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Differential Involvement of IB Kinases and β in Cytokine- and Insulin-Induced Mammalian Target of Rapamycin Activation Determined by Akt¹

Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599

	Abstract

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The mammalian target of rapamycin (mTOR) is a mediator of cell growth, survival, and energy metabolism at least partly through its ability to regulate mRNA translation. mTOR is activated downstream of growth factors such as insulin, cytokines such as TNF, and Akt-dependent signaling associated with oncoprotein expression. mTOR is negatively controlled by the tuberous sclerosis complex 1/2 (TSC1/2), and activation of Akt induces phosphorylation of TSC2, which blocks the repressive TSC1/2 activity. Previously, we showed that activation of mTOR in PTEN-deficient cancer cells involves IB kinase (IKK) , a catalytic subunit of the IKK complex that controls NF-B activation. Recently, a distinct IKK subunit, IKKβ, was shown to phosphorylate TSC1 to promote mTOR activation in an Akt-independent manner in certain cells stimulated with TNF and in some cancer cells. In this study, we have explored the involvement of both IKK and IKKβ in insulin- and TNF-induced mTOR activation. Insulin activation of mTOR requires Akt in a manner that involves IKK, preferentially to IKKβ, and TSC2 phosphorylation. TNF, in most cells examined, activates Akt to use IKK to control mTOR activation. In MCF7 cells, TNF does not activate Akt and requires IKKβ to activate mTOR. The results show that Akt-dependent signaling, induced by cytokines or insulin, alters the IKK subunit-dependent control of mTOR.

	Introduction

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The mammalian target of rapamycin (mTOR)³ is a ser/thr kinase that is activated downstream of increases in amino acid levels, by exposure of cells to growth factors such as insulin, by expression/activation of certain oncoproteins, and by the loss of the PTEN tumor suppressor (1, 2, 3, 4). Additionally, it has been reported that cytokines, such as TNF, induce mTOR activation (5, 6, 7). mTOR controls cell growth and survival, at least partly, through its ability to phosphorylate S6K and 4EBP1, key regulators of mRNA translation. The control of mTOR downstream of insulin-dependent signaling involves the Ser/Thr kinase Akt, which regulates mTOR through the ability of this kinase to phosphorylate and inactivate tuberous sclerosis complex 2 (TSC2) (1, 8, 9). TSC1 and 2 form a complex that exhibits GTPase-activating protein activity, blocking the activity of Rheb, itself a GTPase that controls mTOR activation (1, 10). Additionally, PRAS40 has been identified as a negative regulator of mTOR, which is phosphorylated by Akt downstream of insulin-induced signaling to relieve the PRAS40-controlled inhibitory effect on mTOR kinase activity (11, 12).

mTOR forms a rapamycin-sensitive complex with Raptor (TORC1) that controls mTOR downstream functions that include phosphorylation of S6K and 4E-BP1 (13, 14). A second mTOR complex (TORC2) contains the protein Rictor, and this complex has been shown to function as the PDK2 activity to phosphorylate Akt on Ser473 (15). Raptor has been shown to be required for mTOR function in the TORC1 complex (13, 14). An additional protein termed GβL associates with mTOR to control mTOR kinase activity via stabilization of mTOR-Raptor interaction (16).

Akt/PKB is a Ser/Thr kinase that is activated downstream of growth factor-induced signaling, where it controls suppression of apoptosis, cell growth and proliferation, and energy metabolism (17, 18, 19, 20). Additionally, cytokines induce Akt activation (21). In cancers, Akt is constitutively activated downstream of growth factor receptor signaling, through activating mutations in PI3K, or following PTEN loss of expression or mutation (17, 18, 19, 20, 22). The initiating step in Akt activation is the binding of PIP3 to the PH domain of Akt, leading to translocation of Akt to the cell membrane where it is activated by phosphorylation through PDK1 (23, 24) and PDK2 (15).

The transcription factor NF-B is activated in response to inflammatory mediators and growth factor pathways (25, 26). NF-B activation in most signal transduction cascades uses the IKK complex, containing IKK, IKKβ, and IKK. IKK and IKKβ comprise the catalytic activity of IKK, with IKKβ appearing to be dominant in inflammatory-mediated pathways and IKK functioning downstream in the so-called alternative pathway (25, 27). The IKK complex phosphorylates the inhibitory IB proteins leading to their ubiquitination and subsequent proteasome-dependent degradation. This allows efficient NF-B nuclear accumulation and binding to target sequences in the promoters and regulatory sequences of genes encoding cytokines, chemokines, and regulators of apoptosis (25). Interestingly, it has been reported that Akt can activate NF-B transcriptional activity (28, 29) and nuclear accumulation (30) through a pathway involving IKK. Recently, we described a requirement for IKK in controlling Akt-dependent activation of mTOR in PTEN-deficient cancer cells (31).

In this study, we have explored a potential relationship between the IKK family of proteins and the control of mTOR activation downstream of insulin-induced and cytokine-induced signaling. Our data indicate that the IKK subunit of IKK is required for efficient mTOR activation in the insulin pathway, which activates Akt in all cells tested. Insulin promotes a robust interaction between mTOR and IKK that is suppressed by inhibition of the PI3K/Akt pathway. As previously reported (1, 8), insulin induces an Akt-dependent phosphorylation of TSC2. In this pathway, TSC2 inhibition appears to play an important role in mTOR activation. In the TNF response, IKK largely controls mTOR activation if cells induce an Akt-response. TNF induction of mTOR requires IKKβ in cells that do not initiate Akt activation. The results presented here demonstrate that an insulin or TNF response that uses Akt-dependent signaling activates IKK to induce mTOR activity, whereas a TNF response that does not involve Akt activates IKKβ to activate mTOR. Thus, IKK-specific pathways are differentially regulated by Akt and activate mTOR through distinct mechanisms.

	Materials and Methods

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Abs and reagents

Abs were obtained from the following sources. Abs against IKK-, IKK-β, Akt, and mTOR were obtained from Upstate Biotechnology. Ab against phosphoserine/threonine is from BD Transduction Lab. Anti-HA and anti-Flag Abs were obtained from Roche and Sigma-Aldrich, respectively. Anti-actin was obtained from Calbiochem. Anti-Xpress was from Invitrogen. The anti-myc (9E-10), anti-IB, anti-Tubulin, anti-S6K and control rabbit IgG, as well as HRP-labeled anti-mouse and anti-rabbit secondary Abs, were from Santa Cruz Biotechnology. All other Abs were from Cell Signaling. Other reagents were obtained from the following sources. Insulin was from Invitrogen. Protease and phosphatase inhibitor mixtures were from Roche. Recombinant human TNF- was from Promega. CHAPS was from Pierce. Protein A and protein G-agarose beads were from Invitrogen. Compound A was provided by Dr. Karl Ziegelbauer, Bayer HealthCare (Wuppertal, Germany).

Cell lines, cell culture, and transfection

IKK wild-type, IKK^–/–, and IKKβ^–/– mouse embryonic fibroblasts (MEFs) were provided by I. Verma (Salk Institute, La Jolla, CA) and M. Karin (University of California, San Diego, CA). HEK293 was provided by J. Q. Cheng (University of South Florida, Tampa, FL). All cells were maintained in DMEM supplemented with 10% FBS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin (Invitrogen). Transfections were performed using Polyfect Transfection reagent (Qiagen) or Lipofectamine and Plus (Invitrogen) following the manufacturer's instructions. In brief, 3–4 h after transfection, cells were recovered in full serum for 36 h or in full serum for 24 h and then serum-starved for 16–24 h as indicated.

RNA interference

Short interfering RNA (siRNA) SMARTpool IKK (Catalog no. M-003473), IKKβ (Catalog no. M-003503), Akt1 (Catalog no. M-003000), and Akt1 (Catalog no. M-003000) were from Dharmacon. Each of these represents four pooled, SMART selected siRNA duplexes that target the indicated gene. HEK293 and HeLa cell lines were transfected with indicated SMARTpool siRNA or nonspecific control pool using DharmaFECT 1 reagent (Dharmacon) according to the manufacturer's instructions. In brief, 20 nM final concentration of siRNA was used to transfect cells at 60–70% confluency. Twenty-four hours after transfection, cells were recovered in full serum or were serum-starved 16 h before harvest. Cells were harvested 48–72 h after siRNA transfection.

Cell lysis, immunoblotting, and coimmunoprecipitations

Cells were lysed and immunoblotted as described in Ref. 17 with minor modification. In brief, cells growing in 100-mm dishes were rinsed twice with cold PBS and then lysed on ice for 20 min in 1 ml of lysis buffer (40 mM HEPES (pH 7.5), 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.5 mM orthovanadate, and EDTA-free protease inhibitors (Roche)) containing 1% Triton X-100. After centrifugation at 13,000 x g for 10 min, samples containing 20–50 µg of protein were resolved by SDS-PAGE and proteins transferred to Pure Nitrocellulose Membrane (Bio-Rad), blocked in 5% nonfat milk, and blotted with the indicated Abs. For immunoprecipitation experiments, the lysis buffer contained 0.3% CHAPS instead of 1% Triton. A total of 4 µg of the indicated Abs was added to the cleared cellular lysates and incubated with rotation for 6–16 h. Then 25 µl of protein G-agarose was added and the incubation continued for 1 h. Immunoprecipitates captured with protein G-agarose were washed three times with the CHAPS lysis buffer, two times by wash buffer A (50 mM HEPES (pH 7.5), and 150 mM NaCl), and boiled in 4x SDS samples buffer for Western blot.

	Results

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Akt is required for insulin-induced mTOR activation in different cells types

To analyze a requirement for Akt in controlling mTOR activation downstream of insulin-induced signaling, HEK293, HeLa, and MCF-7 cells were pretreated with the PI3K antagonist LY294002 and then exposed to insulin. Phosphorylation of S6K was determined by the use of an Ab that recognizes S6K phospho-Thr389. In these studies, LY suppressed both constitutive and insulin-induced S6K phosphorylation (data not shown), as expected. Given the potential that PI3K antagonists can directly suppress mTOR kinase activity (32), we used siRNA to Akt1 in HEK 293 cells and measured mTOR activity in response to insulin treatment. The results from these experiments demonstrate that siRNA to Akt1 suppressed insulin-induced mTOR activation as measured via S6K phosphorylation (Fig. 1A). Note that Akt2 remains expressed in these cells and likely contributes to the remaining mTOR activity. These studies indicate, as expected, that the control of insulin-induced activation of mTOR in several cell types requires Akt activity.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. A, HEK 293 cells were transfected with siRNA control or Akt1 siRNA. Cells were serum-starved for 24 h, after 36 h of transfection. Cells were left untreated or treated with insulin (100 nM; Invitrogen) for 30 min. Extracts were electrophoresed and blotted with Abs to phospho-S6K, S6K, Akt, and β-actin. B, HeLa cells were transfected with siRNA control, IKK, or IKKβ siRNA. Cells were serum starved for 24 h after 36 h of transfection and stimulated with insulin (100 nM) for 30 min. Western blot shows the level of endogenous phosphorylation of S6K and levels of S6K, IKK, IKKβ, and β-tubulin. Scanned values for this experiment were compared with control levels, which were set at 1. Bar graph shows the average of three different experiments with error bars. C, Serum-starved MEFs WT, IKK–/–, IKKβ–/–, and IKKβ DKO cells were stimulated with insulin (100 nM) for 30 min as described above. The levels of endogenous phosphorylation of S6K and levels of S6K, IKK, IKKβ, mTOR, and β-actin were determined by immunoblotting with the indicated Abs. Quantitation was as described in B. D, MEFs IKK–/– cells were transfected with HA-S6K or cotransfected with wild-type IKK as indicated. The cells were serum starved for 16 h, and then stimulated with insulin and lysed. The HA immunoprecipitate and total lysates were electrophoresed and blotted with Abs to phospho-S6K, HA, and IKK β-actin. E, IKKβ DKO cells were transfected as the indicated. The cells were serum-starved, stimulated with insulin, lysed, and HA immunoprecipitates and total lysates were electrophoresed and blotted with Abs to phospho-S6K, HA, IKK, and β-actin as in D. F, Serum-starved HEK 293 and HeLa cells were left untreated or pretreated with the IKKβ inhibitor (Cmpd. A, 1 µM) for 3 h and then stimulated with insulin (100 nM) for 30 min before lysis. Lysates were electrophoresed and blotted with Abs to phospho-S6K, phospho-Akt, S6K, Akt, IB, and β-actin. Results presented are representative of at least three experiments.

IKK is required for efficient induction of mTOR activation in response to insulin

Based on previous experiments indicating the involvement of IKK downstream of Akt in controlling mTOR activation in PTEN-deficient cells (31), we asked whether IKK is involved in insulin-induced activation of mTOR. siRNA to IKK in HeLa cells strongly reduced the ability of insulin to activate mTOR, as measured through endogenous S6K phosphorylation (Fig. 1B). siRNA to IKKβ only weakly reduced insulin-induced mTOR activation (Fig. 1B). Similar results were found using siRNA to IKK and IKKβ in HEK 293 cells (data not shown). To extend these findings, IKK –/– and IKKβ –/– MEFs were analyzed for their responses to insulin. Knockout of IKK reduced insulin-induced activation of mTOR by 75%, whereas knockout of IKKβ was significantly less effective at blocking mTOR activation (Fig. 1C). The use of IKK double knockout cells (IKK DKO) showed that both IKK and IKKβ are required for the normal, high level activation of mTOR induced by insulin but loss of these subunits does not completely eliminate inducible S6K phosphorylation (Fig. 1C). HA-tagged S6K was transfected into IKK–/– cells, without or with IKK, and phosphorylation of S6K was measured following HA-dependent immunoprecipitation from extracts of untreated or insulin-treated cells. In this experiment, the reduced level of insulin-induced S6K phosphorylation seen in IKK–/– cells was increased with expression of IKK (Fig. 1D). In a similar experiment, expression of IKK elevated insulin-induced S6K phosphorylation in IKK DKO cells whereas IKKβ only weakly affected this response (Fig. 1E). To further address a possible role for IKKβ relative to insulin-induced signaling, a highly specific inhibitor of IKKβ (compound A;33) was tested. Compound A could not suppress insulin-induced mTOR activation in HEK 293 or HeLa cells (Fig. 1F), although IKKβ inhibition suppressed basal S6K phosphorylation in HeLa cells. Further evidence for the efficacy of compound A in blocking IKK activity is shown in Fig. 3F. Additionally, inhibition of IKKβ enhances insulin-induced mTOR activity, suggesting a potential shift to IKK under these conditions. These results indicate that IKK, and to a lesser degree IKKβ, is important for induction of mTOR activity downstream of insulin-induced signaling.

View larger version (42K): [in this window] [in a new window]   FIGURE 3. A, HEK 293 cells were transfected with siRNA control or Akt1 siRNA. Cells were serum-starved for 24 h after 36 h of transfection. Cells were left untreated or treated with TNF- (15 ng/ml, 30 min). Extracts were electrophoresed and blotted with the indicated Abs. B, HEK 293 and HeLa cells were transfected with siRNA control, IKK, or IKKβ siRNA. Cells were serum starved for 24 h after 36 h of transfection and stimulated with TNF- for 30 min. Western blot shows the level of endogenous phosphorylation of S6K and levels of S6K, IKK, IKKβ, and β-actin. Scanning and quantitation was performed as described in Fig. 1B (both for the presented experiment and for the bar graph which shows values from three experiments). C, Serum-starved MEFs WT, IKK–/–, IKKβ–/–, and IKKβ DKO cells were stimulated with TNF- for 30 min. The levels of endogenous phosphorylation of S6K and levels of S6K, IKK, IKKβ, mTOR, and β-actin were determined by immunoblotting with the indicated Abs. Scanning and quantitation was performed as described in Fig. 1B (both for the presented experiment and for the bar graph which shows values from three experiments). D, MEFs IKK–/– cell were transfected with HA-S6K or cotransfected with WT IKK as indicated. The cells were serum starved for 16 h, and then stimulated with TNF- and lysed. The HA immunoprecipitates and total lysates were electrophoresed and blotted with Abs to phospho-S6K, HA, IKK and actin. E, IKKβ DKO cells were transfected as indicated. The cells were serum starved, stimulated with TNF-, lysed, immunoprecipitated, and then HA immunoprecipitates and total lysates were electrophoresed and blotted with Abs to phospho-S6K, HA, IKK, and actin as in Fig. 2D. F, Serum-starved HEK 293 cells were left untreated or pretreated with the IKKβ (Cmpd. A, 1 µM) for 3 h and then stimulated with TNF- for 20 min before lysis. Lysates were electrophoresed and blotted with Abs to phospho-S6K, phospho-Akt, S6K, Akt, IB, and actin. Quantified results are as in Fig. 1B. Results are representative of at least three different experiments.

IKK is induced to associate with mTOR in the insulin-induced pathway in a manner dependent on PI3K-signaling

To determine whether insulin responses lead to an alteration in association between IKK and mTOR (or an mTOR-associated protein), HEK 293 and HeLa cells were pretreated with the PI3K antagonist LY294002 and then stimulated with insulin for 30 min. Extracts were prepared and immunoprecipitation studies were conducted using the IKK Ab. The immunoprecipitate was electrophoresed on SDS gels, transferred to membrane, and probed with anti-mTOR. The results show (Fig. 2A) that, in both cell types, insulin induces a robust interaction between IKK and mTOR. LY294002 pretreatment blocks this induced association, indicating that insulin induces an association between IKK and mTOR in a manner dependent on the PI3K/Akt-associated pathway. To address a potential involvement of IKKβ in controlling the insulin-induced IKK/mTOR association, IKKβ–/– and IKK–/– cells were treated with insulin with or without LY294002. Immunoprecipitation of IKK from IKKβ–/– cells shows that insulin induces IKK and mTOR interaction, whereas immunoprecipitation of IKKβ from IKK–/– cells shows that IKKβ does not associate with mTOR (Fig. 2B). These results suggest that IKK is the critical IKK subunit in controlling interaction with mTOR.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. A, Serum-starved 293 and HeLa cells were pretreated with the PI3K antagonist LY294002 (LY) (10 µM) for 30 min and then stimulated with insulin (100 nM) for 30 min before lysis. Immunoprecipitates of endogenous IKK prepared from indicated cells were blotted with mTOR and IKK Abs. Results shown are typical of three experiments. B, IKKβ–/– and IKK–/– cells were treated with insulin with or without LY294002 (LY) as described in A. Either IKK or IKKβ was immunoprecipitated and analyzed for association with mTOR by immunoblotting.

TNF-induced mTOR activity is controlled by IKK and IKKβ in a cell-type, signaling-dependent manner

mTOR has been shown to be induced by TNF in several cell types (5, 6, 7). In their study using two breast cancer cell lines (MCF7 and MDA-MB-453), Lee and coworkers (6) concluded that TNF-induced mTOR activation was IKKβ dependent and Akt independent. In contrast with that general conclusion, TNF was shown to induce mTOR activity in HEK293 cells and this induction was blocked by the PI3K antagonist LY294002 (data not shown) and by knockdown of Akt (Fig. 3A). In HEK 293 cells, knockdown of IKK suppressed TNF-induced mTOR activation by greater than 50% (Fig. 3B, left panel), whereas knockdown of IKKβ had less of an effect. In HeLa cells, IKK and IKKβ appear to equally contribute to mTOR activation induced by TNF (Fig. 3B, right panel). Using wild-type and IKK knockout MEFs, IKK was found to be significantly more important in controlling mTOR activation downstream of TNF-induced signaling as compared with IKKβ (Fig. 3C). To analyze direct effects on S6K, as a measure of mTOR activity, HA-tagged S6K was transfected into IKK–/– MEFs and phosphorylation was analyzed following immunoprecipitation. As shown in Fig. 3D, expression of IKK in IKK–/– MEFs largely restores the normal level of S6K phosphorylation induced by TNF. A similar result was found in IKK DKO cells. In this experiment, expression of IKKβ yielded a less robust response to TNF as compared with IKK expression (Fig. 3E). In HEK 293 cells, the IKKβ inhibitor (Cmpd A) described above reduced the basal level of mTOR activity but only weakly blocked the fold-induction of mTOR controlled by TNF treatment (Fig. 3F). These studies indicate that in several cell types, IKK plays an important role in the control of mTOR activation downstream of TNF-induced signaling.

TNF induces an association between IKK and mTOR that is suppressed by PI3K antagonist

To determine whether TNF-signaling induces an association between IKK and mTOR, two approaches were taken. First, 293 and HeLa cells were transfected with HA-tagged IKK and myc-tagged mTOR, and subsequently treated with TNF. IKK was immunoprecipitated with the HA Ab, and the precipitate was electrophoresed and probed with anti-myc. The results showed that TNF induces an approximate 4-fold increase in association between IKK and mTOR or an mTOR-associated protein (Fig. 4A). Similar results were obtained in HeLa cells (Fig. 4A, right panel). To determine whether TNF induces an association between endogenous IKK and mTOR, HEK 293 cells were left untreated or were treated with TNF for different times. One set of cells was pretreated with the PI3K antagonist LY294002. IKK was immunoprecipitated and associated mTOR was measured following electrophoresis and probing with the mTOR Ab. These results demonstrate that TNF induces an association between endogenous mTOR (or an associated protein) and IKK, which is blocked by LY294002 (Fig. 4B). We then performed a similar experiment to the one shown in Fig. 2B, to determine a potential requirement for IKKβ in controlling IKK association with mTOR downstream of TNF-induced signaling. TNF induced an association between IKK and mTOR in IKKβ–/– cells, which is blocked by LY, whereas IKKβ was not inducibly associated with mTOR in IKK–/– cells (Fig. 4C). These results indicate that IKK is critical for the induced association of IKK and mTOR.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. A, HEK 293, and HeLa cells were cotransfected with HA-IKK and myc-mTOR. Cells were serum-starved for 16 h and were pretreated with the PI3K antagonist LY294002 (LY, 10 µM) for 30 min and then stimulated with TNF- for different times as indicated, before lysis. Immunoprecipitates of HA were blotted with myc and HA Abs. B, Serum-starved HEK 293 cells were left untreated or pretreated by LY294002, and then stimulated with TNF- for different times. Immunoprecipitates of IKK were blotted with mTOR and IKK Abs. Quantified results are performed as described above for Fig. 1B. Results are representative of three different experiments. C, Experiment is identical with that described in Fig. 2B, except that cells were stimulated with TNF instead of insulin.

Lee and coworkers (6) showed that IKKβ phosphorylates TSC1 to activate mTOR. Previously, it was shown that insulin induces Akt-dependent phosphorylation of TSC2 at Serine 939 and Threonine 1462, as part of the mTOR activation response (8). We asked whether TNF induces phosphorylation of TSC2 at these positions. HEK 293 cells were left untreated or treated with TNF for varying times. Extracts were prepared and analyzed by immunoblotting using Abs for TSC2 phosphorylated at S939 or T1462. Results from this experiment show that TNF induces TSC2 phosphorylation at positions known to be phosphorylated by Akt (Fig. 5A). Importantly, TNF effectively induces Akt activation in HEK 293 cells as indicated by increased reactivity of Akt to the phospho-S473 Ab (Fig. 5A). To determine whether this phosphorylation is important for the ability of TNF to induce mTOR activity, either wild-type TSC2 or a form of TSC2 mutated in multiple Akt sites (34), was transfected into HEK 293 cells and analyzed for effects on the ability of TNF to induce mTOR activity, as measured through S6K phosphorylation. Results from this experiment demonstrate that the mutant form of TSC2, which is inhibited relative to its ability to be phosphorylated by Akt, suppresses mTOR activation induced by TNF, whereas the wild-type (WT) TSC2 does not (Fig. 5B). These data indicate that the ability of TNF to induce mTOR activation is generated, at least partly, through the ability of TNF-induced Akt to phosphorylate TSC2.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. A, Serum-starved HEK 293 cells were stimulated with TNF- for different times, as shown. The lysates were electrophoresed and blotted with Abs as indicated. B, HEK 293 cells were transfected with vectors, TSC2-WT or TSC2 mutant plasmid as indicated. The cells were serum starved for 16 h and then left untreated or treated by TNF- for 30 min. Extracts were electrophoresed and blotted with the indicated Abs. Results are representative of at three different experiments.

TNF requires IKKβ to control mTOR activity in cells where Akt is not induced, but insulin uses IKK in these same cells

Lee and coworkers (6) showed that TNF does not induce Akt activation in MCF7 cells, and that IKKβ is critical for TNF-induced mTOR activation in these cells. In agreement with that report, our results demonstrates that TNF does not induce Akt phosphorylation over that basal levels in MCF7 cells (Fig. 6A). LY294002 blocks basal levels of Akt phosphorylation (Fig. 6A). In contrast with the results observed in other cells used in our study, siRNA-directed knockdown of IKK has only a modest affect on TNF-induced mTOR activity in MCF7 cells, whereas knockdown of IKKβ strongly suppressed TNF-induced mTOR activity (Fig. 6B), consistent with the results of Lee and coworkers (6). We asked whether the effect observed in MCF7 cells relative to TNF-induced mTOR activation was also true for insulin-induced mTOR activity in these cells. Opposite to the result obtained with TNF treatment of MCF7 cells, knockdown of IKK suppressed insulin-induced mTOR activation, whereas knockdown of IKKβ led to a minimal effect on insulin-induced mTOR activation (Fig. 6C). Consistent with these results indicating the role for IKKβ in controlling TNF- but not insulin-induced mTOR activation in MCF7 cells, the IKKβ inhibitor blocked basal and induced levels of mTOR activity in response to TNF, but not in response to insulin treatment (Fig. 6D). Although TNF was shown to induce TSC2 phosphorylation in HEK 293 cells where Akt is activated (Fig. 5A), TNF cannot induce TSC2 phosphorylation in MCF-7 cells (Fig. 6E). In these cells, insulin induces TSC2 phosphorylation, which is not blocked by the IKKβ inhibitor (Fig. 6E). Consistent with the results described above, insulin induces an association between mTOR and IKK in MCF-7 cells, which is blocked by the PI3K antagonist LY294002, whereas TNF does not induce this association in these cells (Fig. 6F). These results demonstrate that within the same cell, the Akt signaling pathway specifies the involvement of a distinct IKK subunit to control mTOR activation.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. A, Serum-starved MCF-7 cells were left untreated or pretreated with LY294002, and then stimulated with TNF- for 30 min before lysis. Lysates were electrophoresed and blotted with Abs to phospho-S6K, phospho-Akt, S6K, Akt, IB, and actin. B and C, MCF-7 cells were transfected with siRNA control, IKK, or IKKβ siRNA. Cells were serum starved for 24 h after 36 h of transfection and stimulated with TNF- (B) or insulin (C) for 30 min. Western blot shows the level of endogenous phosphorylation of S6K and levels of S6K, IKK, IKKβ, and β-actin. D and E, Serum-starved MCF-7 cells were left untreated or pretreated with the IKKβ inhibitor (Cmpd. A) for 3 h, and then stimulated with TNF- or insulin for 30 min before lysis. Western blot shows the level of endogenous phosphorylation of S6K, Akt, and TSC2, and levels of S6K, Akt, TSC2 and Actin. F, Serum-starved MCF-7 cells were pretreated with the PI3K antagonist LY294002 (LY) for 30 min and then stimulated with insulin (100 nM) or TNF- (15 ng/ml) for 30 min before lysis. Immunoprecipitates of endogenous IKK prepared from indicated cells were blotted with mTOR and IKK Abs. Results are representative of at least three experiments.

	Discussion

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The results presented in this manuscript, using a variety of cells, demonstrate that IKK is required for insulin to induce efficient, high level mTOR activity. In this context, insulin induces a robust association between IKK and mTOR (or an mTOR-associated protein) and this response is inhibited by PI3K inhibition (Fig. 3). IKKβ contributes to the insulin-induced mTOR activation with less involvement in MEFs and HEK 293 cells, and approximately equivalent to IKK in HeLa cells. This may relate to the Akt-response in these cells, as hypothesized, or to other cell-type specific mechanisms. Although IKK interaction with mTOR involves both IKK and IKKβ (data not shown, and see Ref. 6), results shown in Fig. 2 indicate that IKKβ is not required for this interaction downstream of insulin-induced, Akt-dependent signaling. The mechanism that IKK plays in enhancing mTOR activation is not understood, but may involve mTOR phosphorylation and/or altered association with key regulatory proteins, such as Raptor. Ongoing experiments attempt to address these potential regulatory mechanisms.

Although pathways associated with the ability of insulin to induce mTOR activity have been studied extensively, there have been less studies on the ability of cytokines, such as TNF, to induce mTOR activity. Previously, Glantschnig et al. (5) and Ozes et al. (7) demonstrated that TNF can induce mTOR activity. As described above, Lee and coworkers (6) showed that TNF induces mTOR activity in two breast cancer cell lines through a pathway that does not involve Akt but requires IKKβ. In this pathway, IKKβ was reported to directly phosphorylate TSC1 to activate mTOR, presumably through the suppression of the inhibitory effect of TSC1/2 on the positive regulator of mTOR, Rheb. We have analyzed the ability of TNF to induce mTOR activity in several cell lines. In our studies, IKK is consistently more important that IKKβ in controlling mTOR activation in the TNF response in cells that induce an Akt response to this cytokine. In these cells, Akt induces phosphorylation of TSC2 (Fig. 5A) and a mutant form of TSC2 that is mutated in Akt target sites blocks the ability of TNF to induce mTOR activity (Fig. 5B). These results indicate that TSC2 phosphorylation is an important regulatory mechanism in the control of TNF-induced mTOR activity in cells where Akt is activated (see Fig. 7 model figure). It is postulated that IKK functions to amplify the mTOR response following TSC2 phosphorylation, and subsequent activation of Rheb (see Fig. 7). In MCF7 cells, where TNF does not induce Akt, IKKβ is significantly more important in the control of mTOR activation (see Fig. 7 model figure), consistent with the results of Lee and coworkers (6). Interestingly, insulin treatment of MCF7 cells requires IKK, as compared with IKKβ, in controlling mTOR activation, indicating that the same cell uses distinct IKK pathways to regulate mTOR activation dependent on the signaling pathway uses. The studies conclude that both IKK and IKKβ serve dedicated functions to contribute to mTOR activation, and that Akt activation directs the control of IKK in this response.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. Model depicting pathways and cell type dependent mTOR activation in response to insulin or TNF-. A, Insulin induces mTOR through an Akt-dependent pathway that activates IKK and induces TSC2 phosphorylation to induce mTOR activation. B, TNF induces mTOR activation through an Akt-dependent pathway, which functions similar to insulin induction, and an Akt-independent pathway that targets TSC1 via IKKβ (see Lee and coworkers, 20 ). This response varies in cell in parallel with the strength of the Akt response. C, In MCF7 cells, TNF largely uses IKKβ and the Akt-independent pathway to induce mTOR activation. See text.

	Acknowledgments

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
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 grants from the National Institutes of Health (AI35098 and CA75080). A postdoctoral fellowship to H.C.D. from the Department of Defense is acknowledged. Additional support was provided the Samuel Waxman Cancer Research Foundation.

² Address correspondence and reprint requests to Dr. Albert S. Baldwin, Lineberger Comprehensive Cancer Center, Campus Box No. 7295, University of North Carolina, Chapel Hill, NC 27599. E-mail address: abaldwin{at}med.unc.edu

³ Abbreviations used in this paper: mTOR, mammalian target of rapamycin; IKK, IB kinase; TSC, tuberous sclerosis complex; siRNA, short interfering RNA; IKK DKO, IKK double knockout; MEF, mouse embryonic fibroblasts.

Received for publication December 13, 2007. Accepted for publication March 25, 2008.

	References

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