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Protein kinase C δ activates IκB-kinase α to induce the p53 tumor suppressor in response to oxidative stress
Cellular Signalling 19 (2007) 2088 – 2097 www.elsevier.com/locate/cellsig
Protein kinase C δ activates IκB-kinase α to induce the p53 tumor suppressor in response to oxidative stress Tomoko Yamaguchi, Yoshio Miki ⁎, Kiyotsugu Yoshida ⁎ Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan Received 10 May 2007; accepted 14 June 2007 Available online 21 June 2007
Abstract Protein kinase C δ (PKCδ) functions as a redox-sensitive kinase in various cell types. Upon exposure to reactive oxygen species (ROS), it is activated by tyrosine phosphorylation, nuclear translocation and caspase-3-mediated cleavage. Activated PKCδ is associated with cell cycle arrest or apoptosis, although its precise mechanism of action is unclear. Previous studies have demonstrated that the transcription factor, nuclear factor κB (NF-κB), functions as a redox-sensitive factor. ROS induce NF-κB signaling pathways including upstream IκB kinases (IKKs), although the mechanisms of ROS-induced activation of IKKs are unknown. Here we show that both PKCδ and IKKα, but not IKKβ, translocate to the nucleus in response to oxidative stress. The results also demonstrate that PKCδ interacts with and activates IKKα. Importantly, our data suggest that, upon exposure to oxidative stress, PKCδ-mediated IKKα activation does not contribute to NF-κB activation; instead, nuclear IKKα regulates the transcription activity of the p53 tumor suppressor by phosphorylation at Ser20. These findings collectively support a novel mechanism in which the PKCδ → IKKα signaling pathway contributes to ROS-induced activation of the p53 tumor suppressor. © 2007 Elsevier Inc. All rights reserved. Keywords: PKCδ; IKKα; p53; ROS; Oxidative stress
1. Introduction The protein kinase C (PKC) family represents serine and threonine kinases that are responsible for a variety of cellular responses such as growth, proliferation, transformation and cell death [1,2]. The PKC family is subdivided into three categories: conventional, novel and atypical PKCs [1,2]. Accumulating lines of evidence have revealed that PKCδ, a novel PKC, plays a crucial role in the cellular response to reactive oxygen species (ROS) [3,4]. Upon exposure to oxidative stress, PKCδ is activated and cleaved by caspase-3 to form a 40 kDa catalytically active fragment. Overexpression of the PKCδ catalytic fragment (PKCδCF) induces chromatin condensation and DNA fragmentation, which supports a role for PKCδ cleavage in the induction of apoptosis [5]. Other studies have shown that PKCδ interacts with the c-Abl tyrosine kinase [6]. c-Abl is a pro⁎ Corresponding authors. Yoshida is to be contacted at Tel.: +81 3 5803 5826; fax: +81 3 5803 0242. Miki, Tel.: +81 3 5803 5825; fax: +81 3 5803 0242. E-mail addresses: [email protected] (Y. Miki), [email protected] (K. Yoshida). 0898-6568/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2007.06.002
apoptotic tyrosine kinase that targets to the nucleus following oxidative stress [7–9]. Importantly, c-Abl-mediated phosphorylation activates PKCδ and induces its translocation to the nucleus [6]. Consistent with these findings, tyrosine phosphorylation of PKCδ is necessary for its nuclear translocation and subsequent caspase-dependent cleavage [2,10,11]. Previous studies have also demonstrated that the nuclear complex of cAbl and Lyn tyrosine kinases includes the protein tyrosine phosphatase SHPTP1 [12,13], and that PKCδ phosphorylates and inactivates SHPTP1 in response to genotoxic stress [14]. Another study showed that cells derived from PKCδ-null transgenic mice were defective in mitochondria-dependent apoptosis induced by hydrogen peroxide [15]. We have recently demonstrated that PKCδ phosphorylates the p53 tumor suppressor to induce apoptotic cell death [16]. These findings collectively support an essential role for PKCδ in the induction of apoptosis in the oxidative stress response [2]. ROS have also been implicated in the induction of the nuclear factor-κB (NF-κB) signaling pathway in various types of cell [17,18]. Activation of NF-κB is regulated by multiple distinct signaling cascades including inhibitors of the NF-κB (IκB) kinase
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(IKK) signalosome [19,20]. IKK phosphorylates IκBα at Ser32 and Ser36 in response to a variety of stimuli, resulting in its ubiquitination and subsequent proteasomal degradation [19,20]. The released NF-κB targets to the nucleus and thereby induces the expression of specific target genes. Previous studies have shown that IKK forms a complex that consists of IKKα, β and γ [21]. Interestingly, IkBα is phosphorylated mainly by IKKβ [21]: IKKα is thought to be less crucial for nuclear targeting of NF-κB in response to cytokine. Rather, recent studies have demonstrated that IKKα specifically translocates to the nucleus and phosphorylates histone H3 to regulate NF-κB-responsive gene transcription [21,22]. Moreover, IKKα forms a complex with transcription coactivators such as AIB1/SRC-3 to modulate gene expression [23]. Other studies have suggested that IKKα is recruited to the chromatin to derepress the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) [24,25]. Taken together, these findings indicate a pivotal role for nuclear IKKα as a regulator of transcription in response to cytokine. With regard to ROS, it remains unclear whether NF-κB activation is IKKα-dependent, although stimulation of cells with hydrogen peroxide induces IKK activity [26,27]. In this context, previous studies have showed that NF-κB activation by ROS is not involved in the IKK phosphorylation of IκBα, but that ROS induce phosphorylation of IκBα at Tyr42 [28,29]. This tyrosine phosphorylation of IκBα represents a proteolysis-independent mechanism of NF-κB activation. In this study, we show that PKCδ and IKKα, but not IKKβ, translocate to the nucleus in response to oxidative stress. We also demonstrate that PKCδ activates IKKα. Importantly, PKCδ-mediated IKKα activation by ROS does not contribute to NF-κB activation. Finally, nuclear IKKα functions in the regulation of p53 transcription activity. These findings support a novel mechanism in which the PKCδ → IKKα signaling pathway contributes to ROS-induced p53 activation. 2. Materials and methods 2.1. Cell culture Human MOLT-4 and HL-60 leukemia cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 μg/ml), and L-glutamine (2 mM). Human U2-OS cells, MCF-7 cells and 293T cells were grown in Dulbecco's modified Eagle's medium containing 10% FBS and antibiotics. Cells were treated with 500 μM H2O2 (Nacalai Tesque), 50 ng/ml TNFα (PeproTech EC) or 5 μM rottlerin (Sigma-Aldrich).
2.2. Plasmids PKCδ expression plasmids have been described previously [14,30]. Constructs of IKKα wild-type and kinase-inactive mutants are described elsewhere [31]. p53 cDNA was amplified by PCR from a human fetal brain cDNA library, then cloned into pcDNA3-Flag or pEGFP-C1 vectors as described elsewhere [16]. Various mutations were introduced by site-directed mutagenesis and were confirmed by sequencing.
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2.4. Immunoprecipitation and immunoblot analysis Cell lysates were prepared as described elsewhere [13,33] and cleared by centrifugation at 12000 g for 15 min. Soluble proteins were incubated with antiFlag (Sigma-Aldrich), anti-PKCδ (Santa Cruz Biotechnology (SCBT)), antiIKKα (SCBT) or anti-p53 (SCBT) antibodies for 2 h at 4 °C followed by a 1 h incubation with protein A- (Amersham Biosciences) or G- (Zymed Laboratories) Sepharose beads. The immune complexes were washed three times with lysis buffer. Cell lysates or immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose filters, which were then incubated with antiFlag, anti-myc (Cell Signaling Technology), anti-GFP (Nacalai Tesque), antiIKKα (SCBT, MBL or Cell Signaling Technology (CST)), IKKβ (CST), antiPKCδ, anti-PCNA (SCBT), anti-tubulin (Sigma-Aldrich), anti-phospho-IKK (CST), anti-IκBα (SCBT), anti-phospho-IκBα (CST), anti-RelA (SCBT), antiRelB (SCBT), anti-p53 (SCBT), anti-phospho-p53 (CST) or anti-GST (Nakalai Tesque). The antigen-antibody complexes were visualized by chemiluminescence (PerkinElmer).
2.5. Reporter gene assays 293 cells stably tranfected with pNF-κB-luc and pTK-hyg (Panomics) were left untreated or pretreated with rottlerin for 30 min followed by the treatment with H2O2. As a control, cells were treated with TNFα. The luciferase activity was determined by the Bright-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol.
2.6. In vitro kinase assays Recombinant active GST-IKKα protein was obtained from Upstate. Purified GST-p53 protein was purchased from Santa Cruz Biotechnology. In vitro kinase assays were performed as described elsewhere [34]. Briefly, GST-IKKα was incubated in kinase buffer (20 mM HEPES pH 7.0, 10 mM MgCl2, 0.1 mM Na3VO4, 2 mM DTT) with GST-p53 and ATP for 10 min at 30°C. Samples were boiled for 5 min and analyzed by SDS-PAGE followed by immunoblotting with anti-phospho-p53 antibodies (CST).
2.7. RT-PCR analysis for gene expression Total cellular RNA was extracted using the RNeasy kit (Qiagen). First-stand cDNA synthesis and the following PCR reactions were performed with 500 ng of total RNA using SuperScript One-Step RT-PCR System (Invitrogen) according to the manufacturer's protocol. For p53 gene expression, the nucleotide sequence 5'ACCTACCAGGGCAGCTACGGTTTC-3' was used as the sense primer, and 5'GCCGCCCATGCAGGAACTGTTACA-3' as the antisense primer. For GADD45α gene expression, the nucleotide sequence 5'-ATGACTTTGGAGGAATTCTCGGCT-3' was used as the sense primer, and 5'-TCACCGTTCAGGGAGATTAATCAC-3' as the antisense primer. For Bax gene expression, the nucleotide sequence 5'-ACCAGCTCTGAGCAGATCATGAAGACAG-3' was used as the sense primer, and 5'-AACCACCCTGGTCTTGGATCCAGCCCAA3' as the antisense primer. For β-actin gene expression, the nucleotide sequence 5'CAGGGCGTGATGGTGGGCA-3' was used as the sense primer, and 5'CAAACATCATCTGGGTCATCTTCTC-3' as the antisense primer. The reaction products were resolved on a 2% agarose gel.
2.8. Subcellular fractionation Subcellular fractionation was performed as described previously [35,36]. Purity of the fractions was monitored by immunoblot analysis with anti-PCNA and anti-tubulin.
2.9. siRNA transfections 2.3. Cell transfections Cell transfections were performed as described [32]. The total DNA concentration was kept constant by inclusion of an empty vector.
siRNA duplexes (siRNAs) were synthesized and purified by Invitrogen (Stealth Select RNAi). Transfection of siRNAs was performed using Lipofectamine RNAiMax (Invitrogen).
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3. Results 3.1. Nuclear translocation of IKKα and PKCδ following oxidative stress Previous studies demonstrated that IKKα and not IKKβ targets to the nucleus in response to tumor necrosis factor α TNFα [21]. Nuclear IKKα phosphorylates histone H3 to transduce NF-κB-responsive transcription [21,22]. Other studies have showed that IKK is activated in response to oxidative stress [26,27]. To investigate whether IKK translocates to the nucleus in response to oxidative stress, we examined the subcellular localization of IKK following treatment of MOLT-4 cells with hydrogen peroxide (H2O2) as a source of ROS. IKKα, but not IKKβ, was detectable in the nuclear fraction after 1 h of H2O2 treatment (Fig. 1A), and similar results were obtained with MCF-7 and U2-OS cells (data not shown). Recent studies have demonstrated that PKCδ translocates to the nucleus upon exposure to genotoxic stress [11,35], and other studies reported that PKCδ is activated in response to oxidative stress [4]. To determine if PKCδ targets to the nucleus following H2O2 exposure, subcellular fractionation assays were performed. Like IKKα, PKCδ was accumulated in the nucleus upon exposure of MOLT-4 cells to H2O2 (Fig. 1A). Similar findings were obtained with MCF-7 and U2-OS cells (data not shown). These results demonstrate nuclear translocation of IKKα and PKCδ in response to oxidative stress. To determine whether the translocation of IKKα is transient, longer periods of H2O2 exposure were examined. Nuclear accumulation of IKKα peaked 4 hours after treatment (Fig. 1B, second panel). Furthermore, activation of nuclear IKKα, monitored by phosphorylation level, peaked at 2 h then declined, reaching baseline level after 8 h (Fig. 1B, top panel). In contrast, there was no activation of nuclear IKKβ detectable following H2O2 exposure (Fig. 1B, top panel). Importantly, there was little phosphorylation of cytoplasmic IKKα and IKKβ (Fig. 1B, top panel). Comparable results were obtained with U2-OS cells (data not shown). Taken together, these findings indicate that IKKα, but not IKKβ, is activated in the nucleus in response to oxidative stress.
with U2-OS cells (data not shown). These results indicate that PKCδCF is responsible for the interaction with IKKα. To determine whether PKCδ phophorylates IKKα, 293T cells were co-transfected with Flag-IKKα and myc vector, myc-PKCδCF or kinase-inactive myc-PKCδCF(K-R), which is the dominantnegative form of PKCδ. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-phospho-IKK, which monitors IKK activation. Phosphorylation of IKKα was increased by expression of kinase-active, but not kinase-dead PKCδ (Fig. 2C), suggesting that PKCδ is involved in the phosphorylation of IKKα. These results demonstrate that PKCδ is required for phosphorylation of IKKα in response to oxidative stress.
3.2. PKCδ associates with and phosphorylates IKKα The finding that both PKCδ and IKKα simultaneously translocate to the nucleus led us to examine a potential association between them. 293T cells were co-transfected with Flag-IKKα and GFP vector, GFP-PKCδ full length (FL), GFPPKCδ catalytic fragment (CF) or GFP-PKCδ regulatory domain (RD). Analysis of anti-Flag immunoprecipitates with anti-GFP revealed the binding of IKKα to PKCδFL and CF, but not RD (Fig. 2A). To extend these findings to endogenous PKCδ and IKKα, anti-IKKα immunoprecipitates from MOLT-4 cells were subjected to immunoblot analysis with anti-PKCδ. The results demonstrated that IKKα and PKCδ form complexes in cells (Fig. 2B). In reciprocal experiments, immunoblot analysis of anti-PKCδ immunoprecipitates with anti-IKKα confirmed the association of PKCδ with IKKα. Similar results were obtained
Fig. 1. PKCδ and IKKα, but not IKKβ, are translocated to the nucleus in response to oxidative stress. A and B, MOLT-4 cells were treated with H2O2 for the indicated times. Nuclear and cytoplasmic lysates were subjected to immunoblot analysis with anti-IKKα, anti-IKKβ, anti-PKCδ (A), anti-phospho-IKK (B), anti-PCNA or anti-tubulin.
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Fig. 2. PKCδ interacts with and phosphorylates IKKα. A, 293T cells were co-transfected with Flag-IKKα and GFP vector, GFP-PKCδFL, GFP-PKCδCF or GFPPKCδRD. Immunoprecipitates of cell lysates with anti-Flag were analyzed by immunoblotting with anti-GFP (upper panel) or anti-Flag (middle panel). Cell lysates were also subjected to immunoblot analysis with anti-GFP (lower panel). B, Lysates from MOLT-4 cells were subjected to immunoprecipitation with anti-IKKα, preimmune rabbit serum (PIRS) or anti-PKCδ. Cell lysates and immunoprecipitates were analyzed by immunoblotting with anti-PKCδ (upper panel) or anti-IKKα (lower panel). C, 293T cells were co-transfected with Flag-IKKα and myc vector, myc-PKCδCF or myc-PKCδCF(K-R). Immunoprecipitates of cell lysates with anti-Flag were analyzed by immunoblotting with anti-phospho-IKK (top panel) or anti-Flag (second panel). Cell lysates were also subjected to immunoblot analysis with antimyc (third panel) or anti-tubulin (bottom panel).
3.3. PKCδ-induced IKKα phosphorylation is independent of NF-κB activation To assess whether phosphorylation of IKKα by PKCδ is involved in NF-κB activation in response to oxidative stress, MOLT-4 cells were treated with H2O2 in the presence or absence of rottlerin, a specific inhibitor of PKCδ [37]. Phosphorylation of IKKα was detectable at 30 min and peaked at 2 h (Fig. 3A). Pretreatment with rottlerin was associated with inhibition of IKKα phosphorylation (Fig. 3A) and there was no detectable phosphorylation of IKKβ following H2O2 treatment (Fig. 3A). Under these experimental conditions, phosphorylation of IκBα was constant and its degradation was hardly observed, indicating that there is little if any ubiquitin-
proteasome modification of IκBα in response to oxidative stress (Fig. 3A). Similar findings were obtained with HL-60 cells (data not shown). As a control, we assessed the activation of NF-κB pathways following exposure to TNFα (Supplementary Fig. S1A). Phosphorylation of IKKα and β peaked at 15 min and then decreased immediately, whether or not there had been rottlerin pretreatment. Importantly, phosphorylation of IKKβ was substantially higher than that of IKKα, suggesting that IKKβ, not IKKα, plays the central role in TNFα-mediated activation of NF-κB pathways (Supplementary Fig. S1A). In addition, IκBα was promptly degraded and then resynthesized in a PKCδ-independent manner (Supplementary Fig. S1A). To further examine whether the PKCδ → IKKα signaling is involved in oxidative-stress-induced NF-κB activation, we
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Fig. 3. PKCδ-dependent activation of IKKα by oxidative stress is independent of NF-κB activation. A and B, MOLT-4 cells were treated with H2O2 in the presence or absence of rottlerin. Whole cell lysates (A) or nuclear lysates (B) were subjected to immunoblot analysis with the indicated antibodies. C and D, 293/NF-κB-luc cells were treated with H2O2 in the presence (closed bars) or absence (open bars) of rottlerin. As a control, cells were treated with TNFα (dotted bars). The luciferase assays were performed and the results were normalized by basal activity of control cells. The data indicate the mean ± S.D. from three independent experiments each performed in triplicate.
analyzed nuclear targeting of RelA/p65 and RelB following H2O2 treatment. The results demonstrated that RelA/p65 and RelB were gradually translocated to the nucleus (Fig. 3B). Furthermore, pretreatment with rottlerin had little effect on nuclear targeting of RelA/p65 and RelB, suggesting that PKCδmediated IKKα activation is independent on NF-κB activation (Fig. 3B). Abrogation of PKCδ activity also had little if any effect on the nuclear targeting of IKKα (Fig. 3B). Similar findings were obtained with HL-60 cells (data not shown). Meanwhile, treatment of MOLT-4 cells with TNFα demon-
strated that nuclear translocation of RelA/p65 peaks after 15 min and then gradually decreases (Supplementary Fig. S1B). Moreover, as shown for H2O2, nuclear targeting of RelA/p65 was not required for PKCδ activity (Supplementary Fig. S1B). Comparable results were obtained with HL-60 cells (data not shown). Under these experimental conditions, nuclear targeting of IKKα and β was hardly detected following exposure to TNFα (Supplementary Fig. S1B). To confirm the independence of the PKCδ→IKKα signaling pathway on NF-κB activation in response to oxidative stress, we performed the luciferase
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reporter assays to measure NF-κB activity. 293 cells stably transfected with the luciferase-reporter vector containing NFκB response elements were left untreated or pretreated with rottlerin for 30 min followed by the treatment with H2O2 for the indicated times. As a control, cells were treated with TNFα. Analysis of luciferase activity demonstrated that there was little if any activation of NF-κB following H2O2 treatment regardless of PKCδ activity (Fig. 3C and D). In contrast, TNFα treatment substantially up-regulated NF-κB activity (Fig. 3D). These results collectively demonstrate that PKCδ-mediated IKKα phosphorylation and activation elicited by oxidative stress are independent of NF-κB activation. 3.4. IKKα interacts with and activates the p53 tumor suppressor in response to oxidative stress The finding that PKCδ → IKKα signaling is not linked to the NF-κB pathways led us to identify novel downstream targets in the nucleus. In this regard, recent studies have demonstrated that PKCδ regulates p53 at both the transcriptional and post-
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translational level upon exposure to genotoxic stress, though its precise mechanism remains unclear [16,38]. Given our discovery that PKCδ activates IKKα in response to oxidative stress, it is conceivable that oxidative-stress-induced IKKα activation is associated with p53 regulation. To examine this possibility, GFP-p53 was co-transfected into 293T cells together with Flag vector, wild-type Flag-IKKα or the Flag-IKKα(K-M) mutant, which is the dominant-negative form of IKKα. Lysates were subjected to immunoprecipitation with anti-Flag followed by immunoblot analysis with anti-GFP. The results demonstrated that IKKα binds to p53 (Fig. 4A). Moreover, this interaction is independent of IKKα kinase activity (Fig. 4A). To assess the possibility that IKKα phosphorylates p53, recombinant kinase-active GST-IKKα was incubated with purified GST-p53. Immunoblot analysis with three anti-phospho-p53 antibodies revealed that Ser20, but not Ser6, Ser9, Ser15, Ser46 or Ser392, is the in vitro phosphorylation site of p53 by IKKα (Fig. 4B and data not shown). To determine the functional significance of the PKCδ→IKKα pathway in induction of p53 activation, we examined possible interaction of endogenous p53
Fig. 4. IKKα interacts with and induces the p53 tumor suppressor. A, 293T cells were co-transfected with GFP-p53 and Flag vector, Flag-IKKα or Flag-IKKα(K-M). Immunoprecipitates of cell lysates with anti-Flag were subjected to immunoblot analysis with anti-GFP (top panel) or anti-Flag (second panel). Lysates were also analyzed by immunoblotting with anti-GFP (third panel) or anti-tubulin (bottom panel). B, GST-p53 was incubated in the presence or absence of kinase-active GSTIKKα. The complexes were resolved in SDS-PAGE and then analyzed by immunoblotting with anti-phospho-p53 (Ser20) or anti-GST. C, MOLT-4 cells were treated with H2O2 in the presence or absence of rottlerin. Anti-p53-immunoprecipitates or cell lysates were subjected to immunoblot analysis with anti-IKKα (top panel), antip53 (second panel), anti-phospho-p53 (Ser20) (third panel) or anti-tubulin (bottom panel). N.S. indicates ‘non-specific’ bands.
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and IKKα. The results demonstrated that endogenous p53 associates with endogenous IKKα following oxidative stress (Fig. 4C). More importantly, this interaction was abrogated by inhibition of PKCδ activity, indicating that activation of the PKCδ→IKKα signaling pathway is required for its proper downstream connection including p53. In this regard, the finding that pretreatment with rottlerin was associated with inhibition of Ser20 phosphorylation further supports a direct link between the PKCδ→IKKα signaling pathway and phosphorylation of p53 at Ser20 in response to oxidative stress
(Fig. 4C). To extend these findings, U2-OS cells were transfected with GFP vector or the GFP-IKKα(K-M) mutant. The level of p53 expression increased following treatment with H2O2 (Fig. 5A). In contrast, co-expression of kinase-inactive IKKα was associated with attenuation of H2O2-induced p53 stabilization (Fig. 5A). In concert with these results, phosphorylation on Ser20 was attenuated in cells with co-expression of dominant-negative IKKα (Fig. 5A). To further define the physiological regulation of p53 expression by IKKα in response to oxidative stress, IKKα was knocked down by transfection
Fig. 5. IKKα is involved in oxidative-stress-induced activation of p53. A, U2-OS cells transfected with GFP vector or GFP-IKKα(K-M) were treated with H2O2 for the indicated times. Lysates were subjected to immunoblot analysis with anti-phospho-p53 (Ser20) (top panel), anti-p53 (second panel), anti-GFP (third panel) or antitubulin (fourth panel). Total RNA was subjected to RT-PCR analysis using primer sets for p53 (fifth panel), GADD45α (sixth panel), Bax (seventh panel) or β-actin (bottom panel). B, U2-OS cells transfected with scrambled siRNA or IKKα siRNA were left untreated or treated with H2O2 for 2 or 4 h. Cell lysates were analyzed by immunoblotting with anti-phospho-p53 (Ser20) (top panel), anti-p53 (second panel), anti-IKKα (third panel), anti-IKKβ (fourth panel) or anti-tubulin (bottom panel). C, U2-OS cells transfected with scrambled siRNA or IKKαsiRNA were left untreated or treated with H2O2 for 4 h. Total RNA was analyzed by RT-PCR as described above.
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Fig. 6. A proposed model of the signaling pathways in response to oxidative stress. Upon exposure to ROS, PKCδ activates IKKα to induce its nuclear translocation. Activated IKKα in turn phosphorylates p53 at Ser20 to induce its stabilization and activation.
with small interfering RNAs (siRNAs) targeting IKKα into U2OS cells. This attenuated H2O2-induced stabilization of endogenous p53 expression and Ser20 phosphorylation (Fig. 5B). Taken together, these results demonstrate that IKKα associates with and stabilizes p53 by Ser20 phosphorylation in response to oxidative stress. To determine whether regulation of p53 by IKKα affects p53 function, U2-OS cells were transfected with GFP vector or the GFP-IKKα(K-M) mutant. 48 h post-transfection, cells were treated with H2O2 or left untreated. RT-PCR analysis revealed that the mRNA levels of p53 remained unchanged regardless of IKKα activity (Fig. 5A). Given that inhibition of IKKα attenuated H2O2 induction of p53 expression (Fig. 5A & B), this regulation might be modulated at the post-translational level. Importantly, GADD45α and Bax, which are regulated by p53 [39], were transcriptionally suppressed with the forced expression of kinase-inactive IKKα (Fig. 5A). To confirm these findings by knocking down IKKα, U2-OS cells were transfected with scramble or IKKα siRNAs then treated with H2O2. Assessment of mRNA levels by RT-PCR demonstrated that silencing IKKα inhibits H2O2-induced up-regulation of GADD45α and Bax by p53 (Fig. 5C). These results collectively support the role for IKKα in a post-translational regulation of p53 in the cellular response to oxidative stress. 4. Discussion 4.1. Activation of IKKα by PKCδ upon exposure to reactive oxygen species NF-κB is a dimeric transcription factor that regulates a variety of genes [20]. Accumulating lines of evidence have revealed that NF-κB is activated by various stimuli such as cytokines, radiation, viral infection and ROS [20]. The IKK complex plays an essential role in the phosphorylation of IκBα, resulting in nuclear translocation and activation of NF-κB. Recent studies have demonstrated that activation of the IKK
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complex by cytokines such as TNFα, IL-1 or IL-6 is regulated by several upstream kinases including NF-κB-inducing kinase, NF-κB-activating kinase, TGF-β activating kinase 1, mitogenactivated protein/ERK kinase kinase 1 (MEKK1) and MEKK3 [40]. However, the molecular mechanism by which the IKK complex is activated in response to oxidative stress is largely unknown. We have demonstrated that IKKα, but not IKKβ, is activated following exposure of cells to ROS, and this activation is, at least in part, regulated by PKCδ. In this regard, ROS induces tyrosine phosphorylation of PKCδ by c-Abl and Lyn [4,30]. Our results support a model in which the activation of IKKα by ROS is mediated in part by the c-Abl/Lyn→PKCδ signaling pathways. Our results further demonstrate that PKCδ is involved in IKKα activation by its phosphorylation. Expression of kinase-active PKCδ was associated with increased IKKα phosphorylation. The finding that a catalytic fragment of PKCδ (PKCδCF) was responsible for binding to IKKα further supports PKCδ phosphorylation of IKKα. Moreover, H2O2-induced phosphorylation of IKKα was defective in PKCδ inhibitor rottlerin-pretreated cells, indicating that PKCδ is at least in part required for IKKα phosphorylation by ROS. However, our results do not exclude the possibility that another kinase may link PKCδ to IKKα. Obviously, additional studies are now needed to determine whether PKCδ directly phosphorylates and activates IKKα in response to ROS. 4.2. Activation of IKKα is independent of NF-κB activation in response to ROS There is evidence to demonstrate that the mechanism of NFκB activation by ROS can be dependent or independent of IKK [26,27]; the difference may depend on the cell type. More importantly, previous studies have shown that H2O2-induced NFκB activation is not involved in the phosphorylation of IκBα at Ser32 and Ser36, but that ROS induce phosphorylation of IκBα at Tyr42 [28,29]. In contrast to serine phosphorylation, tyrosine phosphorylation of IκBα does not lead to its degradation by ubiquitin-proteasome machinery. In agreement with this model, we have revealed that IκBα is not degraded upon exposure of the cells to H2O2. Furthermore, Ser32- and Ser36-phosphorylation of IκBα remained constant throughout the H2O2 treatment. Consistent with these results, there was no detectable activation of IKKβ. Nevertheless, the finding that RelA and RelB targeted to the nucleus following H2O2 exposure regardless of PKCδ activity supports a model in which the mechanism of ROS-induced NFκB activation is independent of both PKCδ and IKK. These results collectively indicate that PKCδ-mediated activation of IKKα by ROS targets to novel effector(s) instead of NF-κB. In this regard, our results demonstrate that both PKCδ and IKKα translocate to the nucleus in response to ROS, suggesting that downstream effector(s) might reside there. 4.3. IKKα is of functional importance in regulation of the p53 tumor suppressor Recent studies have shown that PKCδ stabilizes and activates the p53 tumor suppressor in response to DNA damage
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[16,38]. The signaling mechanisms responsible for this regulation remain largely unknown. Certain insights have been derived from the finding that PKCδ phosphorylates p53 to induce apoptotic cell death upon exposure to genotoxic stress [16]. Moreover, stabilization of p53 was partly dependent on PKCδ by an unknown mechanism [16,38]. These findings led us to examine the possibility that PKCδ-mediated IKKα activation by oxidative stress is associated with p53 stability. The results demonstrate that IKKα stabilizes p53 in a kinaseactivity-dependent manner, suggesting the involvement of IKKα phosphorylation of p53. Indeed, the present study revealed that IKKα is capable of phosphorylation of p53 at Ser20. Interestingly, whereas activation of IKKα by PKCδ peaked at 2 h following oxidative stress, down-regulation of IKKα by expression of dominant-negative IKKα or IKKα siRNA was also associated with attenuation of p53 expression especially at 4 h (Fig. 5A and B, second panels). As results, Ser20 phosphorylation was significantly reduced at 4 h after stimulation. Moreover, the present study demonstrated that endogenous IKKα interacts with endogenous p53 at 4 h following treatment of cells with hydrogen peroxide and this interaction was dependent on PKCδ activity (Fig. 4C), suggesting the involvement of IKKα on p53 expression at 4 h by yet unknown mechanism. Given the previous findings that Ser20 phosphorylation induces p53 stabilization and activation in response to various stimuli [41,42], IKKα phosphorylation of p53 could be important for its transcriptional function in response to oxidative stress (Fig. 6). Alternatively, since IKKα forms a complex with transcription factors [40], IKKα may regulate p53 at a transcriptional level. However, based on our present findings that mRNA levels of p53 remain constant regardless of IKKα activity, modification of p53 by IKKα at a transcriptional level is probably insignificant. Our previous study demonstrated that PKCδ is involved in phosphorylation of p53 at Ser46 in response to DNA damage. However, clearly distinct from Ser20 or Ser15 phosphorylation, Ser46 phosphorylation is induced with a slow kinetics and specifically linked to the induction of apoptosis [16,43]. We also demonstrated that PKCδ is started to cleave at 4–6 h following treatment of cells with genotoxic agents [35] and Ser46 phosphorylation is, at least in part, required for cleavage of PKCδ by caspase-3 to induce its full activation. In this context, we speculate that these two pathways are activated differently upon dose and time of stress. However, it is also plausible that these pathways are synergically activated for p53 modification to determine cell fate. Further studies are needed to clarify this issue. Importantly, activation of IKKα by oxidative stress contributes to p53 stabilization and activation. In this context, the demonstration that both PKCδ and IKKα activate and accumulate in the nucleus following H2O2 exposure further supports the regulation of nuclear p53 by PKCδ → IKKα signaling pathways. The mechanism for the nuclear targeting of these kinases remains obscure and additional experiments to define it are currently underway. Consistent with such regulation, our results demonstrate that IKKα contributes to p53-dependent expression of GADD45α and Bax in response to ROS. Stabilization and activation of p53 as conferred by IKKα could thus contribute to
downstream effectors of p53 and thereby regulate p53-mediated cell fate in response to oxidative stress. Acknowledgements The authors thank Dr. Hiroyasu Nakano for providing FlagIKKα plasmids. This work was supported by grants from the Ministry of Education, Science and Culture of Japan (to K.Y. and Y.M.), Kanae Foundation for Life & Socio-medical Science (to K.Y.), Kowa Life Science Foundation (to K.Y.), the Astellas Foundation for Research on Medical Resources (to K.Y.) and Japan Leukemia Research Fund (to K.Y.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellsig.2007.06.002. References [1] Y. Nishizuka, Nature 334 (1988) 661. [2] K. Yoshida, Cell. Signal. 19 (2007) 892. [3] A.G. Kanthasamy, M. Kitazawa, A. Kanthasamy, V. Anantharam, Antioxid. Redox Signal. 5 (2003) 609. [4] X. Sun, F. Wu, R. Datta, S. Kharbanda, D. Kufe, J. Biol. Chem. 275 (2000) 7470. [5] T. Ghayur, M. Hugunin, R.V. Talanian, S. Ratnofsky, C. Quinlan, Y. Emoto, P. Pandey, R. Datta, Y. Huang, S. Kharbanda, H. Allen, R. Kamen, W. Wong, D. Kufe, J. Exp. Med. 184 (1996) 2399. [6] Z.M. Yuan, T. Utsugisawa, T. Ishiko, S. Nakada, Y. Huang, S. Kharbanda, R. Weichselbaum, D. Kufe, Oncogene 16 (1998) 1643. [7] K. Yoshida, Y. Miki, Cell Cycle 4 (2005) 777. [8] K. Yoshida, T. Yamaguchi, T. Natsume, D. Kufe, Y. Miki, Nat. Cell Biol. 7 (2005) 278. [9] Z.M. Yuan, Y. Huang, T. Ishiko, S. Kharbanda, R. Weichselbaum, D. Kufe, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 1437. [10] M. Blass, I. Kronfeld, G. Kazimirsky, P.M. Blumberg, C. Brodie, Mol. Cell. Biol. 22 (2002) 182. [11] T.A. DeVries, M.C. Neville, M.E. Reyland, EMBO J. 21 (2002) 6050. [12] S. Kharbanda, A. Bharti, D. Pei, J. Wang, P. Pandey, R. Ren, R. Weichselbaum, C.T. Walsh, D. Kufe, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 6898. [13] K. Yoshida, S. Kharbanda, D. Kufe, J. Biol. Chem. 274 (1999) 34663. [14] K. Yoshida, D. Kufe, Mol. Pharmacol. 60 (2001) 1431. [15] M. Leitges, M. Mayr, U. Braun, U. Mayr, C. Li, G. Pfister, N. GhaffariTabrizi, G. Baier, Y. Hu, Q. Xu, J. Clin. Invest. 108 (2001) 1505. [16] K. Yoshida, H. Liu, Y. Miki, J. Biol. Chem. 281 (2006) 5734. [17] N. Li, M. Karin, FASEB J. 13 (1999) 1137. [18] Y. Kabe, K. Ando, S. Hirao, M. Yoshida, H. Handa, Antioxid. Redox Signal. 7 (2005) 395. [19] M. Karin, Oncogene 18 (1999) 6867. [20] M.S. Hayden, S. Ghosh, Genes Dev. 18 (2004) 2195. [21] Y. Yamamoto, U.N. Verma, S. Prajapati, Y.T. Kwak, R.B. Gaynor, Nature 423 (2003) 655. [22] V. Anest, J.L. Hanson, P.C. Cogswell, K.A. Steinbrecher, B.D. Strahl, A.S. Baldwin, Nature 423 (2003) 659. [23] K.J. Park, V. Krishnan, B.W. O'Malley, Y. Yamamoto, R.B. Gaynor, Mol. Cell 18 (2005) 71. [24] J.E. Hoberg, A.E. Popko, C.S. Ramsey, M.W. Mayo, Mol. Cell. Biol. 26 (2006) 457. [25] J.E. Hoberg, F. Yeung, M.W. Mayo, Mol. Cell 16 (2004) 245. [26] I. Jaspers, W. Zhang, A. Fraser, J.M. Samet, W. Reed, Am. J. Respir. Cell Mol. Biol. 24 (2001) 769. [27] H. Kamata, T. Manabe, S. Oka, K. Kamata, H. Hirata, FEBS Lett. 519 (2002) 231.
T. Yamaguchi et al. / Cellular Signalling 19 (2007) 2088–2097 [28] V. Imbert, R.A. Rupec, A. Livolsi, H.L. Pahl, E.B. Traenckner, C. MuellerDieckmann, D. Farahifar, B. Rossi, P. Auberger, P.A. Baeuerle, J.F. Peyron, Cell 86 (1996) 787. [29] S. Singh, B.G. Darnay, B.B. Aggarwal, J. Biol. Chem. 271 (1996) 31049. [30] K. Yoshida, Y. Miki, D. Kufe, J. Biol. Chem. 277 (2002) 48372. [31] H. Nakano, M. Shindo, S. Sakon, S. Nishinaka, M. Mihara, H. Yagita, K. Okumura, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 3537. [32] K. Yoshida, K. Komatsu, H.G. Wang, D. Kufe, Mol. Cell. Biol. 22 (2002) 3292. [33] K. Yoshida, R. Weichselbaum, S. Kharbanda, D. Kufe, Mol. Cell. Biol. 20 (2000) 5370. [34] N. Taira, K. Nihira, T. Yamaguchi, Y. Miki, K. Yoshida, Mol. Cell 25 (2007) 725. [35] K. Yoshida, H.G. Wang, Y. Miki, D. Kufe, EMBO J. 22 (2003) 1431.
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[36] K. Yoshida, T. Yamaguchi, H. Shinagawa, N. Taira, K.I. Nakayama, Y. Miki, Mol. Cell. Biol. 26 (2006) 3414. [37] M. Gschwendt, H.J. Muller, K. Kielbassa, R. Zang, W. Kittstein, G. Rincke, F. Marks, Biochem. Biophys. Res. Commun. 199 (1994) 93. [38] T. Abbas, D. White, L. Hui, K. Yoshida, D.A. Foster, J. Bargonetti, J. Biol. Chem. 279 (2004) 9970. [39] M.B. Kastan, Q. Zhan, W.S. el-Deiry, F. Carrier, T. Jacks, W.V. Walsh, B.S. Plunkett, B. Vogelstein, A.J. Fornace Jr., Cell 71 (1992) 587. [40] Y. Yamamoto, R.B. Gaynor, Trends Biochem. Sci. 29 (2004) 72. [41] A.M. Bode, Z. Dong, Nat. Rev., Cancer 4 (2004) 793. [42] E. Appella, C.W. Anderson, Eur. J. Biochem. 268 (2001) 2764. [43] K. Oda, H. Arakawa, T. Tanaka, K. Matsuda, C. Tanikawa, T. Mori, H. Nishimori, K. Tamai, T. Tokino, Y. Nakamura, Y. Taya, Cell 102 (2000) 849.
Protein kinase C δ activates IκB-kinase α to induce the p53 tumor suppressor in response to oxidative stress Tomoko Yamaguchi, Yoshio Miki ⁎, Kiyotsugu Yoshida ⁎ Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan Received 10 May 2007; accepted 14 June 2007 Available online 21 June 2007
Abstract Protein kinase C δ (PKCδ) functions as a redox-sensitive kinase in various cell types. Upon exposure to reactive oxygen species (ROS), it is activated by tyrosine phosphorylation, nuclear translocation and caspase-3-mediated cleavage. Activated PKCδ is associated with cell cycle arrest or apoptosis, although its precise mechanism of action is unclear. Previous studies have demonstrated that the transcription factor, nuclear factor κB (NF-κB), functions as a redox-sensitive factor. ROS induce NF-κB signaling pathways including upstream IκB kinases (IKKs), although the mechanisms of ROS-induced activation of IKKs are unknown. Here we show that both PKCδ and IKKα, but not IKKβ, translocate to the nucleus in response to oxidative stress. The results also demonstrate that PKCδ interacts with and activates IKKα. Importantly, our data suggest that, upon exposure to oxidative stress, PKCδ-mediated IKKα activation does not contribute to NF-κB activation; instead, nuclear IKKα regulates the transcription activity of the p53 tumor suppressor by phosphorylation at Ser20. These findings collectively support a novel mechanism in which the PKCδ → IKKα signaling pathway contributes to ROS-induced activation of the p53 tumor suppressor. © 2007 Elsevier Inc. All rights reserved. Keywords: PKCδ; IKKα; p53; ROS; Oxidative stress
1. Introduction The protein kinase C (PKC) family represents serine and threonine kinases that are responsible for a variety of cellular responses such as growth, proliferation, transformation and cell death [1,2]. The PKC family is subdivided into three categories: conventional, novel and atypical PKCs [1,2]. Accumulating lines of evidence have revealed that PKCδ, a novel PKC, plays a crucial role in the cellular response to reactive oxygen species (ROS) [3,4]. Upon exposure to oxidative stress, PKCδ is activated and cleaved by caspase-3 to form a 40 kDa catalytically active fragment. Overexpression of the PKCδ catalytic fragment (PKCδCF) induces chromatin condensation and DNA fragmentation, which supports a role for PKCδ cleavage in the induction of apoptosis [5]. Other studies have shown that PKCδ interacts with the c-Abl tyrosine kinase [6]. c-Abl is a pro⁎ Corresponding authors. Yoshida is to be contacted at Tel.: +81 3 5803 5826; fax: +81 3 5803 0242. Miki, Tel.: +81 3 5803 5825; fax: +81 3 5803 0242. E-mail addresses: [email protected] (Y. Miki), [email protected] (K. Yoshida). 0898-6568/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2007.06.002
apoptotic tyrosine kinase that targets to the nucleus following oxidative stress [7–9]. Importantly, c-Abl-mediated phosphorylation activates PKCδ and induces its translocation to the nucleus [6]. Consistent with these findings, tyrosine phosphorylation of PKCδ is necessary for its nuclear translocation and subsequent caspase-dependent cleavage [2,10,11]. Previous studies have also demonstrated that the nuclear complex of cAbl and Lyn tyrosine kinases includes the protein tyrosine phosphatase SHPTP1 [12,13], and that PKCδ phosphorylates and inactivates SHPTP1 in response to genotoxic stress [14]. Another study showed that cells derived from PKCδ-null transgenic mice were defective in mitochondria-dependent apoptosis induced by hydrogen peroxide [15]. We have recently demonstrated that PKCδ phosphorylates the p53 tumor suppressor to induce apoptotic cell death [16]. These findings collectively support an essential role for PKCδ in the induction of apoptosis in the oxidative stress response [2]. ROS have also been implicated in the induction of the nuclear factor-κB (NF-κB) signaling pathway in various types of cell [17,18]. Activation of NF-κB is regulated by multiple distinct signaling cascades including inhibitors of the NF-κB (IκB) kinase
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(IKK) signalosome [19,20]. IKK phosphorylates IκBα at Ser32 and Ser36 in response to a variety of stimuli, resulting in its ubiquitination and subsequent proteasomal degradation [19,20]. The released NF-κB targets to the nucleus and thereby induces the expression of specific target genes. Previous studies have shown that IKK forms a complex that consists of IKKα, β and γ [21]. Interestingly, IkBα is phosphorylated mainly by IKKβ [21]: IKKα is thought to be less crucial for nuclear targeting of NF-κB in response to cytokine. Rather, recent studies have demonstrated that IKKα specifically translocates to the nucleus and phosphorylates histone H3 to regulate NF-κB-responsive gene transcription [21,22]. Moreover, IKKα forms a complex with transcription coactivators such as AIB1/SRC-3 to modulate gene expression [23]. Other studies have suggested that IKKα is recruited to the chromatin to derepress the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) [24,25]. Taken together, these findings indicate a pivotal role for nuclear IKKα as a regulator of transcription in response to cytokine. With regard to ROS, it remains unclear whether NF-κB activation is IKKα-dependent, although stimulation of cells with hydrogen peroxide induces IKK activity [26,27]. In this context, previous studies have showed that NF-κB activation by ROS is not involved in the IKK phosphorylation of IκBα, but that ROS induce phosphorylation of IκBα at Tyr42 [28,29]. This tyrosine phosphorylation of IκBα represents a proteolysis-independent mechanism of NF-κB activation. In this study, we show that PKCδ and IKKα, but not IKKβ, translocate to the nucleus in response to oxidative stress. We also demonstrate that PKCδ activates IKKα. Importantly, PKCδ-mediated IKKα activation by ROS does not contribute to NF-κB activation. Finally, nuclear IKKα functions in the regulation of p53 transcription activity. These findings support a novel mechanism in which the PKCδ → IKKα signaling pathway contributes to ROS-induced p53 activation. 2. Materials and methods 2.1. Cell culture Human MOLT-4 and HL-60 leukemia cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 μg/ml), and L-glutamine (2 mM). Human U2-OS cells, MCF-7 cells and 293T cells were grown in Dulbecco's modified Eagle's medium containing 10% FBS and antibiotics. Cells were treated with 500 μM H2O2 (Nacalai Tesque), 50 ng/ml TNFα (PeproTech EC) or 5 μM rottlerin (Sigma-Aldrich).
2.2. Plasmids PKCδ expression plasmids have been described previously [14,30]. Constructs of IKKα wild-type and kinase-inactive mutants are described elsewhere [31]. p53 cDNA was amplified by PCR from a human fetal brain cDNA library, then cloned into pcDNA3-Flag or pEGFP-C1 vectors as described elsewhere [16]. Various mutations were introduced by site-directed mutagenesis and were confirmed by sequencing.
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2.4. Immunoprecipitation and immunoblot analysis Cell lysates were prepared as described elsewhere [13,33] and cleared by centrifugation at 12000 g for 15 min. Soluble proteins were incubated with antiFlag (Sigma-Aldrich), anti-PKCδ (Santa Cruz Biotechnology (SCBT)), antiIKKα (SCBT) or anti-p53 (SCBT) antibodies for 2 h at 4 °C followed by a 1 h incubation with protein A- (Amersham Biosciences) or G- (Zymed Laboratories) Sepharose beads. The immune complexes were washed three times with lysis buffer. Cell lysates or immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose filters, which were then incubated with antiFlag, anti-myc (Cell Signaling Technology), anti-GFP (Nacalai Tesque), antiIKKα (SCBT, MBL or Cell Signaling Technology (CST)), IKKβ (CST), antiPKCδ, anti-PCNA (SCBT), anti-tubulin (Sigma-Aldrich), anti-phospho-IKK (CST), anti-IκBα (SCBT), anti-phospho-IκBα (CST), anti-RelA (SCBT), antiRelB (SCBT), anti-p53 (SCBT), anti-phospho-p53 (CST) or anti-GST (Nakalai Tesque). The antigen-antibody complexes were visualized by chemiluminescence (PerkinElmer).
2.5. Reporter gene assays 293 cells stably tranfected with pNF-κB-luc and pTK-hyg (Panomics) were left untreated or pretreated with rottlerin for 30 min followed by the treatment with H2O2. As a control, cells were treated with TNFα. The luciferase activity was determined by the Bright-Glo Luciferase Assay System (Promega) according to the manufacturer's protocol.
2.6. In vitro kinase assays Recombinant active GST-IKKα protein was obtained from Upstate. Purified GST-p53 protein was purchased from Santa Cruz Biotechnology. In vitro kinase assays were performed as described elsewhere [34]. Briefly, GST-IKKα was incubated in kinase buffer (20 mM HEPES pH 7.0, 10 mM MgCl2, 0.1 mM Na3VO4, 2 mM DTT) with GST-p53 and ATP for 10 min at 30°C. Samples were boiled for 5 min and analyzed by SDS-PAGE followed by immunoblotting with anti-phospho-p53 antibodies (CST).
2.7. RT-PCR analysis for gene expression Total cellular RNA was extracted using the RNeasy kit (Qiagen). First-stand cDNA synthesis and the following PCR reactions were performed with 500 ng of total RNA using SuperScript One-Step RT-PCR System (Invitrogen) according to the manufacturer's protocol. For p53 gene expression, the nucleotide sequence 5'ACCTACCAGGGCAGCTACGGTTTC-3' was used as the sense primer, and 5'GCCGCCCATGCAGGAACTGTTACA-3' as the antisense primer. For GADD45α gene expression, the nucleotide sequence 5'-ATGACTTTGGAGGAATTCTCGGCT-3' was used as the sense primer, and 5'-TCACCGTTCAGGGAGATTAATCAC-3' as the antisense primer. For Bax gene expression, the nucleotide sequence 5'-ACCAGCTCTGAGCAGATCATGAAGACAG-3' was used as the sense primer, and 5'-AACCACCCTGGTCTTGGATCCAGCCCAA3' as the antisense primer. For β-actin gene expression, the nucleotide sequence 5'CAGGGCGTGATGGTGGGCA-3' was used as the sense primer, and 5'CAAACATCATCTGGGTCATCTTCTC-3' as the antisense primer. The reaction products were resolved on a 2% agarose gel.
2.8. Subcellular fractionation Subcellular fractionation was performed as described previously [35,36]. Purity of the fractions was monitored by immunoblot analysis with anti-PCNA and anti-tubulin.
2.9. siRNA transfections 2.3. Cell transfections Cell transfections were performed as described [32]. The total DNA concentration was kept constant by inclusion of an empty vector.
siRNA duplexes (siRNAs) were synthesized and purified by Invitrogen (Stealth Select RNAi). Transfection of siRNAs was performed using Lipofectamine RNAiMax (Invitrogen).
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3. Results 3.1. Nuclear translocation of IKKα and PKCδ following oxidative stress Previous studies demonstrated that IKKα and not IKKβ targets to the nucleus in response to tumor necrosis factor α TNFα [21]. Nuclear IKKα phosphorylates histone H3 to transduce NF-κB-responsive transcription [21,22]. Other studies have showed that IKK is activated in response to oxidative stress [26,27]. To investigate whether IKK translocates to the nucleus in response to oxidative stress, we examined the subcellular localization of IKK following treatment of MOLT-4 cells with hydrogen peroxide (H2O2) as a source of ROS. IKKα, but not IKKβ, was detectable in the nuclear fraction after 1 h of H2O2 treatment (Fig. 1A), and similar results were obtained with MCF-7 and U2-OS cells (data not shown). Recent studies have demonstrated that PKCδ translocates to the nucleus upon exposure to genotoxic stress [11,35], and other studies reported that PKCδ is activated in response to oxidative stress [4]. To determine if PKCδ targets to the nucleus following H2O2 exposure, subcellular fractionation assays were performed. Like IKKα, PKCδ was accumulated in the nucleus upon exposure of MOLT-4 cells to H2O2 (Fig. 1A). Similar findings were obtained with MCF-7 and U2-OS cells (data not shown). These results demonstrate nuclear translocation of IKKα and PKCδ in response to oxidative stress. To determine whether the translocation of IKKα is transient, longer periods of H2O2 exposure were examined. Nuclear accumulation of IKKα peaked 4 hours after treatment (Fig. 1B, second panel). Furthermore, activation of nuclear IKKα, monitored by phosphorylation level, peaked at 2 h then declined, reaching baseline level after 8 h (Fig. 1B, top panel). In contrast, there was no activation of nuclear IKKβ detectable following H2O2 exposure (Fig. 1B, top panel). Importantly, there was little phosphorylation of cytoplasmic IKKα and IKKβ (Fig. 1B, top panel). Comparable results were obtained with U2-OS cells (data not shown). Taken together, these findings indicate that IKKα, but not IKKβ, is activated in the nucleus in response to oxidative stress.
with U2-OS cells (data not shown). These results indicate that PKCδCF is responsible for the interaction with IKKα. To determine whether PKCδ phophorylates IKKα, 293T cells were co-transfected with Flag-IKKα and myc vector, myc-PKCδCF or kinase-inactive myc-PKCδCF(K-R), which is the dominantnegative form of PKCδ. Anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-phospho-IKK, which monitors IKK activation. Phosphorylation of IKKα was increased by expression of kinase-active, but not kinase-dead PKCδ (Fig. 2C), suggesting that PKCδ is involved in the phosphorylation of IKKα. These results demonstrate that PKCδ is required for phosphorylation of IKKα in response to oxidative stress.
3.2. PKCδ associates with and phosphorylates IKKα The finding that both PKCδ and IKKα simultaneously translocate to the nucleus led us to examine a potential association between them. 293T cells were co-transfected with Flag-IKKα and GFP vector, GFP-PKCδ full length (FL), GFPPKCδ catalytic fragment (CF) or GFP-PKCδ regulatory domain (RD). Analysis of anti-Flag immunoprecipitates with anti-GFP revealed the binding of IKKα to PKCδFL and CF, but not RD (Fig. 2A). To extend these findings to endogenous PKCδ and IKKα, anti-IKKα immunoprecipitates from MOLT-4 cells were subjected to immunoblot analysis with anti-PKCδ. The results demonstrated that IKKα and PKCδ form complexes in cells (Fig. 2B). In reciprocal experiments, immunoblot analysis of anti-PKCδ immunoprecipitates with anti-IKKα confirmed the association of PKCδ with IKKα. Similar results were obtained
Fig. 1. PKCδ and IKKα, but not IKKβ, are translocated to the nucleus in response to oxidative stress. A and B, MOLT-4 cells were treated with H2O2 for the indicated times. Nuclear and cytoplasmic lysates were subjected to immunoblot analysis with anti-IKKα, anti-IKKβ, anti-PKCδ (A), anti-phospho-IKK (B), anti-PCNA or anti-tubulin.
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Fig. 2. PKCδ interacts with and phosphorylates IKKα. A, 293T cells were co-transfected with Flag-IKKα and GFP vector, GFP-PKCδFL, GFP-PKCδCF or GFPPKCδRD. Immunoprecipitates of cell lysates with anti-Flag were analyzed by immunoblotting with anti-GFP (upper panel) or anti-Flag (middle panel). Cell lysates were also subjected to immunoblot analysis with anti-GFP (lower panel). B, Lysates from MOLT-4 cells were subjected to immunoprecipitation with anti-IKKα, preimmune rabbit serum (PIRS) or anti-PKCδ. Cell lysates and immunoprecipitates were analyzed by immunoblotting with anti-PKCδ (upper panel) or anti-IKKα (lower panel). C, 293T cells were co-transfected with Flag-IKKα and myc vector, myc-PKCδCF or myc-PKCδCF(K-R). Immunoprecipitates of cell lysates with anti-Flag were analyzed by immunoblotting with anti-phospho-IKK (top panel) or anti-Flag (second panel). Cell lysates were also subjected to immunoblot analysis with antimyc (third panel) or anti-tubulin (bottom panel).
3.3. PKCδ-induced IKKα phosphorylation is independent of NF-κB activation To assess whether phosphorylation of IKKα by PKCδ is involved in NF-κB activation in response to oxidative stress, MOLT-4 cells were treated with H2O2 in the presence or absence of rottlerin, a specific inhibitor of PKCδ [37]. Phosphorylation of IKKα was detectable at 30 min and peaked at 2 h (Fig. 3A). Pretreatment with rottlerin was associated with inhibition of IKKα phosphorylation (Fig. 3A) and there was no detectable phosphorylation of IKKβ following H2O2 treatment (Fig. 3A). Under these experimental conditions, phosphorylation of IκBα was constant and its degradation was hardly observed, indicating that there is little if any ubiquitin-
proteasome modification of IκBα in response to oxidative stress (Fig. 3A). Similar findings were obtained with HL-60 cells (data not shown). As a control, we assessed the activation of NF-κB pathways following exposure to TNFα (Supplementary Fig. S1A). Phosphorylation of IKKα and β peaked at 15 min and then decreased immediately, whether or not there had been rottlerin pretreatment. Importantly, phosphorylation of IKKβ was substantially higher than that of IKKα, suggesting that IKKβ, not IKKα, plays the central role in TNFα-mediated activation of NF-κB pathways (Supplementary Fig. S1A). In addition, IκBα was promptly degraded and then resynthesized in a PKCδ-independent manner (Supplementary Fig. S1A). To further examine whether the PKCδ → IKKα signaling is involved in oxidative-stress-induced NF-κB activation, we
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Fig. 3. PKCδ-dependent activation of IKKα by oxidative stress is independent of NF-κB activation. A and B, MOLT-4 cells were treated with H2O2 in the presence or absence of rottlerin. Whole cell lysates (A) or nuclear lysates (B) were subjected to immunoblot analysis with the indicated antibodies. C and D, 293/NF-κB-luc cells were treated with H2O2 in the presence (closed bars) or absence (open bars) of rottlerin. As a control, cells were treated with TNFα (dotted bars). The luciferase assays were performed and the results were normalized by basal activity of control cells. The data indicate the mean ± S.D. from three independent experiments each performed in triplicate.
analyzed nuclear targeting of RelA/p65 and RelB following H2O2 treatment. The results demonstrated that RelA/p65 and RelB were gradually translocated to the nucleus (Fig. 3B). Furthermore, pretreatment with rottlerin had little effect on nuclear targeting of RelA/p65 and RelB, suggesting that PKCδmediated IKKα activation is independent on NF-κB activation (Fig. 3B). Abrogation of PKCδ activity also had little if any effect on the nuclear targeting of IKKα (Fig. 3B). Similar findings were obtained with HL-60 cells (data not shown). Meanwhile, treatment of MOLT-4 cells with TNFα demon-
strated that nuclear translocation of RelA/p65 peaks after 15 min and then gradually decreases (Supplementary Fig. S1B). Moreover, as shown for H2O2, nuclear targeting of RelA/p65 was not required for PKCδ activity (Supplementary Fig. S1B). Comparable results were obtained with HL-60 cells (data not shown). Under these experimental conditions, nuclear targeting of IKKα and β was hardly detected following exposure to TNFα (Supplementary Fig. S1B). To confirm the independence of the PKCδ→IKKα signaling pathway on NF-κB activation in response to oxidative stress, we performed the luciferase
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reporter assays to measure NF-κB activity. 293 cells stably transfected with the luciferase-reporter vector containing NFκB response elements were left untreated or pretreated with rottlerin for 30 min followed by the treatment with H2O2 for the indicated times. As a control, cells were treated with TNFα. Analysis of luciferase activity demonstrated that there was little if any activation of NF-κB following H2O2 treatment regardless of PKCδ activity (Fig. 3C and D). In contrast, TNFα treatment substantially up-regulated NF-κB activity (Fig. 3D). These results collectively demonstrate that PKCδ-mediated IKKα phosphorylation and activation elicited by oxidative stress are independent of NF-κB activation. 3.4. IKKα interacts with and activates the p53 tumor suppressor in response to oxidative stress The finding that PKCδ → IKKα signaling is not linked to the NF-κB pathways led us to identify novel downstream targets in the nucleus. In this regard, recent studies have demonstrated that PKCδ regulates p53 at both the transcriptional and post-
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translational level upon exposure to genotoxic stress, though its precise mechanism remains unclear [16,38]. Given our discovery that PKCδ activates IKKα in response to oxidative stress, it is conceivable that oxidative-stress-induced IKKα activation is associated with p53 regulation. To examine this possibility, GFP-p53 was co-transfected into 293T cells together with Flag vector, wild-type Flag-IKKα or the Flag-IKKα(K-M) mutant, which is the dominant-negative form of IKKα. Lysates were subjected to immunoprecipitation with anti-Flag followed by immunoblot analysis with anti-GFP. The results demonstrated that IKKα binds to p53 (Fig. 4A). Moreover, this interaction is independent of IKKα kinase activity (Fig. 4A). To assess the possibility that IKKα phosphorylates p53, recombinant kinase-active GST-IKKα was incubated with purified GST-p53. Immunoblot analysis with three anti-phospho-p53 antibodies revealed that Ser20, but not Ser6, Ser9, Ser15, Ser46 or Ser392, is the in vitro phosphorylation site of p53 by IKKα (Fig. 4B and data not shown). To determine the functional significance of the PKCδ→IKKα pathway in induction of p53 activation, we examined possible interaction of endogenous p53
Fig. 4. IKKα interacts with and induces the p53 tumor suppressor. A, 293T cells were co-transfected with GFP-p53 and Flag vector, Flag-IKKα or Flag-IKKα(K-M). Immunoprecipitates of cell lysates with anti-Flag were subjected to immunoblot analysis with anti-GFP (top panel) or anti-Flag (second panel). Lysates were also analyzed by immunoblotting with anti-GFP (third panel) or anti-tubulin (bottom panel). B, GST-p53 was incubated in the presence or absence of kinase-active GSTIKKα. The complexes were resolved in SDS-PAGE and then analyzed by immunoblotting with anti-phospho-p53 (Ser20) or anti-GST. C, MOLT-4 cells were treated with H2O2 in the presence or absence of rottlerin. Anti-p53-immunoprecipitates or cell lysates were subjected to immunoblot analysis with anti-IKKα (top panel), antip53 (second panel), anti-phospho-p53 (Ser20) (third panel) or anti-tubulin (bottom panel). N.S. indicates ‘non-specific’ bands.
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and IKKα. The results demonstrated that endogenous p53 associates with endogenous IKKα following oxidative stress (Fig. 4C). More importantly, this interaction was abrogated by inhibition of PKCδ activity, indicating that activation of the PKCδ→IKKα signaling pathway is required for its proper downstream connection including p53. In this regard, the finding that pretreatment with rottlerin was associated with inhibition of Ser20 phosphorylation further supports a direct link between the PKCδ→IKKα signaling pathway and phosphorylation of p53 at Ser20 in response to oxidative stress
(Fig. 4C). To extend these findings, U2-OS cells were transfected with GFP vector or the GFP-IKKα(K-M) mutant. The level of p53 expression increased following treatment with H2O2 (Fig. 5A). In contrast, co-expression of kinase-inactive IKKα was associated with attenuation of H2O2-induced p53 stabilization (Fig. 5A). In concert with these results, phosphorylation on Ser20 was attenuated in cells with co-expression of dominant-negative IKKα (Fig. 5A). To further define the physiological regulation of p53 expression by IKKα in response to oxidative stress, IKKα was knocked down by transfection
Fig. 5. IKKα is involved in oxidative-stress-induced activation of p53. A, U2-OS cells transfected with GFP vector or GFP-IKKα(K-M) were treated with H2O2 for the indicated times. Lysates were subjected to immunoblot analysis with anti-phospho-p53 (Ser20) (top panel), anti-p53 (second panel), anti-GFP (third panel) or antitubulin (fourth panel). Total RNA was subjected to RT-PCR analysis using primer sets for p53 (fifth panel), GADD45α (sixth panel), Bax (seventh panel) or β-actin (bottom panel). B, U2-OS cells transfected with scrambled siRNA or IKKα siRNA were left untreated or treated with H2O2 for 2 or 4 h. Cell lysates were analyzed by immunoblotting with anti-phospho-p53 (Ser20) (top panel), anti-p53 (second panel), anti-IKKα (third panel), anti-IKKβ (fourth panel) or anti-tubulin (bottom panel). C, U2-OS cells transfected with scrambled siRNA or IKKαsiRNA were left untreated or treated with H2O2 for 4 h. Total RNA was analyzed by RT-PCR as described above.
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Fig. 6. A proposed model of the signaling pathways in response to oxidative stress. Upon exposure to ROS, PKCδ activates IKKα to induce its nuclear translocation. Activated IKKα in turn phosphorylates p53 at Ser20 to induce its stabilization and activation.
with small interfering RNAs (siRNAs) targeting IKKα into U2OS cells. This attenuated H2O2-induced stabilization of endogenous p53 expression and Ser20 phosphorylation (Fig. 5B). Taken together, these results demonstrate that IKKα associates with and stabilizes p53 by Ser20 phosphorylation in response to oxidative stress. To determine whether regulation of p53 by IKKα affects p53 function, U2-OS cells were transfected with GFP vector or the GFP-IKKα(K-M) mutant. 48 h post-transfection, cells were treated with H2O2 or left untreated. RT-PCR analysis revealed that the mRNA levels of p53 remained unchanged regardless of IKKα activity (Fig. 5A). Given that inhibition of IKKα attenuated H2O2 induction of p53 expression (Fig. 5A & B), this regulation might be modulated at the post-translational level. Importantly, GADD45α and Bax, which are regulated by p53 [39], were transcriptionally suppressed with the forced expression of kinase-inactive IKKα (Fig. 5A). To confirm these findings by knocking down IKKα, U2-OS cells were transfected with scramble or IKKα siRNAs then treated with H2O2. Assessment of mRNA levels by RT-PCR demonstrated that silencing IKKα inhibits H2O2-induced up-regulation of GADD45α and Bax by p53 (Fig. 5C). These results collectively support the role for IKKα in a post-translational regulation of p53 in the cellular response to oxidative stress. 4. Discussion 4.1. Activation of IKKα by PKCδ upon exposure to reactive oxygen species NF-κB is a dimeric transcription factor that regulates a variety of genes [20]. Accumulating lines of evidence have revealed that NF-κB is activated by various stimuli such as cytokines, radiation, viral infection and ROS [20]. The IKK complex plays an essential role in the phosphorylation of IκBα, resulting in nuclear translocation and activation of NF-κB. Recent studies have demonstrated that activation of the IKK
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complex by cytokines such as TNFα, IL-1 or IL-6 is regulated by several upstream kinases including NF-κB-inducing kinase, NF-κB-activating kinase, TGF-β activating kinase 1, mitogenactivated protein/ERK kinase kinase 1 (MEKK1) and MEKK3 [40]. However, the molecular mechanism by which the IKK complex is activated in response to oxidative stress is largely unknown. We have demonstrated that IKKα, but not IKKβ, is activated following exposure of cells to ROS, and this activation is, at least in part, regulated by PKCδ. In this regard, ROS induces tyrosine phosphorylation of PKCδ by c-Abl and Lyn [4,30]. Our results support a model in which the activation of IKKα by ROS is mediated in part by the c-Abl/Lyn→PKCδ signaling pathways. Our results further demonstrate that PKCδ is involved in IKKα activation by its phosphorylation. Expression of kinase-active PKCδ was associated with increased IKKα phosphorylation. The finding that a catalytic fragment of PKCδ (PKCδCF) was responsible for binding to IKKα further supports PKCδ phosphorylation of IKKα. Moreover, H2O2-induced phosphorylation of IKKα was defective in PKCδ inhibitor rottlerin-pretreated cells, indicating that PKCδ is at least in part required for IKKα phosphorylation by ROS. However, our results do not exclude the possibility that another kinase may link PKCδ to IKKα. Obviously, additional studies are now needed to determine whether PKCδ directly phosphorylates and activates IKKα in response to ROS. 4.2. Activation of IKKα is independent of NF-κB activation in response to ROS There is evidence to demonstrate that the mechanism of NFκB activation by ROS can be dependent or independent of IKK [26,27]; the difference may depend on the cell type. More importantly, previous studies have shown that H2O2-induced NFκB activation is not involved in the phosphorylation of IκBα at Ser32 and Ser36, but that ROS induce phosphorylation of IκBα at Tyr42 [28,29]. In contrast to serine phosphorylation, tyrosine phosphorylation of IκBα does not lead to its degradation by ubiquitin-proteasome machinery. In agreement with this model, we have revealed that IκBα is not degraded upon exposure of the cells to H2O2. Furthermore, Ser32- and Ser36-phosphorylation of IκBα remained constant throughout the H2O2 treatment. Consistent with these results, there was no detectable activation of IKKβ. Nevertheless, the finding that RelA and RelB targeted to the nucleus following H2O2 exposure regardless of PKCδ activity supports a model in which the mechanism of ROS-induced NFκB activation is independent of both PKCδ and IKK. These results collectively indicate that PKCδ-mediated activation of IKKα by ROS targets to novel effector(s) instead of NF-κB. In this regard, our results demonstrate that both PKCδ and IKKα translocate to the nucleus in response to ROS, suggesting that downstream effector(s) might reside there. 4.3. IKKα is of functional importance in regulation of the p53 tumor suppressor Recent studies have shown that PKCδ stabilizes and activates the p53 tumor suppressor in response to DNA damage
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[16,38]. The signaling mechanisms responsible for this regulation remain largely unknown. Certain insights have been derived from the finding that PKCδ phosphorylates p53 to induce apoptotic cell death upon exposure to genotoxic stress [16]. Moreover, stabilization of p53 was partly dependent on PKCδ by an unknown mechanism [16,38]. These findings led us to examine the possibility that PKCδ-mediated IKKα activation by oxidative stress is associated with p53 stability. The results demonstrate that IKKα stabilizes p53 in a kinaseactivity-dependent manner, suggesting the involvement of IKKα phosphorylation of p53. Indeed, the present study revealed that IKKα is capable of phosphorylation of p53 at Ser20. Interestingly, whereas activation of IKKα by PKCδ peaked at 2 h following oxidative stress, down-regulation of IKKα by expression of dominant-negative IKKα or IKKα siRNA was also associated with attenuation of p53 expression especially at 4 h (Fig. 5A and B, second panels). As results, Ser20 phosphorylation was significantly reduced at 4 h after stimulation. Moreover, the present study demonstrated that endogenous IKKα interacts with endogenous p53 at 4 h following treatment of cells with hydrogen peroxide and this interaction was dependent on PKCδ activity (Fig. 4C), suggesting the involvement of IKKα on p53 expression at 4 h by yet unknown mechanism. Given the previous findings that Ser20 phosphorylation induces p53 stabilization and activation in response to various stimuli [41,42], IKKα phosphorylation of p53 could be important for its transcriptional function in response to oxidative stress (Fig. 6). Alternatively, since IKKα forms a complex with transcription factors [40], IKKα may regulate p53 at a transcriptional level. However, based on our present findings that mRNA levels of p53 remain constant regardless of IKKα activity, modification of p53 by IKKα at a transcriptional level is probably insignificant. Our previous study demonstrated that PKCδ is involved in phosphorylation of p53 at Ser46 in response to DNA damage. However, clearly distinct from Ser20 or Ser15 phosphorylation, Ser46 phosphorylation is induced with a slow kinetics and specifically linked to the induction of apoptosis [16,43]. We also demonstrated that PKCδ is started to cleave at 4–6 h following treatment of cells with genotoxic agents [35] and Ser46 phosphorylation is, at least in part, required for cleavage of PKCδ by caspase-3 to induce its full activation. In this context, we speculate that these two pathways are activated differently upon dose and time of stress. However, it is also plausible that these pathways are synergically activated for p53 modification to determine cell fate. Further studies are needed to clarify this issue. Importantly, activation of IKKα by oxidative stress contributes to p53 stabilization and activation. In this context, the demonstration that both PKCδ and IKKα activate and accumulate in the nucleus following H2O2 exposure further supports the regulation of nuclear p53 by PKCδ → IKKα signaling pathways. The mechanism for the nuclear targeting of these kinases remains obscure and additional experiments to define it are currently underway. Consistent with such regulation, our results demonstrate that IKKα contributes to p53-dependent expression of GADD45α and Bax in response to ROS. Stabilization and activation of p53 as conferred by IKKα could thus contribute to
downstream effectors of p53 and thereby regulate p53-mediated cell fate in response to oxidative stress. Acknowledgements The authors thank Dr. Hiroyasu Nakano for providing FlagIKKα plasmids. This work was supported by grants from the Ministry of Education, Science and Culture of Japan (to K.Y. and Y.M.), Kanae Foundation for Life & Socio-medical Science (to K.Y.), Kowa Life Science Foundation (to K.Y.), the Astellas Foundation for Research on Medical Resources (to K.Y.) and Japan Leukemia Research Fund (to K.Y.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellsig.2007.06.002. References [1] Y. Nishizuka, Nature 334 (1988) 661. [2] K. Yoshida, Cell. Signal. 19 (2007) 892. [3] A.G. Kanthasamy, M. Kitazawa, A. Kanthasamy, V. Anantharam, Antioxid. Redox Signal. 5 (2003) 609. [4] X. Sun, F. Wu, R. Datta, S. Kharbanda, D. Kufe, J. Biol. Chem. 275 (2000) 7470. [5] T. Ghayur, M. Hugunin, R.V. Talanian, S. Ratnofsky, C. Quinlan, Y. Emoto, P. Pandey, R. Datta, Y. Huang, S. Kharbanda, H. Allen, R. Kamen, W. Wong, D. Kufe, J. Exp. Med. 184 (1996) 2399. [6] Z.M. Yuan, T. Utsugisawa, T. Ishiko, S. Nakada, Y. Huang, S. Kharbanda, R. Weichselbaum, D. Kufe, Oncogene 16 (1998) 1643. [7] K. Yoshida, Y. Miki, Cell Cycle 4 (2005) 777. [8] K. Yoshida, T. Yamaguchi, T. Natsume, D. Kufe, Y. Miki, Nat. Cell Biol. 7 (2005) 278. [9] Z.M. Yuan, Y. Huang, T. Ishiko, S. Kharbanda, R. Weichselbaum, D. Kufe, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 1437. [10] M. Blass, I. Kronfeld, G. Kazimirsky, P.M. Blumberg, C. Brodie, Mol. Cell. Biol. 22 (2002) 182. [11] T.A. DeVries, M.C. Neville, M.E. Reyland, EMBO J. 21 (2002) 6050. [12] S. Kharbanda, A. Bharti, D. Pei, J. Wang, P. Pandey, R. Ren, R. Weichselbaum, C.T. Walsh, D. Kufe, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 6898. [13] K. Yoshida, S. Kharbanda, D. Kufe, J. Biol. Chem. 274 (1999) 34663. [14] K. Yoshida, D. Kufe, Mol. Pharmacol. 60 (2001) 1431. [15] M. Leitges, M. Mayr, U. Braun, U. Mayr, C. Li, G. Pfister, N. GhaffariTabrizi, G. Baier, Y. Hu, Q. Xu, J. Clin. Invest. 108 (2001) 1505. [16] K. Yoshida, H. Liu, Y. Miki, J. Biol. Chem. 281 (2006) 5734. [17] N. Li, M. Karin, FASEB J. 13 (1999) 1137. [18] Y. Kabe, K. Ando, S. Hirao, M. Yoshida, H. Handa, Antioxid. Redox Signal. 7 (2005) 395. [19] M. Karin, Oncogene 18 (1999) 6867. [20] M.S. Hayden, S. Ghosh, Genes Dev. 18 (2004) 2195. [21] Y. Yamamoto, U.N. Verma, S. Prajapati, Y.T. Kwak, R.B. Gaynor, Nature 423 (2003) 655. [22] V. Anest, J.L. Hanson, P.C. Cogswell, K.A. Steinbrecher, B.D. Strahl, A.S. Baldwin, Nature 423 (2003) 659. [23] K.J. Park, V. Krishnan, B.W. O'Malley, Y. Yamamoto, R.B. Gaynor, Mol. Cell 18 (2005) 71. [24] J.E. Hoberg, A.E. Popko, C.S. Ramsey, M.W. Mayo, Mol. Cell. Biol. 26 (2006) 457. [25] J.E. Hoberg, F. Yeung, M.W. Mayo, Mol. Cell 16 (2004) 245. [26] I. Jaspers, W. Zhang, A. Fraser, J.M. Samet, W. Reed, Am. J. Respir. Cell Mol. Biol. 24 (2001) 769. [27] H. Kamata, T. Manabe, S. Oka, K. Kamata, H. Hirata, FEBS Lett. 519 (2002) 231.
T. Yamaguchi et al. / Cellular Signalling 19 (2007) 2088–2097 [28] V. Imbert, R.A. Rupec, A. Livolsi, H.L. Pahl, E.B. Traenckner, C. MuellerDieckmann, D. Farahifar, B. Rossi, P. Auberger, P.A. Baeuerle, J.F. Peyron, Cell 86 (1996) 787. [29] S. Singh, B.G. Darnay, B.B. Aggarwal, J. Biol. Chem. 271 (1996) 31049. [30] K. Yoshida, Y. Miki, D. Kufe, J. Biol. Chem. 277 (2002) 48372. [31] H. Nakano, M. Shindo, S. Sakon, S. Nishinaka, M. Mihara, H. Yagita, K. Okumura, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 3537. [32] K. Yoshida, K. Komatsu, H.G. Wang, D. Kufe, Mol. Cell. Biol. 22 (2002) 3292. [33] K. Yoshida, R. Weichselbaum, S. Kharbanda, D. Kufe, Mol. Cell. Biol. 20 (2000) 5370. [34] N. Taira, K. Nihira, T. Yamaguchi, Y. Miki, K. Yoshida, Mol. Cell 25 (2007) 725. [35] K. Yoshida, H.G. Wang, Y. Miki, D. Kufe, EMBO J. 22 (2003) 1431.
2097
[36] K. Yoshida, T. Yamaguchi, H. Shinagawa, N. Taira, K.I. Nakayama, Y. Miki, Mol. Cell. Biol. 26 (2006) 3414. [37] M. Gschwendt, H.J. Muller, K. Kielbassa, R. Zang, W. Kittstein, G. Rincke, F. Marks, Biochem. Biophys. Res. Commun. 199 (1994) 93. [38] T. Abbas, D. White, L. Hui, K. Yoshida, D.A. Foster, J. Bargonetti, J. Biol. Chem. 279 (2004) 9970. [39] M.B. Kastan, Q. Zhan, W.S. el-Deiry, F. Carrier, T. Jacks, W.V. Walsh, B.S. Plunkett, B. Vogelstein, A.J. Fornace Jr., Cell 71 (1992) 587. [40] Y. Yamamoto, R.B. Gaynor, Trends Biochem. Sci. 29 (2004) 72. [41] A.M. Bode, Z. Dong, Nat. Rev., Cancer 4 (2004) 793. [42] E. Appella, C.W. Anderson, Eur. J. Biochem. 268 (2001) 2764. [43] K. Oda, H. Arakawa, T. Tanaka, K. Matsuda, C. Tanikawa, T. Mori, H. Nishimori, K. Tamai, T. Tokino, Y. Nakamura, Y. Taya, Cell 102 (2000) 849.