling by rapamycin has a cytostatic or cytotoxic effect on cancer cells and was shown to restore the susceptibility of certain resistant cancer cells to chemotherapy [21,36]. Treatment with rapamycin, however, also induced activation of endogenous Akt/PKB in several cancer cell lines [18,19], probably in a negative feedback mechanism that may contribute to drug resistance.

Complementary inhibition by dual inhibitors that targeted Bcr-Abl and Src were shown to be active in case of resistance to the inhibitor of Bcr-Abl, imatinib, used alone in the therapy of chronic myelogenous leukemia [37]. Similarly, the Src kinase inhibitors may be promising agents, probably for dual drug treatment with other inhibitors of mTOR signaling. Therapeutical efficiency might therefore be achieved in human tumors, in which Akt/PKB and/or the mTOR signaling pathway are activated in the regulatory positive or negative feedback mechanism, by suppressing the kinase activity of Src. The fact that nutritional deprivation [38], hypoxia [39], and inhibition of the Src activity (this article) all reduce mTOR signaling indicates that mTOR may function in hypoxic areas of tumors and in tumors with high Src activity. The Src-dependent activation of the mTORC1 signaling to translational control may promote enhanced selective expression of proteins involved in certain malignant processes.

Our data show that besides rapamycin and its analogs, the specific inhibitors of the mTOR signaling pathway currently examined as potent antitumor agents, inhibitors of the Src activity may also display a profound inhibition of mTOR signaling in some type of cells, independently of the activated Akt1. It is possible that Akt1 and Akt2, which are expressed ubiquitously [12], may be redundant in the studied cells, and thus an isoform-specific substrate recognition might explain the Akt/PKB-independent regulation of S6K1 and 4E-BP1 in RSV-transformed cells. Also, akt mutation [40] cannot be excluded. However, the fact that the activity of TSC2, a substrate of the endogenous Akt/PKB of hamster fibroblasts, was regulated by the transduced ectopic Akt1, whereas the activity of mTOR targets was not, indicates that Akt1 is not able to overcome the suppression of mTOR-dependent pathway induced by Src in these cells.

In conclusion, these data suggest that the activity of Src may have an essential role in the Akt1/TSC2-independent regulation of the mTORC1 signaling pathway in RSV-transformed hamster fibroblasts.

Acknowledgments

We thank J. Svoboda, V. Sovová, and V. Kořínek (Institute of Molecular Genetics, Prague) for the critical reading of the manuscript. The excellent technical assistance of M. Čechová and I. Dosoudilová is also acknowledged.

Abbreviations

GST

glutathione S-transferase

HA

haemaglutinin

HT

4-hydroxytamoxifen

mTOR

mammalian target of rapamycin

PH

pleckstrin homology

PKB

protein kinase B

Footnotes

1

This study was supported by grant 301/04/0550 from the Grant Agency of the Czech Republic, by Research Project AVOZ50520514 of the Academy of Sciences of the Czech Republic, and by Institutional Research Project MZO00064211.

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