Transcriptional activation of the NF-kappaB p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1) - PubMed (original) (raw)
Transcriptional activation of the NF-kappaB p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1)
Linda Vermeulen et al. EMBO J. 2003.
Abstract
Nuclear factor kappaB (NF-kappaB) is one of the key regulators of transcription of a variety of genes involved in immune and inflammatory responses. NF-kappaB activity has long been thought to be regulated mainly by IkappaB family members, which keep the transcription factor complex in an inactive form in the cytoplasm by masking the nuclear localization signal. Nowadays, the importance of additional mechanisms controlling the nuclear transcription potential of NF-kappaB is generally accepted. We show that the mitogen-activated protein kinase inhibitors SB203580 and PD98059 or U0126, as well as a potent mitogen- and stress- activated protein kinase-1 (MSK1) inhibitor H89, counteract tumor necrosis factor (TNF)-mediated stimulation of p65 transactivation capacity. Mutational analysis of p65 revealed Ser276 as a target for phosphorylation and transactivation in response to TNF. Moreover, we identified MSK1 as a nuclear kinase for p65, since MSK1 associates with p65 in a stimulus-dependent way and phosphorylates p65 at Ser276. This effect represents, together with phosphorylation of nucleosome components such as histone H3, an essential step leading to selective transcriptional activation of NF-kappaB-dependent gene expression.
Figures
Fig. 1. Regulation of NF-κB-dependent gene expression. (A) L929sA cells were left untreated or were treated with 2000 IU/ml TNF, either in combination with 10 µM H89 or following a 2 h pre-treatment with 10 µM SB203559 and 10 µM U0126, and mRNA was isolated and subjected to northern blot analysis. (B) IL-6 production in various cell lines was tested after 6 h treatment with 2000 IU/ml TNF, pre-treated or not with 10 µM SB203580 and 10 µM U0126. (C) L929sA cells were stably transfected with reporter plasmids for either the E-selectin, the IL-8 or the IL-6 promoter, and pPGKβgeobpA. Transfectants were left untreated or were treated with 2000 IU/ml TNF for 6 h, either in combination with 10 µM H89 or following a 2 h pre-treatment with 10 µM SB203580 and 10 µM PD98059, with 20 µM LY294002 or with 100 nM wortmannin. The induction factor is defined as the amount of luciferase produced in TNF-treated cells after normalization for β-galactosidase expression, compared with the non-induced state. (D) Similar experiments were performed with L929sA cells, stably transfected with synthetic CREB-, AP1- or NF-κB-dependent reporter constructs.
Fig. 2. Effect of kinase inhibitors on TNF-induced NF-κB binding, p65 phosphorylation and transactivation. (A) L929sA cells were left untreated or were treated with 2000 IU/ml TNF for 30 min, either in combination with 10 µM H89 and/or following a 2 h pre-treatment with 10 µM SB203580 and/or 10 µM PD98059. Total cell lysates were incubated with a 32P-labeled IL-6 κB site-containing probe. Complexes formed were analyzed by EMSA. Loading of equal amounts of protein was verified by comparison with the binding activity of the repressor molecule RBP-Jκ (Plaisance et al., 1997). (B) Pools of L929sA cells stably expressing the Gal4 DNA-binding domain, Gal4-VP16, Gal4-p65 or serine-mutated variants thereof were transiently transfected with p(gal4)2hu.IL6-luc+. At 48 h post-transfection, cells were left untreated or were treated with 2000 IU/ml TNF for 6 h. The pools stably expressing Gal4-p65 were also co-treated with 10 µM H89 or pre-treated for 2 h with 10 µM SB203580 and 10 µM PD98059. (C) [32P]orthophosphate-labeled L929sA cells were stimulated with 5000 IU/ml TNF for 15 min in the absence or presence of 10 µM H89 at 2 h before TNF. Total cell lysates were immunoprecipitated with an anti-p65 antibody, analyzed by 10% SDS–PAGE and visualized using PhosphorImager technology. (D) L929sA mouse fibroblasts were starved for 24 h in 0.5% serum. Quiescent cells were treated for 20 min with 2500 IU/ml TNF alone or following a 2 h pre-treatment with 10 µM H89. Top panels, DAPI-stained nuclei; bottom panels, corresponding signals obtained with anti-phospho Ser276 p65 antibody.
Fig. 3. Ser276 is a crucial residue for TNF-mediated transactivation of p65. HEK293 cells were transiently transfected with a combination of expression plasmids, i.e 180 ng of p1168hu.IL6-luc+, 20 ng of pPGKβgeobpA, 2 ng of pRcRSVp65 (or the respective mutant versions) and 100 ng of p300 expression plasmid (pCIp300 or pCIp300ΔHAT). The total amount of DNA was kept constant in all set ups by supplementing empty vector DNA. Cells were lysed 42 h after transfection. Luciferase levels were determined and corrected for transfection efficiency by normalization to β-galactosidase levels. In addition, HEK293 cells were transfected with an increasing amount of pRcRSVp65 or pRcRSVp65 S576C (30–900 ng in a total amount of 12 µg per medium Petri dish). Cytoplasmic (c) and nuclear (n) protein extracts were determined for p65 expression by western blot analysis.
Fig. 4. MSK1 is activated by TNF in vivo and phosphorylates Ser276 of p65 in vitro. (A) L929sA cells were starved for 48 h in serum-free medium and stimulated with 2000 IU/ml TNF. Cells were lysed and endogenous MSK1 was isolated by immunoprecipitation. The activity of MSK1 was assessed by an in vitro kinase assay. (B) After 2 days of serum starvation, L929sA cells were incubated for 4 h in serum-free medium supplemented with 10 µM SB203580, 10 µM PD98059 or a combination. Cells were treated with 2000 IU/ml TNF for 15 min in the presence or absence of these inhibitors. After cell lysis, MSK1 was immunoprecipitated and assayed for its ability to phosphorylate CREBtide. Where indicated, H89 was included in the in vitro reaction. (C) L929sA cells were treated with 2000 IU/ml TNF. The presence of p65 in the nuclear extracts was revealed by western blotting. (D) MSK1 was isolated from HEK293 cells overexpressing either wt MSK1 or a kinase-dead mutant, together with the upstream activators p38 and MKK6. Immunoprecipitates were used in an in vitro kinase reaction with either wt GSTp6512–317 or the corresponding Ser276 mutant (S276C) for 20 min at 30°C, followed by SDS–PAGE. Similar experiments were performed with 15 ng of purified PKAc.
Fig. 5. MSK1 phosphorylates p65 in vivo. (A) HEK293 cells, transiently transfected with the relevant expression vectors, were labeled with [32P]orthophosphate for 4 h. Cell lysates were subjected to immunoprecipitation with anti-p65 antibody. Precipitated proteins were separated by SDS–PAGE and analyzed using PhosphorImager technology. (B) Similar experiment to that in (A), but immunoprecipitation was performed with anti-Flag antibody.
Fig. 6. p65 interacts with activated MSK1 in vivo. (A) HEK293 cells were transiently transfected using expression vectors for p65, Flag-tagged wt MSK1 or C-terminal kinase-dead MSK1 (MSK1-CKD). Activation of MSK1 was achieved by co-transfecting MKK6 and p38 kinase. At 48 h post-transfection, Flag-MSK1 was isolated by immunoprecipitation. Co-precipitating p65 was detected by immunoblotting using an anti-p65 antibody (a). To monitor the activation status of MSK1, blots were stripped and reprobed using an anti-Flag antibody (b). (B) L929sA cells were pre-treated with 10 µM H89 for 2 h, either followed or not by induction with 2000 IU/ml TNF for 15 min. Cytoplasmic and nuclear cell lysates were prepared and subjected to immunoprecipitation with an MSK1 antibody. Co-precipitating p65 was detected by western analysis using an anti-p65 antibody.
Fig. 7. MSK1 and its phosphorylated substrates position at the endogenous IL-6 promoter upon TNF treatment. L929sA mouse fibroblasts were starved for 24–48 h in 0.5% serum. Quiescent cells were treated for 30 min with 2500 IU/ml TNF alone, or following 2 h pre-treatment with the inhibitors SB203580 + PD98059 (10 µM) or H89 (10 µM). ChIP analysis was performed against MSK1, or against phospho NF-κB p65 (Ser276) (A and C) or phospho H3 (Ser10) (C). After reversal of cross-linking, co-immunoprecipitated genomic DNA fragments were analyzed by quantitative PCR for 27 cycles with IL-6 or H4 promoter-specific primer sets. Input reflects the relative amounts of sonicated DNA fragments present before immunoprecipitation and revealed by quantitative PCR with either IL-6- or H4-specific primers. (B) Schematic representation of the results obtained in (A).
Fig. 8. Characterization of MSK1–/–/MSK2–/– MEFs. MSK1–/–/MSK2–/– and wt cells were treated with 2000 IU/ml TNF for the indicated times. Nuclear and total cell extracts were prepared to visualize nuclear import of p65 and IκBα degradation, respectively (A) and NF-κB DNA-binding capacity (B). (C) MSK1/MSK2 double knockout and wt MEFs were transiently transfected with Gal4-p65 expression vector together with p(gal4)2hu.IL6-luc+. At 48 h post-transfection, cells were left untreated or were treated with 2000 IU/ml TNF for 6 h. Corresponding cell lysates were analyzed for luciferase activity and normalized for transfection efficiency by quantifying β-galactosidase levels expressed upon co-transfection of pPGKβgeobpA. (D) Confocal microscope images of TNF-induced wt or MSK1–/–/MSK2–/– MEFs showing Ser276-phosphorylated p65 (green) and PI-stained (red) nuclei. (E) L929sA, wt and MSK1–/–/MSK2–/– MEFs were left untreated or were treated for 4 h with 2000 IU/ml TNF, either pre-treated or not with 10 µM SB203580 and 10 µM U0126 for 2 h. Total RNA was isolated and analyzed using GEArray technology according to the manufacturer’s instructions. Specific mRNA expression was normalized for loading differences.
Fig. 9. Overview of the phosphorylation pathways leading to activation of the IL-6 enhanceosome. Ac, acetyl; AP-1, activator protein-1; BTM, basal transcription machinery; C/EBP, CCAAT enhancer-binding protein; CREB, cAMP-responsive element binding protein; p/CAF, p300/CBP-associated factor; Pol, polymerase; SRC, steroid receptor activator.
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