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

Fang L, Choudhary S, Zhao Y, Edeh CB, Yang C, Boldogh I, Brasier AR - Nucleic Acids Res. (2014)

Bottom Line: In ATM knockdown cells, TNF-induced RelA Ser 276 phosphorylation is significantly decreased.We conclude that ATM is a nuclear damage-response signal modulator of TNF-induced NF-κB activation that plays a key scaffolding role in IκBα degradation and RelA Ser 276 phosphorylation.Our study provides a mechanistic explanation of decreased innate immune response associated with A-T mutation.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Texas Medical Branch (UTMB), 301 University Blvd, Galveston, TX 77555 USA Department of Biochemistry and Molecular Biology, UTMB, Galveston, TX 77555, USA.

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TNF-induced ATM activation and nuclear export. (A) A549 cells were treated with TNF (30 ng/ml) for the indicated time. Equal amounts of nuclear extract (NE) and cytoplasmic extract (CE) were analyzed by WBs to detect the level of ATM in both NE and CE. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (B) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of KU-55933 (10 μM, 1 h). Equal amounts of NE and CE were analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (C) Top panel. A549 cells were treated with TNF (30 ng/ml) or VP-16 (10 μM) for the indicated time and then assayed by Neutral Comet assay. 100 cells from each time interval were quantitated. Bottom panel. Representative images of comet moments. (D) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amounts of NE and CE were analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (E) A549 cells were treated with TNF (30 ng/ml) for 1 h with or without DMSO (2% in vol, 0.5 h) or NAC (15 mM, 1 h) pretreatment. Equal amounts of NE and CE were extracted and analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (F) IKKγ+/+ and IKKγ−/− MEFs were treated with TNF (30 ng/ml) for the indicated time. Equal amounts of CE were analyzed by WB to detect the level of ATM. β-tubulin were detected as internal control. (G) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amount of NE were immunoprecipitated by anti-IKKγ Ab. Interacting ATM were measured by WBs. (H) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amount of NE were immunoprecipitated by Ubiquitin Ab and subjected to SID-SRM-MS analysis of IKKγ protein level. All of the values are presented as the ratios of native to SIS peptides. (I) A549 cells were treated with TNF (30 ng/ml) for the indicated times, Equal amount of NE were immunoprecipitated by anti-IKKγ Ab and assayed by western blot using anti-IKKγ Ab. * Significantly different from TNF (0 h)-treated samples, P < 0.05;** Significantly different from TNF (0 h)-treated samples, P < 0.01; † Significantly different from ATM+/+ samples, P < 0.05; †† Significantly different from ATM+/+ samples, P < 0.01.
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Figure 1: TNF-induced ATM activation and nuclear export. (A) A549 cells were treated with TNF (30 ng/ml) for the indicated time. Equal amounts of nuclear extract (NE) and cytoplasmic extract (CE) were analyzed by WBs to detect the level of ATM in both NE and CE. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (B) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of KU-55933 (10 μM, 1 h). Equal amounts of NE and CE were analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (C) Top panel. A549 cells were treated with TNF (30 ng/ml) or VP-16 (10 μM) for the indicated time and then assayed by Neutral Comet assay. 100 cells from each time interval were quantitated. Bottom panel. Representative images of comet moments. (D) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amounts of NE and CE were analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (E) A549 cells were treated with TNF (30 ng/ml) for 1 h with or without DMSO (2% in vol, 0.5 h) or NAC (15 mM, 1 h) pretreatment. Equal amounts of NE and CE were extracted and analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (F) IKKγ+/+ and IKKγ−/− MEFs were treated with TNF (30 ng/ml) for the indicated time. Equal amounts of CE were analyzed by WB to detect the level of ATM. β-tubulin were detected as internal control. (G) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amount of NE were immunoprecipitated by anti-IKKγ Ab. Interacting ATM were measured by WBs. (H) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amount of NE were immunoprecipitated by Ubiquitin Ab and subjected to SID-SRM-MS analysis of IKKγ protein level. All of the values are presented as the ratios of native to SIS peptides. (I) A549 cells were treated with TNF (30 ng/ml) for the indicated times, Equal amount of NE were immunoprecipitated by anti-IKKγ Ab and assayed by western blot using anti-IKKγ Ab. * Significantly different from TNF (0 h)-treated samples, P < 0.05;** Significantly different from TNF (0 h)-treated samples, P < 0.01; † Significantly different from ATM+/+ samples, P < 0.05; †† Significantly different from ATM+/+ samples, P < 0.01.

Mentions: 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.


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

Fang L, Choudhary S, Zhao Y, Edeh CB, Yang C, Boldogh I, Brasier AR - Nucleic Acids Res. (2014)

TNF-induced ATM activation and nuclear export. (A) A549 cells were treated with TNF (30 ng/ml) for the indicated time. Equal amounts of nuclear extract (NE) and cytoplasmic extract (CE) were analyzed by WBs to detect the level of ATM in both NE and CE. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (B) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of KU-55933 (10 μM, 1 h). Equal amounts of NE and CE were analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (C) Top panel. A549 cells were treated with TNF (30 ng/ml) or VP-16 (10 μM) for the indicated time and then assayed by Neutral Comet assay. 100 cells from each time interval were quantitated. Bottom panel. Representative images of comet moments. (D) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amounts of NE and CE were analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (E) A549 cells were treated with TNF (30 ng/ml) for 1 h with or without DMSO (2% in vol, 0.5 h) or NAC (15 mM, 1 h) pretreatment. Equal amounts of NE and CE were extracted and analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (F) IKKγ+/+ and IKKγ−/− MEFs were treated with TNF (30 ng/ml) for the indicated time. Equal amounts of CE were analyzed by WB to detect the level of ATM. β-tubulin were detected as internal control. (G) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amount of NE were immunoprecipitated by anti-IKKγ Ab. Interacting ATM were measured by WBs. (H) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amount of NE were immunoprecipitated by Ubiquitin Ab and subjected to SID-SRM-MS analysis of IKKγ protein level. All of the values are presented as the ratios of native to SIS peptides. (I) A549 cells were treated with TNF (30 ng/ml) for the indicated times, Equal amount of NE were immunoprecipitated by anti-IKKγ Ab and assayed by western blot using anti-IKKγ Ab. * Significantly different from TNF (0 h)-treated samples, P < 0.05;** Significantly different from TNF (0 h)-treated samples, P < 0.01; † Significantly different from ATM+/+ samples, P < 0.05; †† Significantly different from ATM+/+ samples, P < 0.01.
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Figure 1: TNF-induced ATM activation and nuclear export. (A) A549 cells were treated with TNF (30 ng/ml) for the indicated time. Equal amounts of nuclear extract (NE) and cytoplasmic extract (CE) were analyzed by WBs to detect the level of ATM in both NE and CE. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (B) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of KU-55933 (10 μM, 1 h). Equal amounts of NE and CE were analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (C) Top panel. A549 cells were treated with TNF (30 ng/ml) or VP-16 (10 μM) for the indicated time and then assayed by Neutral Comet assay. 100 cells from each time interval were quantitated. Bottom panel. Representative images of comet moments. (D) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amounts of NE and CE were analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (E) A549 cells were treated with TNF (30 ng/ml) for 1 h with or without DMSO (2% in vol, 0.5 h) or NAC (15 mM, 1 h) pretreatment. Equal amounts of NE and CE were extracted and analyzed by WBs to detect the level of pATM and ATM, respectively. Lamin B and β-tubulin were also detected as internal control for NE and CE, respectively. (F) IKKγ+/+ and IKKγ−/− MEFs were treated with TNF (30 ng/ml) for the indicated time. Equal amounts of CE were analyzed by WB to detect the level of ATM. β-tubulin were detected as internal control. (G) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amount of NE were immunoprecipitated by anti-IKKγ Ab. Interacting ATM were measured by WBs. (H) A549 cells were treated with TNF (30 ng/ml) for the indicated time with or without the pretreatment of DMSO (2% in vol, 0.5 h). Equal amount of NE were immunoprecipitated by Ubiquitin Ab and subjected to SID-SRM-MS analysis of IKKγ protein level. All of the values are presented as the ratios of native to SIS peptides. (I) A549 cells were treated with TNF (30 ng/ml) for the indicated times, Equal amount of NE were immunoprecipitated by anti-IKKγ Ab and assayed by western blot using anti-IKKγ Ab. * Significantly different from TNF (0 h)-treated samples, P < 0.05;** Significantly different from TNF (0 h)-treated samples, P < 0.01; † Significantly different from ATM+/+ samples, P < 0.05; †† Significantly different from ATM+/+ samples, P < 0.01.
Mentions: 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.

Bottom Line: In ATM knockdown cells, TNF-induced RelA Ser 276 phosphorylation is significantly decreased.We conclude that ATM is a nuclear damage-response signal modulator of TNF-induced NF-κB activation that plays a key scaffolding role in IκBα degradation and RelA Ser 276 phosphorylation.Our study provides a mechanistic explanation of decreased innate immune response associated with A-T mutation.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Texas Medical Branch (UTMB), 301 University Blvd, Galveston, TX 77555 USA Department of Biochemistry and Molecular Biology, UTMB, Galveston, TX 77555, USA.

Show MeSH
Related in: MedlinePlus