Inactivation of BAD by IKK inhibits TNFα-induced apoptosis independently of NF-κB activation - PubMed (original) (raw)
Inactivation of BAD by IKK inhibits TNFα-induced apoptosis independently of NF-κB activation
Jie Yan et al. Cell. 2013.
Abstract
The IκB kinase complex (IKK) is a key regulator of immune responses, inflammation, cell survival, and tumorigenesis. The prosurvival function of IKK centers on activation of the transcription factor NF-κB, whose target gene products inhibit caspases and prevent prolonged JNK activation. Here, we report that inactivation of the BH3-only protein BAD by IKK independently of NF-κB activation suppresses TNFα-induced apoptosis. TNFα-treated Ikkβ(-/-) mouse embryonic fibroblasts (MEFs) undergo apoptosis significantly faster than MEFs deficient in both RelA and cRel due to lack of inhibition of BAD by IKK. IKK phosphorylates BAD at serine-26 (Ser26) and primes it for inactivation. Elimination of Ser26 phosphorylation promotes BAD proapoptotic activity, thereby accelerating TNFα-induced apoptosis in cultured cells and increasing mortality in animals. Our results reveal that IKK inhibits TNFα-induced apoptosis through two distinct but cooperative mechanisms: activation of the survival factor NF-κB and inactivation of the proapoptotic BH3-only BAD protein.
Copyright © 2013 Elsevier Inc. All rights reserved.
Figures
Figure 1
IKK Is Able to Inhibit TNFα-induced Apoptosis through NF-κB-independent Mechanism. (A, B, and C) WT, _Ikk_β−/−, _Rel_A−/− fibroblasts were transfected with siRNA against cRel (sicRel) or control siRNA (siCtrl) as indicated for 24 hr, followed by stimulation with or without TNFα (5 ng/ml). Cleavage of Casp-3 substrate PARP and expression of RelA, cRel, and IKKβ were analyzed by immunoblotting (A), measurement of Casp-3 activity (B), and apoptotic cells, which were identified by Annexin V and PI staining followed by flow cytometric analysis (C). (D, E, and F) _Rel_A−/− MEFs were transfected with sicRel, along with siIKKβ or siCtrl for 24 hr, followed by stimulation with or without TNFα (5 ng/ml). PARP cleavage and expression of IKKβ and cRel were analyzed (D). Casp-3 activity (E) and apoptotic cell death (F) were measured as described in (B) and (C), respectively. (G, H, and I) _Ikk_β−/− MEFs were transfected with siRelA plus sicRel, or siCtrl for 24 hr, followed by stimulation with or without TNFα (5 ng/ml). PARP cleavage and expression of RelA and cRel were analyzed (G). Casp-3 activity (H) and apoptotic cell death (I) were measured as in (B) and (C), respectively. The results in (B), (C), (E), (F), (H), and (I) are presented as means ± standard error and represent three individual experiments. *, p<0.05, **, p<0.01, as analyzed by the Student t test. See also Figure S1.
Figure 2
IKK But not NF-κB Suppresses BAD Pro-apoptotic Activity Upon TNFα Stimulation (A, B, and C) _Ikk_β−/− or _Rel_A−/− MEFs were transfected with siBad, sicRel, or siCtrl as indicated for 24 hr, followed by stimulation with or without TNFα (5 ng/ml). PARP cleavage and expression levels of RelA, cRel, BAD, BCL-XL, and β-actin (A and B), as well as Casp-3 activity (C) were determined. (D) WT and _Ba_d−/− MEFs were pre-treated with or without the special IKKβ inhibitor PS-1145 (10 μM) for 2 hr, followed by stimulation with or without TNFα (5 ng/ml). Cleavage of pro-Casp-3 and PARP, and expression levels of BAD, IκBα, and β-actin were determined. The data in (A–D) represent 2–3 individual experiments with similar results. (E and F) WT and _Ba_d−/− mice were sensitized with D-GalN and then treated with TNFα (see “Experimental Procedure” for details). Dying animals were pre-moved and the livers were extracted for H&E staining and Tunnel staining (E). Mortality rate was determined, p<0.05; n=5 (F), as analyzed by the log-rank (Mantel-Cox) test. See also Figure S2.
Figure 3
IKK Is a BAD Kinase. (A) WT MEFs were stimulated with or without TNFα (5 ng/ml). IKK activity was determined by immune complex kinase assays with purified GST-IκBα (5 μg) or GST-BAD (5 μg) as substrate. CBB, Coomassie brilliant blue staining. (B) WT, _Ikk_β−/−, and _Ikk_β+ MEFs, in which _Ikk_β−/− MEFs were infected with adenoviral vector-encoding HA-IKKβ (300 MOI; 24 hr), were stimulated with or without TNFα (5 ng/ml) for 10 min. IKK activity was determined as in (A). (C) In vitro phosphorylation of purified GST-IκBα, GST-BAD, and GST-c-Jun (5 μg each) by constitutively active IKKβ(EE) (24 ng), in which Ser177 and Ser181 were replaced by glutamic acids (EE) and has been purified to near homogeneity. (D) Phosphorylated GST-BAD proteins were subjected to two-dimensional tryptic phosphopeptide mapping, as described (Lin et al., 1992). Spot b is the major tryptic peptide phosphorylated by basal IKK; spots a and c represent the peptides whose phosphorylation was augmented by active IKK or IKKβ(EE). (E) Phosphorylated BAD or the phosphopeptide a was subjected to phosphoamino acid analysis, as described (Lin et al., 1992). PS, phosphoserine. The data in (A–C) represent 2–3 individual experiments with similar results.
Figure 4
IKK Is Necessary and Sufficient to Phosphorylate BAD at Ser26 In Vitro and In Vivo. (A and B) Phosphorylation of GST-BAD and GST-BAD(S26A) mutant proteins by active IKK (A) or purified IKKβ(EE) proteins (B), as described in Figure 3B. (C) Two-dimensional phosphopeptide mapping of active IKK-phosphorylated GST-BAD and GST-BAD(S26A) mutant proteins, as described in (3D). (D) GST-BAD proteins phosphorylated by active IKK in vitro were tryptic digested and then analyzed by mass spectrometry. Insert, the recovered phosphorylated peptide fragment corresponding to S26. (E) GST-BAD and GST-BAD(S26A) proteins were phosphorylated by active IKK in the presence of non-radioactive ATP (17 μM) and analyzed by immunoblotting using anti-phospho-Ser26 antibody. (F) WT and _Ikk_β−/− MEFs were stimulated with or without TNFα (5 ng/ml). Ser26-phosphorylation of BAD and expression levels of BAD, IKKβ, IκBα, and β-actin were determined. (G) _Ikk_β−/− MEFs were transfected with WT HA-IKK, constitutive active HA-IKKβ(EE), or empty vector (1 μg each), followed by stimulation with or without TNFα (5 ng/ml, 10 min). Ser26-phosphorylation of BAD, and expression levels of HA-IKKβ, HA-IKKβ(EE), and IκBα were determined. The data in (A, B, E, F, and G) represent 2–3 individual experiments with similar results. See also Figure S3.
Figure 5
Phosphorylation of BAD by IKKβ Inhibits Its Pro-apoptotic Activity. (A) WT and _Ikk_β−/− MEFs were treated with or without TNFα (5 ng/ml) and then separated into cytosol and mitochondrial fractions. Subcellular localization of BAD, Ser26-phosphorylated BAD, 14–3–3, BCL-XL, and IKKβ was analyzed by immunoblotting. β-actin and COX-II were used as cytosol and mitochondrial markers, respectively. The amount of BAD in cytosol and mitochondrial fractions in the same numbers of _Ikk_β−/− MEFs were quantitated by the Image J Program. (B and C) WT and _Ikk_β−/− MEFs were treated with or without TNFα (5 ng/ml) and fractionated, as described in (A). 14–3–3- and BCL-XL-associated BAD or Ser26-phosphorylated BAD were analyzed by immunoprecipitation in combination with immunoblotting. BAD Ser26- phosphorylation, expression levels of BAD, 14–3–3, BCL-XL, β-actin, and COX-II were determined. (D and E) WT, _RelA_−/−, and _Ikk_β−/− MEFs were stimulated with or without TNFα (5 ng/ml, 1 hr) and then separated into cytosol (D) and mitochondria (E) fractions. 14–3–3- and BCL- XL-associated BAD or Ser26-phosphorylated BAD, BAD Ser26-phosphorylation, expression levels of BAD, 14–3–3, BCL-XL, and COX-II were determined, as described in (B and C). All data represent 2–3 individual experiments with similar results. See also Figure S4.
Figure 6
IKK Primes BAD Phosphorylation at the “Regulatory Serines”. (A) WT and _Ikk_β−/− MEFs were treated with or without TNFα (5 ng/ml). Phosphorylation of BAD at various serines (Ser26, Ser112, Ser136, and Ser155) and expression levels of BAD, IκBα, and β-actin were analyzed by immunoblotting. (B, C, and D) _Ba_d−/− MEFs stably expressing were HA-BCL-XL transfected with WT M2-Bad [Bad(WT)+] or M2-Bad(3SA) mutant, in which Ser112, Ser136, and Ser155 have been replaced by analines [Bad(3SA)+], and then infected with adenoviral vector encoding HA-IκBα(AA), in which Ser32 and Ser36 were replaced by alanines, for 24 hr. Cells were treated with or without TNFα (5 ng/ml) and either directly harvested (B) or further separated into cytosol and mitochondria fractions (C). Phosphorylation of Ser26 and the “regulatory serines”, and expression levels of M2-BAD, HA-IκBα(AA), HA-BCL-XL, and β-actin were determined (B and C). At each time point, the sum of cytoplasmic and mitochondrial Ser26-phosphorylated BAD(3SA) was calculated as 100% (C). Apoptotic cell death was determined (D), as described in (1C). The data in (A–D) represent 2–3 individual experiments with similar results. (E) WT and _Ba_d3SA/3SA knockin mice were sensitized by D-GalN and then injected intraperitoneally with TNFα, as described in Figure 2E and 2F. Dying animals were pre-moved and mortality rate was determined, p<0.001; n=6 (F), as analyzed by log-rank (Mantel-Cox) test. See also Figure S5.
Figure 7
Elimination of Ser26-phosphorylation Promotes the Pro-apoptotic Activity of BAD in Vitro and in Vivo. _Ba_d−/− MEFs stably expressing WT M2-Bad [Bad(WT)+] or M2-BAD(S26A) mutant [Bad(S26A)+] along with HA-BCL-XL were established, as described in “Experimental Procedure”. (A, B, C, and D) Bad(WT)+ and Bad(S26A)+ MEFs were infected with adenoviral vector encoding HA-IκBα(AA) for 24 hr, and then treated with or without TNFα (5 ng/ml), as indicated. PARP cleavage, expression of M2-BAD, HA-BCL-XL, HA-IκBα(AA), β-actin, and Ser26-phosphorylation of M2-BAD were analyzed (A). Apoptotic cell death was analyzed by Annexin V/PI staining, followed by flow cytometric analysis (B) and Casp-3 activity was determined (C), as described in Figure 1C. Phosphorylation of M2-BAD at various serine residues (Ser26, Ser112, Ser136, and Ser155), and expression levels of M2-BAD, HA-BCL-XL, HAIκBα(AA), and β-actin were analyzed by immunoblotting (D). The data in (A–D) represent 2–3 individual experiments with similar results. (E) _Ba_d−/− mice were injected intravenously with Ad/WT Bad, Ad/Bad(3SA) mutant, or Ad/Ctrl (see “Experimental Procedures” for details) and then challenged with TNFα and D-GalN, as described in Figure 2E and 2F. Mortality rate was determined, p<0.001; n=10 (E), as analyzed by log-rank (Mantel-Cox) test. (F) A schematic presentation of the mechanism by which IKKinhibits TNF α-induced apoptosis through activation of NF-κB and inhibition of BAD. See also Figure S6.
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