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IKBKE Phosphorylation and Inhibition of FOXO3a: A Mechanism of IKBKE Oncogenic Function

Forkhead box O (FOXO) transcription factors are emerging as key regulators of cell survival and growth. The transcriptional activity and subcellular localization of FOXO are tightly regulated by post-translational modifications. Here we report that IKBKE regulates FOXO3a through phosphorylation of FOXO3a-Ser644. The phosphorylation of FOXO3a resulted in its degradation and nuclear-cytoplasmic translocation. Previous studies have shown that IKBKE directly activates Akt and that Akt inhibits FOXO3a by phosphorylation of Ser32, Ser253 and Ser315. However, the activity of Akt-nonphosphorytable FOXO3a-A3 (i.e., converting 3 serine residues to alanine) was inhibited by IKBKE. Furthermore, overexpression of IKBKE correlates with elevated levels of pFOXO3a-S644 in primary lung and breast tumors. IKBKE inhibits cellular function of FOXO3a and FOXO3a-A3 but, to a much less extent, of FOXO3a-S644A. These findings suggest that IKBKE regulates FOXO3a primarily through phosphorylation of SerS644 and that IKBKE exerts its cellular function, at least to some extent, through regulation of FOXO3a.

We also showed that elevated expression of IKBKE is involved in chemo-and tamoxifen-resistance [18].
FoxO transcription factor family is a key player in an evolutionary conserved pathway, which consists of FOXO1, 3, 4 and 6 in mammals. Four members of FOXO share high similarity in their structure, function and regulation. They are involved in diverse cellular and physiological processes including cell survival, proliferation, cell cycle and metabolism as well as reactive oxygen species (ROS) response and longevity. A number of target genes of FOXOs have been identified which include Bim and FasL for inducing apoptosis [19,20]; p27 kip1 and cyclin D for cell cycle control [21,22], GADD45a for DNA repair [23] and G6Pase for glucose metabolism [24,25]. Accumulating studies demonstrated that these FOXOs are predominantly regulated by post-translational modifications, including phosphorylation, acetylation, methylation and ubiquitination [6,[26][27][28]. For instance, FOXO3a has been shown to be phosphorylated by IKKa/b at Ser644 [26], Akt at Ser32, Ser253 and Ser315 [29], and ERK1/2 at Ser294, Ser344 and Ser425 [30,31], resulting in either decrease of FOXO3a DNA binding activity or/and protein stability.
In the present study, we show that IKBKE inhibits FOXO3a and FOXO3a-A3, an Akt-nonphosphorylatable form, function by direct phosphorylation of FOXO3a. While the kinase domain of IKBKE is distinct from IKKa and IKKb [5], it also phosphorylates FOXO3a-Ser644. As a result, IKBKE induces FOXO3a degradation and nuclear-cytoplasmic translocation leading to abrogation of FOXO3a cellular function. (B) FOXO3a-and FOXO3a-A3-induced p27 transcription was reduced by IKBKE. H1299 cells were transfected with indicated plasmids. The p27 mRNA levels were determined by semi-quantitative RT-PCR (upper panels). GAPDH was used as control. Western blot shows the expression of transfected plasmids (panels 3 and 4). Bottom panel is a loading control. (C) IKBKE inhibits FOXO3a and FOXO3a-A3 DNA-binding activity. H1299 cells were transfected with indicated plasmids. ChIP assay was performed as described in ''Experimental Procedures''. Anti-HA antibody was used for chromatin IP. IgG was served as negative control. The DNA prior to the IP was used as positive controls (input). (D) IKBKE inhibition of FOXO3a depends on its kinase activity. MCF7 cells were transfected with pGL3-FHRE-Luc (e.g., 3

Cell Lines, Lung Tumor Specimens, Antibodies and Recombinant Protein
The non-small cell lung cancer (NSCLC) cell lines were provided by Moffitt Cancer Center Lung Cancer Cell Core. Breast cancer cell lines (MCF7, MDA-MB435 and T47D), HEK293 and HeLa were purchased from ATCC. These cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin. Ikbke-knockout mouse fibroblast (MEF-Ikbke 2/2 ) and wild type MEF (MEF-W) were kindly provided by Tom Maniatis (Harvard University). Doxycycline-inducible IKBKE cell line was established by transfection of HeLa tet-on cell (Clontech) with pTRE-Tight-IKBKE.
Fifty primary human NSCLC and 57 breast tumor specimens were collected from 1998 to 2005 at the Moffitt Cancer Center, approved by the Institutional Review Boards (IRB) of University of South Florida (#17121). All participants provided written consent and data were de-identified. Consent forms were kept on file and the IRB approved of the consent procedure under Moffitt Cancer Center Total Cancer Control protocol. The tissues were snap frozen and stored at 270uC.
Antibodies against IKBKE, Myc and HA were purchased from Sigma. Anti-FOXO3a and -p27 antibodies were from Santa Cruz Biotechnology. Antibodies of Bim and GFP were from Cell Signaling. Anti-phospho-FOXO3a-S644 antibody was kindly provided by Mickey C.-T Hu (Stanford University). Recombinant protein IKBKE was purchased from Cell Signaling.

In Vitro Kinase Assay and In Vivo [ 32 P]Pi Cell Labeling
In vitro IKBKE kinase assay was performed as previously described [18,32]. Briefly, recombinant IKBKE was incubated with GST-FOXO3a in the presence of 10 mCi of [c-32 P]ATP (NEN) and 3 mM cold ATP in a kinase buffer. After incubation at 30uC for 30 min, the reaction was stopped and separated in SDS-PAGE gels. Each experiment was repeated three times.
For in vivo labeling, H1299 cells were transfected with HA-FOXO3a-A3 or HA-FOXO3a-A3-S644A together with and without myr-IKBKE. After serum starvation overnight, cells werelabeled with [ 32 P]P i (0.5 mCi/ml) in phenol red-free MEM without phosphate for 4 hours. FOXO3a was immunoprecipitated with HA antibody, separated on SDS-PAGE and transferred to membrane. Phosphorylated FOXO3a was detected by autoradiography and quantified.
Western Blot, Co-immunoprecipitation (co-IP) and Immunofluorescence Western blot, co-IP and immunofluorescence were performed as previously described [33]. Briefly, cell lysates were prepared in a lysis buffer and subject to immunoprecipitation and immunoblot analysis. To determine if the FOXO3a band shift is due to the phosphorylation, a part of cell lysate were treated with CIP (1 unit/mg protein) for 30 min at 37uC prior to Western blot. For immunofluorescence, MCF7 cells were transfected with myc-IKBKE and GFP-FOXO3a using LipofectinH reagent (Invitrogen). After 48 hours, cells were stained with Alexa FluorH 555 conjugated (red) Myc antibody and DAPI and observed under fluorescence microscope.

Cell Viability and Programmed Cell Death
Cell viability was evaluated using CellTiter-GloH Luminescent Cell Viability Agent according to manufacturer's protocol (Promega). Apoptosis was determined by caspase 3/7 activity and TUNEL assay [34]. Briefly, cells were plated into 96-well plate with 1610 4 cells/well and then transfected with different constructs. Following incubation for 48 hours, caspase 3/7 activity was measured using the Caspase-Glo 3/7 Assay Systems (Promega). TUNEL assay was performed with the TUNEL Apoptosis Detection Kit (Millipore).

Statistic Analysis
For luciferase activity and cell survival, the experiments were repeated at least three times in triplicate. The data are represented by means 6 SD. Differences between control and testing cells were evaluated by Student's t test; the correlation of IKBKE expression with phosphorylation of FOXO3a-S644 was analyzed by Chi-square test, all analyses were completed with SPSS software, version 10.0. P,0.05 was considered statistically significant.

IKBKE Represses Transcription and DNA-binding Activity of FOXO3a and FOXO3a-A3
Previous studies showed that FOXO3a functions as a tumor suppressor and inhibits cell survival and growth [35]. Akt was identified as a key regulator of FOXO3a by phosphorylation of three serine residues, Ser32, Ser253 and Ser315 [29]. We have recently shown that IKBKE functions as an Akt-T308 and -S473 kinase and directly activates Akt independent of PI3K/PDK1 and mTORC2 [15]. These findings prompted us to examine whether IKBKE regulated FOXO3a function. As an initial step, we assessed the effect of IKBKE on the transcription activity of FOXO3a and FOXO3a-A3, an Akt-nonphosphorylatable form in which 3 serine residues were converted to alanine. In agreement with previous reports [29,35], p27 promoter activity was induced by FOXO3a or FOXO3a-A3. Unlike Akt, which only inhibited FOXO3a, IKBKE abrogated both FOXO3a-and FOXO3a-A3induced p27 promoter activities ( Fig. 1A and Fig. S1). FOXO3aand FOXO3a-A3-induced p27 mRNA levels were also inhibited by IKBKE (Fig. 1B). Moreover, ChIP assay showed that IKBKE inhibited DNA binding activity of FOXO3a and FOXO3a-A3 (Fig. 1C). In addition to p27 promoter, we examined the effect of IKBKE on FHRE-Luc, which is a luciferase reporter driven by the promoter containing three repeats of FOXO3a binding consensus motif [29]. Fig. 1D shows that constitutively active (Myr)-IKBKE reduced whereas DN-IKBKE enhanced FOXO3a transcription activity. Furthermore, knockdown of IKBKE increased FHRE-Luc activity (Fig. 1E). Together, these findings suggest that IKBKE inhibition of FOXO3a activity is independent of Akt.

Direct Phosphorylation of FOXO3a-S644 by IKBKE
We next investigated if IKBKE interacts with and phosphorylates FOXO3a. Co-immunoprecipitation revealed no interaction between IKBKE and FOXO3a (data not shown). However, when IKBKE and FOXO3a were co-expressed in H1299 cells, we observed remarkable mobility shift of FOXO3a as well as decrease of FOXO3a and p27 protein levels ( Fig. 2A). The mobility shift was inhibited by treatment of cell lysate with calf intestinal alkaline protein phosphatase CIP but not by treatment of cells with Akt inhibitor MK2206 (Fig. 2B). These data suggest that IKBKE could phosphorylate FOXO3a via an Akt-independent manner.
To define the phosphorylation site(s), we mutated individual serine/threonine residue into alanine within FO3-7 region. In vitro kinase assay revealed that phosphorylation of GST-FO3-7/ S644A by IKBKE was significantly reduced compared to the wild-type and other mutant fusion proteins (Fig. 3A). Sequencing analysis revealed that FOXO3a-S644 partially fits a putative IKBKE phosphorylation consensus motif [7] and is also conserved among different species (Fig. 3B). To further examine if FOXO3a-S644 was phosphorylated by IKBKE, in vivo labeling was performed by transfection of wild type FOXO3a, FOXO3a-A3 and FOXO3a-A3-S644A together with and without myr-IKBKE. Fig. 3C showed that IKBKE phosphorylated FOXO3a-A3 and that the phosphorylation level was significantly reduced by mutation of Ser644 (FOXO3a-A3-S644A). Western blot analysis with specific antibody against phospho-FOXO3a-S644 further showed that IKBKE phosphorylated FOXO3a (Fig. 3D and 3E). Furthermore, expression levels of p27 and Bim, 2 representative targets of FOXO3a, were reduced by expression of IKBKE or myr-IKBKE but were increased by knockdown of IKBKE (Fig. 3E).
To determine if this event occurred in vivo, we examined 50 NSCLC specimens for protein expression of IKBKE and pFOXO3a-S644 (Fig. 3F). Of the 50 lung tumors, 27 had overexpression of IKBKE and 28 had elevated pFOXO3a-S644. Of the 28 tumors with elevated pFOXO3a-S644, 20 (71.4%) also had elevated IKBKE (p = 0.006; Fig. 3G). The other 8 cases with elevated pFOXO3a-S644 could result from activation of IKKa or/and IKKb, which have been shown to also phosphorylate FOXO3a-S644 [26]. Similar results were obtained by evaluating additional 57 breast cancer specimens (Fig. S2). Collectively, these data suggest that FOXO3a-S644 is directly phosphorylated by IKBKE. In addition, we noted that IKBKE still induced FOXO3a-S644A mobility shift which was not affected by Akt inhibitor MK2206 (Fig. 3H). These data suggest that IKBKEinduced FOXO3a mobility shift could result from IKBKE regulation of other kinase(s) in addition to Akt.

IKBKE Phosphorylation of FOXO3a-S644 Results in FOXO3a Nuclear-cytoplasmic Translocation and Loss of Transcription Activity
We also examined the effects of phosphorylation of Ser644 on FOXO3a subcellular localization and transcription activity. H1299 cells were transfected with GFP-FOXO3a or GFP-FOXO3a-S644A together with constitutively active IKBKE. Following incubation for 48 hours, the subcellular localization of FOXO3a was determined and quantified. Figs. 4A and 4B showed that GFP-FOXO3a and GFP-FOXO3a-S644A were predominantly located in the nucleus. Expression of IKBKE led to FOXO3a translocation from the nucleus to cytoplasm. However, FOXO3a-S644A remained in the nucleus (Figs. 4A  and 4B). In addition, p27 reporter assay was carried out to assess the effect of phosphorylation of Ser644 on FOXO3a transcription activity. As shown in Fig. 4C, expression of myr-IKBKE repressed FOXO3a-induced p27 promoter activity but had insignificant effect on p27 promoter activity induced by FOXO3a-S644A (e.g., 20% reduction; p.0.05). Based on these data, we concluded that IKBKE phosphorylation of Ser644 leads to FOXO3a nuclear-cytoplasmic translocation and loss of its transcription activity.

IKBKEinduces FOXO3a Degradation in a p-Ser644dependent Manner
We also noticed that expression of IKBKE decreased FOXO3a protein level (Figs. 2A, 2B and 3E). To further examine the effect of IKBKE on FOXO3a expression, we transfected H1299 cells with IKBKE and found that ectopic expression of IKBKE reduced FOXO3a protein but not mRNA level in a dose-dependent manner (Fig. 5A). Furthermore, increase of FOXO3a protein level was detected in IKBKE-depletion cells (Figs. 5B and 5C).
To determine if the observed IKBKE-promoted reduction in FOXO3a expression levels depends on the phosphorylation of FOXO3a-S644, we compared the degradation rate of FOXO3a and FOXO3a-S644A in the absence or presence of IKBKE. Following transfection of FOXO3a or FOXO3a-S644A together with and without IKBKE, H1299 cells were treated with cycloheximide (CHX) for different times. Immunoblot analysis unraveled that FOXO3a-S644A was more stable than FOXO3a (Figs. 5D and 5F). Expression of IKBKE induced more significant degradation of FOXO3a than FOXO3a-S644A (Figs. 5E and 5F). Similar result was observed in doxycycline-inducible IKBKE cell line (Fig. 5G). Moreover, IKBKE-induced FOXO3a degradation was largely abrogated by treatment with proteasome inhibitor MG132 (Fig. 5H). Based on these results, we conclude that IKBKE induces FOXO3a protein degradation primarily via phosphorylation of Ser644.

IKBKE Protects Cells from FOXO3a-induced Apoptosis Primarily through Phosphorylation of FOXO3a-S644
Since IKBKE directly phosphorylates FOXO3a-S644 leading to FOXO3a nuclear-cytoplasmic translocation and protein degradation, IKBKE could suppress FOXO3a cellular function via a Ser644 phosphorylation-dependent manner. To this end, cell viability was assessed in H1299 cells following transfection of FOXO3a, FOXO3a-S644A or FOXO3a-A3 together with and without myr-IKBKE. As shown in Fig. 6A, expression of FOXO3a alone induced cell death. The effects of FOXO3a and FOXO3a-A3 on cell death were largely abrogated by expression of constitutively active IKBKE. In addition, caspase 3/7 and TUNEL assays revealed that myr-IKBKE inhibited the programmed cell death induced by FOXO3a and FOXO3a-A3 (Figs. 6B and 6C). However, FOXO3a-S644A induced cell death was only inhibited by IKBKE at approximately 20%, which did not reach statistical significance (p.0.05; Figs. 6A and 6B). These data indicate that IKBKE inhibits FOXO3a largely through phosphorylation of Ser644 and, to a much less extent, through IKBKE-activated Akt phosphorylation of Ser32, Ser253 and Ser315 (Fig. 7).

Discussion
IKBKE has essential role as a regulator of innate immunity by modulating interferon and NF-kB signaling [1,2]. Recent studies have also implicated IKBKE in malignant transformation [15,17]. We and others have shown IKBKE induction of cell survival, growth and chemoresistance [16,18,36]. However, underlying molecular mechanism remains elusive. In this study, we show that IKBKE directly mediates phosphorylation of FOXO3a-S644 and induces FOXO3a nuclear-cytoplasmic translocation and protein degradation. As a result, FOXO3a cellular function was inhibited by IKBKE. Furthermore, overexpression of IKBKE significantly correlated with phospho-FOXO3a-S644 in primary lung tumors examined. These findings indicate that FOXO3a is a bona fide substrate of IKBKE and that negative regulation of FOXO3a by IKBKE is a key mechanism for promoting cell survival.
Accumulating studies show that FOXO3a regulates a wide range of biological processes, including inhibition of cell survival and proliferation, protection against oxidative stress, and metabolism [37,38,39]. The biological activity of FOXO3a is regulated predominantly by post-translational modifications, including phosphorylation, acetylation, and ubiquitination. Consistent with this notion, one of the first and potentially most important control mechanisms characterized for FOXO3a is its regulation by Akt, where the phosphorylation of FOXO3a at Ser32, Ser253 and Ser315 by Akt results in the cytoplasmic accumulation and subsequent degradation of this transcription factor [29]. We recently reported that IKBKE activated Akt by direct phosphorylation of Akt-T308 and -S473, which is independent of PDK1 and mTORC2 [15]. Thus, IKBKE could regulate FOXO3a through both indirect (e.g., Akt) and direct (e.g., phosphorylation of Ser644) mechanism. Interestingly, our data show that IKBKE phosphorylation of Ser644 residue, while it locates in transactivation domain (Fig. 2C), is sufficient to promote FOXO3a nuclearcytoplasmic translocation and degradation (Figs. 4 and 5). Moreover, IKBKE inhibits Akt-nonphosphorylatable FOXO3a-A3 transcription and DNA binding activities as well as FOXO3a-A3-induced cell death. While IKBKE also suppresses FOXO3a-S644A function towards p27 and apoptosis (approximately ,20%), it did not reach statistical significance (Figs. 4C and 6A/B). These findings suggest that IKBKE represses FOXO3a primarily through direct phosphorylation of Ser644 and that phosphorylation of FOXO3a by IKBKE-Akt axis has a less important role.
While IKBKE is a member of IKK family, its kinase domain shares ,27% amino acid identity to IKKa and IKKb [5]. Even though a majority of IKBKE substrates could not be phosphorylated by IKKa and IKKb, which include Akt, IRF3/7 and STAT1 [13,15], IKKa/b andIKBKEshare some common substrates including IkB. IKKa/b have been shown toactivate NF-kB by phosphorylation of IkB at Ser32/Ser36 whereas IKBKE induces NF-kB by phosphorylation of IkB-Ser36 [40]. Previous studies have shown that FOXO3a-S644 is phosphorylated by IKKa and IKKb, which leads to FOXO3a cytoplasmic accumulation and degradation in a manner of independent of Akt and ERK signaling [26]. Thus, our study provided an additional substrate that is shared by IKBKE and IKKa/b. Furthermore, a recent study shows that IKKa and IKKb inhibit Akt through phosphorylation of p85a in response to starvation [41]. However, we observed activation of Akt by IKBKE but not by IKKa/b in normal condition, which induces p-FOXO3a-S253, -S32 and -S315 ( Fig. S3 and data not shown), while IKBKE and IKKa/b all phosphorylate FOXO3a-S644 (Fig. S3).
Based on our findings, we propose a model IKBKE-dependent repression of FOXO3a that promotes cell survival, growth and tumorigenesis. While IKBKE directly activates Akt, IKBKE phosphorylation of Ser644 plays a predominant role in repression of FOXO3a (Fig. 7). Our study suggests that restoration of FOXO3a activity could be an attractive therapeutic strategy for human tumors expressing elevated levels of IKBKE. In addition, a recent report showed that phosphorylation of FOXO3a by IKBKE regulates INFb expression suggesting the role of FOXO3 in immune response control [42]. Further investigation is required for defining the mechanism by which Ser644 regulates FOXO3a subcellular localization as well as the significance of Ser644 in FOXO3a tumor suppressor function in animal model.