DNA-PK: gatekeeper for IKKγ/NEMO nucleocytoplasmic shuttling in genotoxic stress-induced NF-kappaB activation
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The transcription factors of the nuclear factor κB (NF-κB) family play a pivotal role in the cellular response to DNA damage. Genotoxic stress-induced activation of NF-κB differs from the classical canonical pathway by shuttling of the NF-κB Essential Modifier (IKKγ/NEMO) subunit through the nucleus. Here, we show that DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA double-strand break (DSB) repair, triggers the phosphorylation of NEMO by genotoxic stress, thereby enabling shuttling of NEMO through the nucleus with subsequent NF-κB activation. We identified serine 43 of NEMO as a DNA-PK phosphorylation site and point mutation of this serine to alanine led to a complete block of NF-κB activation by ionizing radiation (IR). Blockade of DNA-PK by a specific shRNA or by DNA-PKcs-deficient cells abrogated NEMO entry into the nucleus, as well. Accordingly, SUMOylation of NEMO, a prerequisite of nuclear NEMO, was abolished. Based on these observations, we propose a model in which NEMO phosphorylation by DNA-PK provides the first step in the nucleocytoplasmic trafficking of NEMO.
KeywordsSignal transduction Inflammation NF-κB pathway DNA-PK IKKγ/NEMO
NF-B essential modifier
DNA-dependent protein kinase
DNA-dependent protein kinase (DNA-PK) is a serine–threonine protein kinase that is ubiquitously expressed in all mammalian cells. This kinase consists of a catalytic subunit (DNA-PKcs) and two regulatory subunits (Ku70 and Ku80) . DNA-PK is mainly known as a critical component in the nonhomologous end-joining (NHEJ) pathway of double-strand break (DSB) repair in mammalian cells . Furthermore, DNA-PK was proposed to be a molecular sensor for DNA damage that increases DSB repair mechanisms by phosphorylation of several downstream targets [3, 4]. In addition to its role in DNA repair, DNA-PK is involved in signalling pathways other than the DNA repair pathway. For example, the enzyme triggers apoptosis after DNA damage caused by ionizing radiation (IR) . Additionally, it has antiproliferative effects through the phosphorylation of p53 in collaboration with GSK-3 .
A central regulator of the immune system is the transcription factor nuclear Factor κB (NF-κB). NF-κB is involved in the regulation of cellular survival, immune responses and inflammation . Two signalling pathways lead to activation of NF-κB, namely, the canonical pathway and the alternative pathway [8, 9]. The canonical NF-κB pathway is mediated by the IKK complex, which consists of IKKα, IKKβ and the regulatory subunit IKK-γ/NF-κB essential modulator (NEMO) . IKKα and IKKβ, but not the IKKγ/NEMO dimer, are catalytic subunits [10, 11]. Although devoid of catalytic activity, IKKγ/NEMO is strictly required for canonical NF-κB activation . In contrast, the alternative pathway plays a pivotal role in the cellular response to DNA damage, such as IR-induced damage [13, 14, 15]. This pathway is tightly regulated by NEMO, which shuttles through the nucleus by undergoing different post-translational modifications, such as phosphorylation, SUMOylation and ubiquitination [16, 17, 18], before binding to the IKK complex and subsequent NF-κB activation. The DNA damage response is coordinated by members of the phosphoinositide-3-kinase-related protein kinase (PIKK) family composed of ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR) and DNA-PK. Others have shown a requirement for NEMO phosphorylation by ATM following DNA damage, which is important for NF-κB activation .
Although regulation of NF-κB by DNA-PK had already been reported , an assumption due to the observation that IR-induced NF-κB activation in DNA-PK-deficient cells, M059J, was completely blocked , but the exact mechanism is not clear. In our study, we identified DNA-PK as an enzyme involved in the first step of NEMO translocation into the nucleus during genotoxic stress.
Materials and methods
All culture media and supplements were purchased from PAA Laboratories (Germany). M059K, M059J, HEK293 and NIH3T3 cells and peripheral blood mononuclear cells (PBMNCs) were grown as described . All procedures involving human materials were approved by the local ethics committee of Magdeburg University in compliance with the principles of the Declaration of Helsinki. PBMNCs were isolated as described . Adherent cells were further cultivated for 14 days to obtain monocyte-derived macrophages.
The full-length cDNA of human NEMO was cloned into pTagGFP-N (Evrogen). Site-directed mutagenesis of NEMO (serine to alanine) was performed using the QuikChange Site-Directed Mutagenesis Kit from Stratagene (La Jolla, CA). The NEMO-S43A and NEMO-S85A mutants were generated by PCR using the wild-type construct as a template. Mutations were verified by DNA sequence analysis. Reporter constructs containing five copies of NF-κB (NF-κB response element) were purchased from Promega (Mannheim, Germany).
Reagents and antibodies
The following antibodies were used: NF-κB Pathway Sampler Kit (#9936) (New England BioLabs, Frankfurt/Main, Germany), anti-pSer-43-NEMO (Abgent, Heidelberg, Germany), phospho-specific anti-pSer-43-NEMO (Eurogentec, Köln, Germany), anti-Ku70 (#sc-1487), anti-DNA-PKcs (#sc-5282) (Santa Cruz Biotechnology, Heidelberg, Germany), anti-Ku70/Ku80 (Abcam, Cambridge, UK), anti-ubiquitin from DAKO (Hamburg, Germany), anti-IKKγ/NEMO (#I5032), anti-IKKγ/NEMO (#WH0008517M1), anti-β-actin (Sigma-Aldrich, Germany), and anti-UK (Invitrogen, Germany). Recombinant human TNFα was purchased from Miltenyi (Bergisch Gladbach, Germany), and the DNA-PK inhibitor NU7026 was purchased from Calbiochem (La Jolla, CA, USA).
HEK293 cells stably transfected with 5xNF-κB-RE (Promega, Mannheim, Germany) were washed with phosphate-buffered saline (Mg2 + and Ca2 + free) and lysed in 150 µl/well luciferase cell culture lysis reagent (Promega, Mannheim, Germany). Luciferase assays were performed using the luciferase assay system from Promega, according to the manufacturer’s instructions, and quantified with a luminometer (LB9506, Berthold, Bad Wildbad, Germany).
Preparation of nuclear extracts
Preparation of nuclear extracts has been described previously .
Immunoprecipitation was performed as described previously . In addition, for isolation of GFP-tagged proteins, we used the GFP Isolation Kit (#130-091-125), according to the manufacturer’s instructions (Miltenyi, Bergisch Gladbach, Germany).
Transfection of human mononuclear cells with siRNA was performed using Viromer (Lipocalyx, Halle, Germany) according to the manufacturer’s instructions. The siRNA oligonucleotides with 3′-TT overhangs were purchased from MWG-BIOTECH AG (Ebersberg, Germany). The following siRNA sequences were used: siDNA-PKcs 1: 5′- GAUCGCACCUUACUCUGU UTTdTdT-3; siGL-3: 5′- CUUACGCUGAGUACUUCGATTdTdT-3. The concentration of siRNAs was 20 nmol/l during transfection. In most experiments, siRNA transfection was repeated after 24 h.
In vitro phosphorylation assay
Recombinant human NEMO (Abnova, Heidelberg, Germany) or purified GST-NEMO fusion proteins (wild-type and mutant) were incubated with DNA-PK (Promega, Mannheim, Germany) at 30 °C in a total volume of 30 μl of DNA-PK kinase assay buffer containing 10 μCi of [γ-32P]ATP (5000 Ci/mmol). Phosphoprotein products were detected by PAGE (10% gel).
Ionizing irradiation was performed using a gamma-irradiation device from the BIOBEAM 8000 series (Gamma-Service Medical, Leipzig, Germany).
Staining of cell cultures for STED microscopy
Fixation and staining of the cells for stimulated emission depletion (STED) microscopy have been described previously . Incubation with the primary antibody was performed overnight at 4 °C. The following antibodies were used: anti-Ku70 (#ab 83501) (Abcam, Berlin, Germany) and anti-IKKγ/NEMO (#SAB1404591) (Sigma-Aldrich, Taufkirchen, Germany). The following secondary antibodies were used: anti-rabbit ATTO 647N (dilution 1:200) and anti-mouse Chromeo 494 (dilution 1:50) (Active Motif, La Hulpe, Belgium).
Multichannel stacks of immunocytochemically stained macrophages were sequentially recorded using a Leica SP8 confocal microscope (Leica Microsystems, Wetzlar) equipped with a 405-nm diode laser, a supercontinuum white light laser source and sensitive hybrid photomultipliers (HYD). Cells were scanned using a 63 × oil immersion objective with a numerical aperture of 1.4 (HCX PL APO CS 63.0 × 1.40 OIL UV). The diameter of the confocal pinhole was set to 95.5 µm (= Airy 1), and images were taken at a 2048 × 2048 pixel resolution with zoom factor 3 and a scan rate of 700 Hz with a line average of 8. Eighteen optical planes were scanned in the axial direction with a step size between two focal planes of 0.3 µm. These settings resulted in an image volume of 61.48 µm (x) × 61.48 µm (y) × 5.07 µm (z) with a voxel size of 30 nm (x) × 30 nm (y) × 300 nm (z) at 8 bit greyscale resolution.
For spectral filtering, the laser microscope was equipped with acoustooptic modulators (AOTF) and an acoustooptical beam splitter (AOBS) that separates the detection beam path from excitation. The fluorophores of the secondary antibodies were sequentially excited with wavelengths of 488 nm, 553 nm and 405 nm for image acquisition of FITC in the spectral range of 503 nm to 531 nm (channel 1), CY3 in the spectral range of 562–593 nm (channel 2) and DAPI in the spectral range of 412–475 nm (channel 3). With this sequential excitation setting, any crosstalk between channels could be excluded.
For determination of colocalization between Ku 70/80 and NEMO in the nucleus and cytoplasm, image stacks were also assessed in the xz or yz direction. For image improvement and contrast enhancement, we used Huygens deconvolution software (SVI, Hilversum, The Netherlands) and ImageJ (NIH, USA).
Statistical analysis was performed by ANOVA. Post-test multiple comparison was performed by the Bonferroni method. All experiments were independently repeated at least three times.
DNA-PK is involved in NF-κB activation
DNA-PK phosphorylates NEMO at Ser43
We then studied the impact of Ser43 phosphorylation on NF-κB transcriptional activity. For this purpose, NEMO-WT and a NEMO-Ser43A mutant were overexpressed in HEK293 cells stably transfected with a 5xNF-κB response element, and NF-κB activity was determined. Mutation of Ser43 of NEMO reduced the activation of NF-κB, indicating that Ser43 is required for full transcriptional activation of NF-κB by genotoxic stress, such as IR (Fig. 2e). Furthermore, using a phospho-specific antibody recognizing Ser43, we confirmed that irradiation resulted in phosphorylation of this site, which was abolished when the mutant of NEMO, where serine was changed to alanine, was overexpressed (Fig. 2f).
DNA-PK-mediated phosphorylation induces NEMO nuclear translocation
Interaction of DNA-PK with NEMO in macrophages
DNA-PK/NEMO interaction modulates the alternative NF-κB pathway
Previous studies have demonstrated the involvement of DNA-PK in NF-κB activation. For example, the Ku subunit of DNA-PK can interact with the NF-κB p50 promoter in gastric cancer cells and, in this way, acts as a positive regulator of p50 protein expression . Additionally, DNA-PK can modulate the NF-κB subunit p65 . Furthermore, activation of the alternative genotoxic stress-induced pathway through DNA-PK has also been proposed because IR-triggered NF-κB activation was completely abolished in DNA-PK-deficient M059J cells . However, the mechanism remains unclear. A central protein in the genotoxic activation of NF-ĸB is NEMO, which takes an alternative route through the nucleus before binding to the IKK complex and activating NF-κB . In this report, we identified NEMO as a new DNA-PK substrate that is phosphorylated at Ser43 located within the N-terminal domain. This serine residue is involved in the control of NF-κB activation by genotoxic stress. We propose a model in which DNA-PK serves as a gatekeeper for NEMO to enter the nucleus, as abrogation of the Ser43 phosphorylation site results in accumulation of the protein within the cytoplasm and reduced NEMO phosphorylation, SUMOylation and ubiquitination, the prerequisites of nuclear NEMO shuttling (Fig. 5b).
We were surprised to identify Ser43 as a DNA-PK phosphorylation site because DNA-PK usually phosphorylates nuclear proteins at serine or threonine residues followed by glutamic acid . However, a previous report showed that DNA-PK, in response to DNA damage, phosphorylates cytoplasmic GOLPH3 even when glutamic acid is absent . A study describing phosphorylation of Ser43 in NEMO by IKKβ lacks a functional explanation regarding the importance of Ser43 in NEMO for NF-kB activation . Previously, we identified GSK-3β as a kinase that phosphorylates Ser8, 17, and 31 of NEMO during TNFα-induced NF-κB activation . In this study, Ser43 was also found to be phosphorylated. However, overexpression of a Ser43 mutant had no effect on TNFα-induced NF-κB activity, excluding a significant involvement in the canonical NF-κB activation pathway. This observation is concordant with a previous report demonstrating a reduction in IR-induced NF-κB activation in DNA-PKcs-deficient cells, while the response to TNFα was not impaired .
Endogenous NEMO is localized predominantly in the cytoplasm. However, following exposure to DNA damage, NEMO translocates to the nucleus . The mechanism underlying this first step of its translocation is not clear, since NEMO lacks a classical nuclear localization signal that would be recognized by importins [32, 33]. However, a recent report by Hwang and colleagues demonstrated that NEMO interacts with the importin β family member IPO3 and translocates to the nucleus . At present, we can only speculate but favour a model in which the Ku70/NEMO interaction modulates NEMO translocation to the nucleus. This assumption is based on the established role of Ku70 in shuttling, for example, in Rickettsia conorii , and on the nuclear translocation of Ku70 upon exposure of cells to DNA damage [36, 37, 38]. How exactly Ku70 can be utilized by cytosolic NEMO to gain entry into the nucleus needs further investigation. Ku70 and NEMO may associate with the nuclear localization receptor importin-α, which enables nuclear translocation . In a recent study , phosphorylation of an S/T-P-S/T domain operated as a general nuclear translocation signal, as is the case for Ser43 in NEMO. Therefore, this site may be directly responsible for NEMO nuclear localization. Genotoxic activation of NF-κB requires a series of nuclear post-translational modifications of NEMO that are critical for NF-κB activation following genotoxic stress (reviewed in ). N-terminal fusion of NEMO with SUMO-1 targeted NEMO from the cytoplasm to the nucleus with subsequent ubiquitination of NEMO [16, 17]. Subsequent work has identified PIASy as the major SUMO E3 ligase for NEMO that is required for NF-κB activation by multiple genotoxic agents . Interestingly, this report shows the nuclear interaction of PIASy and NEMO, postulating nuclear NEMO SUMOylation. The time course analysis suggested that SUMOylation of NEMO occurs earlier and transiently before ubiquitination of NEMO is observed . The induction of DNA DSBs leads to phosphorylation of NEMO by ATM, and this event seems to occur after SUMOylation and before ubiquitination of NEMO . In our experiments, we observed not only a reduction in NF-κB activation by overexpression of mutant NEMO but also a reduction in NEMO SUMOylation. Future experiments including the fusion of SUMO-1 with the NEMO mutant will explore the significance of NEMO phosphorylation for NEMO SUMOylation and whether SUMOylated NEMO is able to overcome the inability of the NEMO S43A mutant to translocate to the nucleus.
In summary, our data imply a gatekeeper function of DNA-PK for the entry of NEMO into the nucleus under genotoxic stress. This novel mechanism of NEMO regulation by DNA-PK highlights the role of the post-translational modifications of NEMO in NF-κB activation.
Activation of the genotoxic stress-induced NF-κB pathway was completely abolished in DNA-PK-deficient M059J cells.
Open Access funding provided by Projekt DEAL. We thank C. Zufelde for valuable technical assistance. Support from a Grant from the Deutsche Forschungsgemeinschaft (SFB854/A02) and GCI3, Medical Center, Otto-von-Guericke, is greatly appreciated.
SM and RCB designed the project. SM, WZ, BL, TN, SW and MP performed a majority of the experiments. TK performed the mass spectrometric analysis. SM and RCB wrote the manuscript. All authors analysed the data.
Compliance with ethical standards
Conflict of interest
The authors declare no competing financial interests.
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