ATM regulates NF-κB-dependent immediate-early genes via RelA Ser 276 phosphorylation coupled to CDK9 promoter recruitment

Cell cultures and reagents

Human A549 pulmonary type II epithelial cells (American Type Culture Collection) were cultured in F-12K medium (Gibco, Invitrogen) with 10% fetal bovine serum, 1.0-mM sodium pyruvate, penicillin (100 U/ml) and streptomycin (100 g/ml) at 37°C in a 5% CO₂ incubator. ATM^+/+ and ATM^−/− MEFs are gifts from Dr Zhao-hui Wu and were cultured in Dulbecco's modified Eagle's medium (Gibco, Invitrogen) with 10% fetal bovine serum, 0.1-mM nonessential amino acids, 1.0-mM sodium pyruvate, penicillin (100 U/ml) and streptomycin (100 g/ml) at 37°C in a 5% CO₂ incubator.

ATM, pATM and pRelA (Ser 276) antibodies (Ab Cat. No. 3037) were purchased from Cell Signaling Technology (Beverly, MA, USA). Lamin B Ab was purchased from EMD Millipore (Darmstadt, Germany). b-tubulin, IKKg, IkBa, pIkBa, b-TrCP, RelA, PKAc and CDK9 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NF-kB2/p100, pIKKb and pPolII antibodies were purchased from Abcam (Cambridge, UK).

Nuclear and cytoplasmic extraction

Cytoplasmic and nuclear extracts were prepared as described earlier (16). Briefly, cells were harvested in PBS and centrifuged to collect pellets. Pellets were resuspended in double cell volume of solution A (50 mM HEPES (pH 7.4), 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 0.1 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor, 10 μg/ml aprotinin and 0.5% IGEPAL-630) and centrifuged to obtain the supernatants as cytoplasmic fraction. The nuclear pellets were purified on sucrose cushions (buffer A with 1 M sucrose). After high salt extraction in buffer C (10% glycerol, 50 mM HEPES (pH 7.4), 400 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor and 10 g/ml aprotinin), the nuclei were centrifuged at 12 000g at 4°C for 20 min. The supernatants were saved as NE. The protein concentrations were measured by Bradford protein assay (Protein Reagent, Bio-Rad).

Western immunoblots

After SDS-PAGE electrophoresis, proteins were transferred to PVDF membrane and blocked in 5% milk/TBST buffer for 1 h. The membranes were incubated with specific primary antibody for overnight at 4°C followed by washing with TBST and incubation with secondary antibody for 1 h. Membranes were exposed by ECL western blotting solution (Amersham) and the films were developed by Kodak machine.

Protein–protein interaction by co-immunoprecipitation

Cellular extracts (nuclear, cytoplasmic or whole cell extracts prepared in RIPA buffer) were precleared by incubating with protein A sepharose CL-4B (Sigma) beads for 45 min at 4°C. One milligram protein from precleared samples were incubated with 5 μg of specific antibody for overnight at 4°C. Immune complexes were captured by addition of protein A sepharose CL-4B (Sigma) for 2 h at 4°C. Following incubation, the beads were washed four times with TBS-T (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA and 0.05% Triton-x100) followed by the extraction of the bound protein boiling the beads in SDS-PAGE buffer containing b-mercaptoethanol for 5 min. The bound proteins were identified by performing western blot using specific antibodies.

PKAc activity assay

Cells were lysed in a buffer containing 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, pH 8.0, 1 mM PMSF, 10 μM leupeptin and 10 μM aprotinin. The cell suspension was sonicated for 30 s × 3, centrifuged and supernatant was collected. PKAc activity was measured in the supernatant using PKA-specific peptide (LRRASLG) according to the supplier's recommendations (PepTag assay, Promega, Madison, WI, USA). Phosphorylated and nonphosphorylated peptides were separated by electrophoresis on a 0.8% agarose gel and quantitated by measuring absorbance at 570 nm. Data were presented as percentage of absorbance relative to 0 h.

q-RT-PCR

Total cellular RNA was extracted by Tri Reagent (Sigma-Aldrich). cDNA was synthesized from 1 μg of RNA using iScript cDNA Synthesis Kit (Bio-Rad). Three μl of cDNA products was amplified in 20 μl reaction volume containing 10 μl iQ SYBR Green Super Mix (Bio-Rad) and 400 nM primer mix. All reactions were processed in MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) and results were analyzed by IQ5 program (Bio-Rad).

Chromatin immunoprecipitation

Two-step cross-link chromatin immunoprecipitation (ChIP) was performed as described previously (17). Briefly, A549 cells were washed twice with PBS and incubated with disuccinimidyl glutarate (2 mM for 45 min) for protein–protein cross-linking. Subsequently, protein–chromatin cross-linking was performed by incubating the cells with 1% formaldehyde for 30 min. Equal amounts of sheared chromatin were immunoprecipitated overnight with 4 ug of the indicated antibody in ChIP dilution buffer at 4°C. Immunoprecipitates were collected with 40 ul protein A magnetic beads (Invitrogen), washed and associated chromatin was eluted in 250 ul elution buffer for 15 min at room temperature. Samples were de-cross-linked in 0.2M NaCl at 65°C for 2–4 h. The precipitated DNA was phenol-chloroform extracted, precipitated with 100% ethanol, and dried. The promoter-specific primers used for quantitative PCR of each gene are as follows: Gro-β: forward: 5′- TCGCCTTCCTTCCGAACTC-3′, reverse: 5′- CGAACCCCTTTTATGCATGGT-3′; IκBα: forward: 5′- GACGACCCCAATTCAAATCG-3′, reverse: 5′- TCAGGCTCGGGGAATTTCC-3′; IL-8: forward: 5′- AGGTTTGCCCTGAGGGGATG-3′, reverse: 5′-GGAGTGCTCCGGTGGCTTTT-3′.

Stable isotope dilution (SID)-selected Reaction Monitoring (SRM)-mass spectrometry (MS) assays

The SRM assays for total RelA, IKKγ and phospho-Ser 536 RelA were described previously (18). The SRM assay for phospho-Ser 276 RelA was developed using a workflow described previously (18). The signature peptides and MS parameters of each SRM assay are listed in Table 1. Stable isotope standard (SIS) peptides were chemically synthesized incorporating isotopically labeled [13C615N4] Arginine or [13C615N4] Lysine to a 99% isotopic enrichment (Thermo Scientific).

Ubiquitin-associated IKKγ or RelA were immunoprecipitated from native extracts using anti-Ubiquitin or RelA Ab and immune complexes captured by protein A magnetic beads (Dynal Inc). For the measurement of the level of ubiquitin-conjugated IKKγ and phospho-Ser 276 RelA, the proteins on the beads were digested with trypsin as described previously (19). Briefly, the beads were washed with PBS for three times and then resuspended in 30 μl of 50 mM ammonium hydrogen carbonate (pH 7.8) and 20 μl of 0.1 μg/ μl of trypsin were added. The samples were mixed and trypsinized by gentle vortexing overnight at 37°C. After digestion, the supernatant was collected. The beads were washed with 50 μl of 50% acetonitrile (ACN) three times and the supernatant was pooled, and dried. The tryptic digests were then reconstituted in 30 μl of 5% formic acid-0.01% TFA for MS analysis. For the measurement of the level of phosphor-Ser 536 RelA, the protein on the beads were digested with Glu-C (Indianapolis, IN) as described previously (18). The peptides were extracted from the beads as described above and then reconstituted in 30 μl of 5% formic acid-0.01% TFA for MS analysis. An aliquot of 10 μl of diluted SIS peptides were added to each digest before LC-SRM-MS analysis.

LC-SRM-MS analysis was performed with a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA) equipped with nanospray source (Thermo Scientific, San Jose, CA) as described before. The online desalting and chromatography were performed using an Eksigent NanoLC-2D HPLC system (AB SCIEX, Dublin, CA). An aliquot of 10 μl of each of tryptic digests were injected on a C18 peptide trap (Agilent, Santa Clara, CA), desalted with 0.1% formic acid at a flow rate of 2 μl/min for 45 min. Peptides were eluted from the trap and separated on a reversed phase nano-HPLC column (PicoFrit™, 75 μm x 10 cm; tip ID 15 μm) packed in house using Zorbax SB-C18 (5-μm diameter particles, Agilent, Santa Clara, CA). Separations were performed using a flow rate of 500 nl/min with a 20-min linear gradient from 2–40% mobile phase B (0.1% formic acid-90% ACN) in mobile phase A (0.1% formic acid), followed by 0.1-min gradient from 40–90% mobile phase B and 5-min 90% mobile phase B. The TSQ Vantage was operated in high-resolution SRM mode with Q1 and Q3 set to 0.2 and 0.7-Da Full Width Half Maximum. All acquisition methods used the following parameters: 1800 V ion spray voltage, a 275°C ion transferring tube temperature, a collision-activated dissociation pressure at 1.5 mTorr and the S-lens voltage used the values in S-lens table generated during MS calibration. All SRM data were manually inspected to ensure peak detection and accurate integration. The chromatographic retention time and the relative product ion intensities of the analyte peptides were compared to those of the SIS peptides. The variation of the retention time between the analyte peptides and their SIS counterparts were within 0.05 min, and no significant difference in the relative product ion intensities of the analyte peptides and SIS peptides were observed. The peak area in the extract ion chromatography of the native and SIS version of each signature peptide were integrated using Xcalibur® 2.1. The default values for noise percentage and base-line subtraction window were used. The ratio between the peak area of native and SIS version of each peptide were calculated.

Comet assays

DNA double-strand breaks were quantitated by a Comet assay (Trevigen, Inc, Gaithersburg, MD) as we described previously (20). Briefly, cells were suspended in 0.6% low melting-point agarose. The cell/agarose suspension (80 μl) was applied onto pre-coated microscope slides (Trevigen Inc). Slides with solidified agarose were placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Trish-HCl (pH 10), 1% sodium sarcosinate and 1% Triton X-100) for 24 h. Slides were placed in pre-cooled (4°C) electrophoresis tanks and electrophoresis was carried out at pH 7.8 in a buffer provided by the manufacturer (Trevigen Inc). After electrophoresis at 1.25 V per cm) at 4°C, preparations were air-dried and DNA stained by SYBER green. Comets were analyzed by using the Comet Assay IV v4.2 system (Perceptive Instruments, Suffolk, UK) image analysis software (4). The fold change in tail comet moments are expressed as the mean (± SD) from three parallel experiments.

ShRNA-mediated ATM knockdown in A549 and HeLa cells

ATM and control shRNA are gifts from Dr Sankar Mitra (Methodist hospital, Houston, TX). Plasmids were transfected to A549 and HeLa cells using FuGENE HD kit (Promega, Madison, WI). Five μg plasmid was mixed with 15 μl of FuGENE HD Transfection Reagent for 5 min. Afterward, the mixture was added to a 10-cm cell plate and mixed gently. Forty eight hours later, cells were selected in puromycin (6 μg/ml) for 2 weeks.

TNF-induced ATM nuclear export requires IKKγ ubiquitination.

In unstimulated cells, ATM resides in the nucleus as an inactive form. Following DNA double-strand breaks (DSBs), it undergoes autophosphorylation at Ser 1981 (21). ATM activation has been reported to be coupled with nuclear export in the DSB-induced DNA repair pathway (8). Therefore, we investigated whether a similar phenomenon happens upon TNF stimulation. Nuclear (NE) and cytoplasmic extract (CE) were prepared from cells stimulated with TNF and assayed by Western immunoblot. First, Lamin B and β-tubulin were detected to characterize subcellular enrichment. Here, we observed that although the nuclear fractions stained strongly with Lamin B, the cytosolic fractions did not, indicating that CEs were largely devoid of nuclear contamination (Figure 1A). We next examined the distribution of ATM using anti-ATM Ab. Under unstimulated conditions, an ∼350 kDa ATM band is primarily located in the nucleus and present in lower abundance in the cytosol. By contrast, after 0.25 h of TNF stimulation, ATM is detected in the cytoplasmic fraction where it continues to accumulate until 1 h of stimulation (Figure 1A). From this experiment, we concluded that TNF induces ATM nuclear-to-cytoplasmic transport.

In order to determine whether ATM kinase activity is required for TNF-induced nuclear export, we utilized the specific ATM kinase inhibitor, KU-55933 (22). A549 cells were pre-treated with the KU-55933 (10 μM, 1 h) before TNF exposure. NE and CE were prepared and assayed by western immunoblot. In the NE, phospho-Ser 1981 ATM (pATM) was observed in the absence of stimulation, whose abundance was induced within 0.25 h of TNF stimulation (Figure 1B). By contrast, a lower abundance of pATM was seen in cells treated with KU-55933 only, and no TNF-inducible accumulation was observed (Figure 1B, top right panel). Correspondingly, the appearance of ATM in the CE was detected upon TNF stimulation while KU-55933 pretreatment completely blocked its cytoplasmic accumulation (Figure 1B, third panel). Our observations that KU-55933 pretreatment completely blocks TNF-induced pATM formation as well as cytoplasmic accumulation suggest that ATM kinase activity is prerequisite for its TNF-inducible nuclear export.

We have reported a significant and transient induction of ROS generation after TNF treatment as measured by H2DCF oxidation, increased in 8-oxoguanine (8-oxoG) DNA lesions, and by protein carbonylation (5). Although elevated ROS and 8-oxoG formation is usually associated with ssDNA breaks, we evaluated the possibility whether TNF treatment is sufficient to induce DNA double strand breaks (DSBs) and provide a potential mechanism of TNF-induced ATM activation and nuclear export. A549 cells were TNF treated for various times intervals followed by Neutral Comet assay, an assay that specifically detects DSBs (23). Etoposide (VP-16), which is a traditional drug to induce significant DSBs, is used as a positive control. A significant, but transient, 2-fold increase in DSB formation was observed after 0.25 h of TNF exposure that further increased to 2.5-fold at 0.5 h before declining to untreated levels by the end of 1 h exposure (Figure 1C, top panel). On the other hand, VP-16 induced much higher (∼42-fold) level of DSBs than that induced by TNF. Representative microscopic images of comet moment after TNF stimulation is shown in Figure 1C (bottom panel). These data suggest that TNF induces DSBs, albeit at much lower levels than that induced by VP-16.

To ascertain whether TNF-induced ROS generation is essential for pATM formation and its subsequent nuclear export, A549 cells were pretreated with the free radical scavenger, DMSO (2% for 0.5 h), at concentrations that we have previously shown to be sufficient to block TNF-induced ROS formation without apparent cytotoxicity (5). CE and NE were prepared from a TNF time course in the presence or absence of DMSO. In NE, we observed an induction of pATM upon TNF stimulation, but surprisingly, DMSO pretreatment did not affect pATM formation (Figure 1D, top panel). By contrast, DMSO pretreatment blocked the rapid kinetics of cytoplasmic accumulation of ATM. Here, we noted that in the absence of DMSO, cytoplasmic ATM is detectable within 0.25 h, whereas in the presence of DMSO, cytoplasmic ATM is markedly reduced (Figure 1D, middle panel). To further validate our surprising findings, we utilized another chemically unrelated ROS inhibitor, NAC (N-acetyl cysteine) (24), to see whether pretreatment affects ATM phosphorylation and ATM export. Similar to the results of the DMSO pretreatment, NAC pretreatment blocked ATM export without affecting pATM formation (Figure 1E). This result suggests that ROS affects ATM cytoplasmic translocation at a step downstream of pATM formation (25).

Previous work showed that DSB-induced ATM export is IKKγ-dependent, where nuclear complex formation of pATM and IKKγ is prerequisite for NF-κB activation. In this mechanism, activated ATM phosphorylates IKKγ on Ser 85 to promote its ubiquitination (Ub) and nuclear export (8). Cytoplasmic export of the ATM·Ub-IKKγ complex allows it to associate with and activate IKK, the rate-limiting step in NF-κB liberation from cytoplasmic stores. Therefore, we assessed if TNF-induced ATM nuclear export also is IKKγ-dependent. For this purpose, IKKγ^+/+ and IKKγ^−/− MEFs were stimulated by TNF for various length times, CE were prepared and immunoblotted with anti-ATM antibody (Ab). Although ATM translocation into the cytosol was observed in IKKγ^+/+ MEFs, ATM export was completely blocked in IKKγ^−/− cells suggesting that IKKγ is essential for TNF-induced nuclear export of ATM (Figure 1F).

Next, we evaluated the effect of ROS on ATM·IKKγ complex formation. Nuclear IKKγ was immunoprecipitated from TNF treated A549 cells in the absence or presence of DMSO pretreatment. Levels of IKK-associated ATM were quantitated in the immunoprecipitates by Western blot. We noted that TNF induced ATM·IKKγ complex formation in a kinetic that matches with pATM phosphorylation and nuclear export. However, a distinct pattern was observed with DMSO pretreatment. Here, the ATM·IKKγ association was observed in DMSO only treated cells, and upon TNF stimulation, the abundance of the ATM·IKKγ complex reduced over time (Figure 1G). These data suggest that TNF-inducible ATM·IKKγ interaction is ROS-dependent (Figure 1G). Previous studies have shown that IKKγ ubiquitination at Lys 285 is essential for ATM-NF-κB pathway (26). Therefore, to further understand the role of TNF-induced ROS on ATM·IKKγ binding and subsequent nuclear export, we assessed its effect on IKKγ ubiquitination. For this measurement, NEs from A549 cells stimulated with TNF with or without DMSO pretreatment were used. Ubiquitinated nuclear proteins were enriched by immunoprecipitation in native extracts using an ubiquitin-specific Ab. IKKγ was quantified by a quantitative, selected reaction monitoring-mass spectrometry (SID-SRM-MS) assay using a prototypic peptide that detects unmodified IKKγ. Within 0.25 h of TNF treatment, Ub-associated IKKγ levels increased by 2.5-fold over untreated cells and persisted for 1 h. However, DSMO pretreatment completely blocked Ub association with IKKγ (Figure 1H). Previous studies have shown that unanchored polyubiquitin chains existing in the cells play essential roles in innate cell signaling pathways (27,28). We therefore investigated whether the increased level of Ub associated with IKKγ comes from conjugated or unconjugated polyubiquitin. NEs were immunoprecipitated with anti-IKKγ Ab and the formation of ubiquitin adducts measured by western blot using anti-ubiquitin Ab. No adducts could be detected (data not shown). We additionally probed the western blot using anti-IKKγ Ab. Only a single IKKγ band was detected without any additional higher molecular weight species (Figure 1I). These data suggest that ROS induce IKKγ binding to free polyubiquitin chains, or the abundance of the covalently linked IKKγ is below the limit of detection of our assay. Overall, our data suggests that TNF-induced DNA DSBs activates ATM and nuclear export. Additionally our data suggest that TNF-induced ROS is required for ATM export through affecting Ub-IKKγ-complex formation and ATM association.

Cytoplasmic ATM is essential for TNF-α-induced IκBα degradation through β-TrCP recruitment.

In our earlier report, we observed a delay in TNF-inducible IκBα proteolysis and decreased RelA nuclear translocation in ATM^−/− MEFs, suggesting a novel role of cytoplasmic ATM in facilitating IκBα degradation (15). Here, we further extend this observation and sought to elucidate the mechanism by which ATM alters IκBα degradation. We first sought to reproduce the findings using shRNA-mediated knockdown of ATM in human lung epithelial cells (A549). ATM-directed shRNA decreased ATM expression by 80% (Figure 2A). TNF-induced IκBα degradation was measured in CEs using western immunoblot. In control A549 cells, there was no marked IκBα degradation after 0.25 h; however, a rapid disappearance was observed 0.5 h after TNF treatment followed by IκBα resynthesis within 1 h. By contrast, in the ATM-depleted cells IκBα degradation was significantly delayed, not being apparent until after 1 h of TNF treatment (Figure 2B). Similar findings were observed in experiments using shRNA mediated ATM knockdown in human cervical cancer epithelial (Hela) cells. IκBα was not affected after 0.25 h, but rapidly degraded 0.5 h in control shRNA transfected cells. This proteolysis was blocked after ATM knockdown (Figure 2C and D). Together we conclude that ATM affects TNF-induced IκBα degradation.

Previous work has shown that TNFα activates the IKK complex, a rate-limiting kinase responsible for IκBα phosphorylation at Ser residues 32 and 36 in its NH2 terminal regulatory domain. Phospho-Ser 32/36 IκBα (pIκBα) is specifically bound by the β-TrCP ubiquitin ligase, triggering recruitment to the 26S proteasome and its degradation (29,30). We first investigated if ATM affects IκBα phosphorylation. pIκBα was detected in whole cell extracts (WCE) from time course of TNF stimulation in control or ATM knockdown A549 cells. In control shRNA transfected cells pIκBα were detectable in unstimulated cells and was depleted by 0.5 h, similar to the profiles for total IκBα degradation (Figure 2B). In contrast, pIκBα accumulates immediately after TNF stimulation in ATM shRNA transfected cells, and persists throughout the time course (Figure 2B). Similar findings were observed in shRNA mediated HeLa cells (Figure 2D). These results demonstrate that pIκBα is stabilized in ATM-deficient cells.

To confirm the effects of ATM on stabilizing pIκBα levels, WCEs from a time course of TNF stimulation in ATM^+/+ and ATM^−/− MEFs were prepared and total IκBα was immunoprecipitated. The level of pIκBα was then detected by western immunoblot. ATM^+/+ MEFs showed a low level of pIκBα in untreated cells. By contrast, there was an accumulation of pIκBα in ATM^−/− MEFs (Figure 2E). Although the level of pIκBα was difficult to detect in ATM^+/+ cells due to its proteolysis, the abundance of pIκBα in ATM^−/− MEFs showed marked accumulation compared to that of ATM^+/+ MEFs (Figure 2E). To further extend these findings, the same experiment was done using control shRNA and ATM shRNA transfected A549 cells. A similar result was observed (Supplementary Figure S1). These data further suggest that pIκBα stabilization is a consistent finding for ATM^−/− MEFs and ATM knockdown human cell lines.

To exclude the potential role of ATM in upstream effects on the TNF signaling pathway, we examined pIKKβ activation, the kinase directly upstream of IκBα (4). WCE from TNF stimulated control- and ATM knockdown A549 cells were assayed by western blot using anti-pIKKβ Ab. We observed indistinguishable induction of pIKKβ in both cell lines (Figure 2F). We therefore conclude that TNF-induced pIKKβ formation is ATM-independent.

Recruitment of β-TrCP to pIκBα is essential for its ubiquitination and proteolysis. One explanation could be that the differences in IκBα proteolysis were due to differences in β-TrCP expression. To address this possibility, CEs from TNF stimulated ATM^+/+ and ATM^−/− MEFs were prepared and assayed for steady-state β-TrCP levels. We observed that the level of β-TrCP protein expression was identical in ATM^+/+ and ATM^−/− MEFs (Figure 2G). Therefore, we assessed whether ATM influenced formation of the IκBα·β-TrCP complex. In this experiment, ATM^+/+ and ATM^−/− MEFs pre-treated with MG132 (to block proteosomal activity) were stimulated with TNF. IκBα complexes were enriched by immunoprecipitation and β-TrCP level detected by immunoblot. In ATM^+/+ cells, β-TrCP was engaged with IκBα at a low level in the absence of stimulation and transiently increased after 0.25 h of treatment (Figure 2H). A subsequent decline in the abundance of the complex was seen after 0.5 h. By contrast, the basal IκBα·β-TrCP interaction was significantly decreased in ATM^−/− MEFs, and was not detectable until after 1 h of stimulation (Figure 2H). To extend these findings, this experiment was also conducted in control- and ATM shRNA-transfected HeLa cells, where different kinetics of β-TrCP and IκBα association was observed (Supplementary Figure S2).

Since β-TrCP also targets NF-κB2/p100 for ubiquitin-mediated proteolysis, we asked whether ATM deficiency affects NF-κB2/p100 binding in response to noncanonical pathway activation (31). For this purpose, control- or ATM shRNA- transfected A549 cells were pre-treated with the proteasome inhibitor MG132 and subsequently TNF stimulated for 1 and 3 h, time points reported to induce p100 processing and non-canonical pathway activation in A549 cells (32). NF-κB2/p100-β-TrCP association was measured by NF-κB2/p100 western blot of β-TrCP immunoprecipitates. We detected induction of NF-κB2/p100-β-TrCP association at equivalent levels in both ATM replete and ATM knockdown cells (Supplementary Figure S3). Taken together, we conclude that cytoplasmic ATM is specifically required for β-TrCP- pIκBα complex formation, essential for rapid pIκBα proteolytic degradation in response to TNF treatment.

We next tested whether ATM participates in the IκBα·β-TrCP complex. β-TrCP was immunoprecipitated from CEs from TNF-exposed A549 cells and the bound ATM was detected by western blot. In the absence of stimulation, little ATM is associated with β-TrCP. However, TNF induced a rapid induction of ATM binding (Figure 2I). In this assay, p53, a protein known to bind ATM upon VP-16 addition (33), served as a positive control. The same experiment was also conducted in ATM-replete HeLa cells; similar results were obtained (Supplementary Figure S4).

Since different kinetics of IκBα degradation was observed in control shRNA and ATM shRNA transfected A549 cells, we further investigated the effects of ATM depletion on RelA nuclear translocation. For this experiment, NE from TNF stimulated control- or ATM shRNA-transfected A549 cells were prepared and assayed for RelA by western blot. In control shRNA-transfected A549 cells, a significant induction of RelA translocation was observed within 0.25 h, peaking within 0.5 h. By contrast, in ATM shRNA-transfected A549 cells, a decreased, but still obvious induction was observed (Figure 2J). The different kinetics of RelA nuclear translocation is consistent with the different kinetics of IκBα degradation in these cell lines (c.f. Figure 2B and D).

Taken together, these findings clearly suggest a novel role of cytoplasmic ATM to bind and recruit β-TrCP to pIκBα and explains the differences of kinetics in IκBα degradation, reduced β-TrCP·IκBα binding and stabilization of pIκBα in ATM-deficient cells.

ATM is required for TNF-induced NF-κB phosphorylation on Ser ²⁷⁶ through the PKAc pathway

Once released from IκBα inhibition, RelA is required for induction of downstream genes (5,34). In this process, RelA is phosphorylated at Ser residues 276 and/or 536 by distinct pathways (35,36). In particular, we have observed that TNF-induced phospho-Ser 276 RelA formation is ROS dependent, whereas phospho-Ser 536 RelA formation is ROS-independent (5). In order to examine whether ATM affects RelA phosphorylation on either Ser 276 or Ser 536, A549 cells were TNF-stimulated in the absence or presence of KU-55933 pretreatment. WCE were prepared and total RelA enriched by immunoprecipitation. Phospho-RelA was then quantitated in the immunoprecipitates by SID-SRM-MS using a proteotypic peptide corresponding to phospho-Ser 276 RelA (Materials and Methods). We observed that TNF induced an 8-fold induction of phospho-Ser 276 RelA formation after 0.5 h of treatment in control cells, whereas KU-55933 pretreatment significantly blocked phospho-Ser 276 RelA formation at all time points (Figure 3A). Quantitation of phospho-Ser 536 RelA formation showed that TNF induced an ∼4.5-fold induction of phospho-Ser 536 RelA in a manner that was not KU-55933 sensitive (Figure 3B). A western blot also showed the same result upon TNF stimulation with or without the pretreatment of KU-55933 (Figure 3C).

To further validate the role of ATM in RelA phosphorylation, we measured phospho-Ser 276 RelA levels in ATM knockdown cells. Either empty shRNA-transfected or ATM shRNA-transfected A549 cells were treated with TNF for indicated times. WCE were collected and RelA was immunoprecipitated. Phospho-Ser 276 RelA formation was quantitated by immunoblot. As expected, TNF treatment increased phospho-Ser 276 RelA formation in control shRNA transfectants starting from 0.25–1 h. However, TNF-induced phospho-Ser 276 RelA formation was significantly reduced in the ATM shRNA transfected cells (Figure 3D).

To ensure the antibody specificity for detection of phospho-Ser 276 RelA (37), WCE from control or TNF stimulated RelA^+/+ and RelA^−/− MEFs were prepared and assayed by western blot. We noted a significant induction of phospho-Ser 276 RelA band in ATM^+/+ MEFs while no signal was detected in ATM^−/− MEFs. The blot with anti-RelA Ab proved the knockout of RelA in ATM^−/− MEFs (Figure 3E).

Furthermore, we used RelA^+/+ and RelA^−/− MEFs to validate the specificity of the IP-SRM assay for phospho-Ser 276 RelA. WCE from control or TNF stimulated RelA^+/+ and RelA^−/− MEFs were prepared and immunoprecipitated by pan anti-RelA Ab. The abundanace of RelA and phospho-Ser 276 RelA were separately quantitated in the immunoprecipitates by SID-SRM-MS. Relative to that of RelA^+/+ MEFs, very little RelA signal was detected (<0.1%) in RelA^−/− MEFs (Figure 3F, left panel). In RelA +/+ MEFs, phospho-Ser 276 RelA signal was detected and increased in response to TNF. Importantly, there was no signal of phospho-Ser 276 RelA detected in the RelA^−/− MEFs (Figure 3F, right panel). These data support the specificity of IP-SRM to measure phospho-Ser 276 RelA.

Earlier we reported that RelA Ser 276 phosphorylation is mediated by redundant ribosomal S6 kinases, including mitogen-stress related kinase-1 (MSK1) and catalytic subunit of active cAMP-dependent protein kinase A (PKAc) (5,38). Since PKAc is an ROS- sensitive kinase that mediates phospho-Ser 276 RelA formation, we investigated if PKAc activity is regulated by ATM. For this purpose, a PKAc kinase activity assay was performed in TNF stimulated ATM^+/+ and ATM^−/− MEFs. In ATM^+/+ MEFs, TNF induced a small, but significant increase in incorporation into the PKAc peptide at 0.25 and 0.5 h of stimulation (Figure 3G). When we examined the effect of ATM knockdown on MSK1 activation (phosphorylation at Ser 276), we did not observe any difference between wild type and ATM knockdown cells (data not shown). Together these data indicate that ATM is required for rapid induction of PKAc kinase activity in response to TNF treatment.

To investigate how ATM affects PKAc activity, we assessed whether ATM complexes with PKAc. PKAc was immunoprecipitated from control or TNF stimulated A549 cells and the immunoprecipitates were immunoblotted using anti-ATM Ab. In unstimulated cells, there was no detectable ATM. However, a strong ATM signal was observed 0.25 h after TNF stimulation peaking at 0.5 h (Figure 3H). p53, which is known to bind ATM upon VP-16 addition (33), serves as a positive control here. Overall, these data suggest that cytoplasmic ATM is essential for TNF-induced PKAc activation leading to RelA Ser 276 phosphorylation via a mechanism involving complex formation.

ATM is required for a NF-κB-dependent immediate-early cytokine gene expression

Previously, we have reported that ROS-inducible RelA phosphorylation on Ser 276 is a post-translationally modified state required for the transcription of a subset of rapidly inducible NF-κB-dependent genes including the cytokines IL-8 and Gro-β, but not IκBα (5). Since ATM selectively affects RelA Ser 276 phosphorylation, we asked whether ATM is required for expression of these immediate-early genes. For this purpose, A549 cells were pre-treated in the absence or presence of KU-55933, TNF stimulated and expression of NF-κB-dependent gene expression performed by q-RT-PCR. In the absence of KU-55933, TNF induced a 15-fold increase in Gro-β expression at 0.5 and 1 h. This induction was significantly decreased by KU-55933 pretreatment (Figure 4A). A time dependent increase in IL-8 expression from 10-fold in 0.25 h to 70-fold after 1 h of TNF exposure was significantly blocked in cells pretreated with KU-55933 (Figure 4A). By contrast, IκBα, a gene whose expression is phospho-Ser 276 RelA independent (34), was induced to similar magnitude in control or KU-55933 pre-treated cells (Figure 4A). A similar effect on Gro-β and IL-8 expression was observed in ATM-shRNA transfected A549 cells, with IκBα being unaffected (Figure 4B). An identical pattern was seen in HeLa cells transfected with ATM shRNA (Figure 4C).

Previous studies have shown that the physiological level of TNFα in human plasma is much lower than the levels used in our experimental design to produce maximum responses (39,40). Therefore we next tested the requirement for ATM on NF-κB-dependent gene expression over a broad TNF dose-response range. For this experiment, control shRNA or ATM shRNA- transfected A549 cells were stimulated by escalating TNF doses (0.3, 3, 30 ng/ml) and IL-8 gene expression measured by q-RT-PCR. We observed a very sharp induction of IL-8 expression between 3 and 30 ng/ml, consistent with the known steep dose response of TNF signaling. However, at all doses of TNF, IL-8 expression was reduced in ATM shRNA transfected A549 cells (Supplementary Figure S5). Therefore, we conclude that ATM is required for TNF induced NF-κB-dependent gene expression over a broad physiological concentration range.

Overall, these studies demonstrated that ATM is required for the expression of a subset of phospho-Ser 276 RelA immediate-early genes.

ATM is required for recruiting RelA and co-activators to immediate-early gene promoters

Earlier we have reported that phospho-Ser 276 RelA dependent gene expression is regulated via its interaction with cyclin-dependent kinase 9 (CDK9) resulting in RNA polymerase II (RNA Pol II) phosphorylation on Ser 2 of the heptad repeat in the C terminal domain (CTD), a biochemical event necessary for expression of Gro-β and IL-8 (but not IκBα) genes (34). We therefore investigated the role of ATM on targeting of CDK9 and RNA Pol II on IL-8, Gro-β and IκBα-containing chromatin by ChIP assay. For this purpose, A549 cells transfected with either control or ATM-specific shRNA were treated with TNF for the indicated times; chromatin was cross-linked and subjected to immunoprecitpitation with anti-RelA, CDK9 or phospho-Ser 2 Pol II Abs. Anti-rabbit IgG was used as a negative control. As expected, in control shRNA transfected A549 cells, TNF induced a 6-fold recruitment of RelA (after 0.5 h) and a 12-fold recruitment (after 1 h) on the Gro-β promoter. However, in cells transected with ATM-shRNA, RelA recruitment was significantly inhibited (less than 2-fold at these time points, Figure 5A). Similarly, TNF induction of CDK9 and phospho-Ser 2 RNA pol II recruitment was significantly induced in control shRNA transfectants and significantly inhibited in the ATM-shRNA transfectants (Figure 5A, middle and right panels).

A similar pattern of inhibition was observed for the IL-8 gene, whereas protein recruitment to the IκBα promoter was not affected (Figure 5B and C). These results provide a potential mechanism by which ATM-induced RelA posttranslational modifications regulate expression of a highly inducible subset of NF-κB-dependent genes.

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Supplementary Materials