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Am J Physiol Cell Physiol 291: C1336-C1345, 2006. First published July 5, 2006; doi:10.1152/ajpcell.00552.2005
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Ribosomal S6 kinase-1 modulates interleukin-1beta-induced persistent activation of NF-{kappa}B through phosphorylation of I{kappa}Bbeta

Shanqin Xu, Hossein Bayat, Xiuyun Hou, and Bingbing Jiang

Vascular Biology Unit, Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts

Submitted 28 October 2005 ; accepted in final form 3 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of NF-{kappa}B requires the phosphorylation and degradation of its associated inhibitory proteins, I{kappa}B. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1beta to induce persistent activation of NF-{kappa}B in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-{kappa}B activation. In cultured rat VSMCs, IL-1beta activated ERK and induced degradation of both I{kappa}B{alpha} and I{kappa}Bbeta, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-{kappa}B p65. RSK1, a downstream kinase of ERK, was associated with an I{kappa}Bbeta/NF-{kappa}B complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1beta decreased I{kappa}Bbeta in the RSK1/I{kappa}Bbeta/NF-{kappa}B complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1beta-induced I{kappa}Bbeta decrease without influencing ether ERK phosphorylation or the earlier I{kappa}B{alpha} degradation. By using recombinant wild-type and mutant I{kappa}Bbeta proteins, both active ERK2 and RSK1 were found to directly phosphorylate I{kappa}Bbeta, but only active RSK1 phosphorylated I{kappa}Bbeta on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates I{kappa}Bbeta and contributes to the persistent activation of NF-{kappa}B by IL-1beta.

extracellular signal-regulated kinase; in vitro phosphorylation assay; recombinant proteins; small interference RNA; vascular smooth muscle cell

NUCLEAR FACTOR (NF)-{kappa}B is an important transcription factor in regulating the expression of numerous inducible genes involved in inflammation, cell differentiation, proliferation, and apoptosis (1, 2, 4). Activation of NF-{kappa}B can occur transiently or persistently, which may result in different gene expression patterns. In the vessel wall, NF-{kappa}B is not ordinarily activated, but activation has been detected in human atherosclerotic lesions and animal arteries after injury (3, 18, 19, 23, 25). Cytokines, growth factors, and angiotensin II, which either promote or retard the development of vascular lesions, have been shown to regulate the expression of NF-{kappa}B-dependent genes in part by regulating the duration of NF-{kappa}B activation (13, 15).

In most cells without inflammatory stimulation, NF-{kappa}B is sequestered in the cytoplasm by inhibitory proteins I{kappa}B ({alpha}, beta, etc.). Stimulation of cells with cytokines such as interleukin (IL)-1beta and tumor necrosis factor (TNF)-{alpha} leads to the phosphorylation and ubiquitination-dependent proteasomal degradation of I{kappa}B, accompanied by NF-{kappa}B translocation to the nucleus, where it binds to consensus sequences in the promoter region of target genes and activates gene transcription (1, 2, 4, 5, 8, 20, 22). I{kappa}B{alpha} and I{kappa}Bbeta have different kinetics for degradation and synthesis in response to cytokine stimulation. I{kappa}B{alpha} is degraded rapidly after cytokine stimulation and is then rapidly resynthesized because of the presence of NF-{kappa}B-binding motifs in the promoter of the I{kappa}B{alpha} gene (17). This autoregulatory loop limits NF-{kappa}B activation in a transient manner (17, 27). I{kappa}Bbeta is slowly but persistently decreased after cytokine stimulation, a process that contributes to persistent NF-{kappa}B activation (27). Two I{kappa}B kinases, IKK{alpha} and IKKbeta (also known as IKK1 and IKK2), are capable of phosphorylating I{kappa}B and inducing NF-{kappa}B activation (16, 21, 30). Both IKK{alpha} and IKKbeta phosphorylate Ser32 and Ser36 of I{kappa}B{alpha} but phosphorylate I{kappa}Bbeta weakly (24, 30). The mechanism causing the phosphorylation and degradation of I{kappa}Bbeta and the subsequent persistent activation of NF-{kappa}B is not fully understood.

Extracellular signal-regulated kinase (ERK) regulates several NF-{kappa}B-dependent events, including inducible nitric oxide synthase (iNOS) expression and human immunodeficiency virus-1 long terminal repeat expression (9, 10, 14, 29). In previous studies using cultured rat aortic vascular smooth muscle cells (VSMCs), we noted that inhibition of ERK activation by selective inhibitors of ERK kinases (MEK) attenuated the prolonged activation of NF-{kappa}B without influencing the early (transient) activation of NF-{kappa}B induced by IL-1beta (11, 13, 14). Inhibition of ERK activation did not affect IL-1beta-induced I{kappa}B{alpha} degradation but attenuated IL-1beta-induced I{kappa}Bbeta degradation, suggesting that an I{kappa}Bbeta-mediated mechanism is involved in the ERK-dependent persistent activation of NF-{kappa}B (11, 14). This observation provided an important clue for the signaling mechanism that controls the duration of NF-{kappa}B activation. Ribosomal S6 kinase (RSK)1 is a downstream kinase of ERK that is able to phosphorylate I{kappa}B{alpha} on Ser32 (7). It is unknown whether RSK1 could also phosphorylate I{kappa}Bbeta and eventually contribute to the persistent activation of NF-{kappa}B. In the present study, we have demonstrated that RSK1, but not ERK, directly associates with the I{kappa}Bbeta/NF-{kappa}B complex and targets I{kappa}Bbeta for degradation by phosphorylation on Ser19 and Ser23. This finding suggests that in the ERK signaling cascade, RSK1 is a key component that directly contributes to the regulation of I{kappa}Bbeta phosphorylation and degradation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. DMEM/Ham's F-12 medium (DMEM/F-12) and FCS were purchased from Life Technologies. Recombinant human IL-1beta (specific activity 1.9 x 107 U/mg) was kindly provided by Dr. Aurigemma (Biological Resources Branch Preclinical Repository, National Cancer Institute). Recombinant human platelet-derived growth factor (PDGF, BB homodimer), PD-98059, and U0126 were from Calbiochem. Activated recombinant IKKbeta and RSK1 were obtained from Upstate. Active and inactive ERK2, polyclonal antibodies against phospho-p90RSK (Ser380 and Thr359/Ser363), p44/42 MAPK (ERK1/2), and phospho-I{kappa}Bbeta (Ser19/23), as well as monoclonal antibodies against phospho-p44/42 MAPK (Thr202/Tyr204) and phospho-I{kappa}B{alpha} (Ser32/36), were obtained from Cell Signaling Technologies. Monoclonal antibody against RSK was obtained from Transduction Laboratories. NF-{kappa}B consensus oligonucleotide agarose conjugate, recombinant I{kappa}B{alpha}, and polyclonal antibodies against RSK1, I{kappa}Bbeta, NF-{kappa}B p50, and NF-{kappa}B p65 were obtained from Santa Cruz Biotechnology. NF-{kappa}B consensus oligonucleotide was obtained from Promega. [{gamma}-32P]ATP was obtained from DuPont-New England Nuclear.

Cell culture. VSMCs, isolated from the thoracic aorta of 8-wk-old male Wistar rats (10), were cultured as described previously. Cells were used between passages 5 and 9. Unless specified, the cells at confluence were washed with serum-free medium and then maintained in DMEM/F-12 with 0.1% FCS for 24–48 h. The medium was refreshed before treatment. The cells were then incubated with or without additions (cytokines, inhibitors, or vehicle) for designated times as indicated.

Transfection of small interference RNA. Small interference RNA (siRNA)-Rsk1 (Rn_Rps6ka1_2_HP siRNA, targeting rat Rps6ka1, gene accession no. NM_031107; target sequence AAG GCC TTT CTA ATA AAC CTA) and nonsilencing control siRNA (siRNA-Ctl) were obtained from Qiagen. Rat VSMCs were cultured in DMEM/F-12 with 10% FCS (no antibiotics) in six-well plates for 1 day (reaching ~70% confluence) and then transfected with 240 pmol of siRNA (an optimized amount) mixed with 12 µl of HiPerFect transfection reagent (Qiagen) in 0.6 ml of serum-free DMEM/F-12 per well. After 3 h of transfection, 2.4 ml of DMEM/F-12 with 0.1% FCS was added. The medium was changed next day with 3 ml of DMEM/F-12 with 0.1% FCS and was further cultured for 48 h before IL-1beta treatment. The transfection under these conditions did not cause observable changes in cell shape and adherence.

Immunoprecipitation and Western blot analysis. To immunoprecipitate active RSK1 from cell lysates, we treated the rat VSMCs with PDGF (10 ng/ml) for 10 min before harvesting the cell lysates with lysis buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerolphosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM PMSF, and 1% Triton X-100. The cell lysates (1 mg of cell protein) were incubated with 8 µg of anti-RSK1 (C-21) for 3 h at 4°C, followed by addition of 80 µl of protein A beads and rotation for 2 h at 4°C. After being washed five times with TBS-T (20 mM Tris·HCl, pH 7.6, 0.8% NaCl, and 0.05% Tween 20) and twice with a kinase buffer (25 mM Tris, pH 7.5, 5 mM beta-glycerolphosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2), the beads were suspended in the kinase buffer and in aliquots of 20 µl each (equal to starting from ~100 µg cell lysate proteins). The immunoprecipitates were directly used for further phosphorylation assay. To determine the association of RSK1 with I{kappa}Bbeta/NF-{kappa}B complex, we performed immunoprecipitation with either monoclonal antibody against RSK or polyclonal antibody against NF-{kappa}B p65. To exclude the possible nonspecific immunoprecipitation, in some experiments, we used the same amounts of normal mouse or rabbit IgG for negative controls as indicated. The precipitates were washed six times with TBS-T and then dissociated from the protein A beads by adding 2x SDS sample buffer and heating in boiling water for 5 min. Western blot analyses were performed as described previously (12). When cell lysates were used, protein content of the cell lysates was determined with BCA protein assay reagent (Pierce), with BSA used as a standard. The images were obtained and analyzed using a model GS-700 Imaging Densitometer (Bio-Rad).

Preparation of I{kappa}Bbeta/NF-{kappa}B complex. I{kappa}Bbeta/NF-{kappa}B complex was prepared from whole cell lysates with an NF-{kappa}B consensus oligonucleotide agarose conjugate. Rat VSMCs were either untreated or treated with IL-1beta for 6 h. Whole cell lysates (100 µg of proteins) were incubated with the oligonucleotide agarose conjugate in 1/3x lysis buffer containing 1 mM DTT and 2 µg/ml of poly(dIdC) at room temperature for 2 h. After being washed three times with 1/3x lysis buffer (1 ml each), the I{kappa}Bbeta/NF-{kappa}B complex was eluted from the oligonucleotide agarose conjugate with 200 µl of 1x lysis buffer. The complex was further used for Western blot analysis and phosphorylation assay.

Recombinant I{kappa}Bbeta. Full-length cDNA encoding wild-type rat I{kappa}Bbeta was generated by RT-PCR with forward primer 5'-AAG CTT GGA TCC GAG GTG GCA GGG GCA ATG-3' and reverse primer 5'-CTC GAG AGA TCT TCA GGC AGG GTT GGG GTC-3', containing start and stop codes (underlined), respectively. After the sequence was verified, the cDNA was cut with BamHI and BglII and inserted into the cloning sites of pTrcHis A vector (Invitrogen). Point mutations with the pTrcHis-I{kappa}Bbeta for Ser19Ala, Ser23Ala, and Ser19/23Ala mutants were generated by PCR with the QuikChange site-directed mutagenesis kit (Stratagene), and the sequences were verified. The primers (shown are sense sequences with underlined AG->GC and T->G changes) were 5'-C GAT GAA TGG TGC GAC GCT GGC CTG GGC TC-3' for Ser19Ala mutation, 5'-C AGT GCC CTG GGC GCT CTA GGT CCC GAC GC-3' for Ser23Ala mutation, and 5'-C GAT GAA TGG TGC GAC GCT GGC CTG GGC GCT CTA GGT CCC GAC G-3' for Ser19/23Ala double mutation. The plasmids were transformed into Escherichia coli TOP-10 cells. The TOP-10 cells were grown in SOB (super optimal broth) medium containing ampicillin to an optical density at 600 nm of 0.6. Isopropyl-beta-D-thiogalactopyranoside was added to a final concentration of 1 mM to induce expression of wild-type and mutant I{kappa}Bbeta proteins. The His-I{kappa}Bbeta proteins were purified from the cell lysates by ProBond columns (Invitrogen) and confirmed by 10% SDS-PAGE, followed by silver staining and Western blot analysis with an I{kappa}Bbeta antibody.

In vitro phosphorylation assay. To determine whether ERK or RSK1 would phosphorylate I{kappa}Bbeta, we used either recombinant ERK2 and RSK1 or RSK1 immunoprecipitated from cell lysates in an in vitro phosphorylation assay, which was performed in the kinase buffer containing 100 µM ATP and 5 µCi [{gamma}32P]ATP in a final 50-µl total volume at 30°C for 30 min. When recombinant kinases were used, the reaction was started by adding the kinases and I{kappa}Bbeta/NF-{kappa}B complex (eluted from the oligonucleotide agarose conjugate) or purified I{kappa}Bbeta, stopped by adding 25 µl of 3x SDS-sample buffer and heating in boiling water for 5 min, and then subjected to 10% SDS-PAGE. When active RSK1 from cell lysates was used, the phosphorylation reaction was performed by incubation of the RSK1 precipitates with recombinant I{kappa}Bbeta in the above kinase buffer at 30°C for 30 min. After centrifugation at 13,000 g at 4°C for 30 s, the supernatant (without RSK1) was mixed with 25 µl 3x SDS-sample buffer, and the beads (with RSK1) were mixed with 60 µl 1x SDS-sample buffer, heated in boiling water for 5 min, and subjected to 10% SDS-PAGE. The proteins were then transferred to nitrocellulose membranes. The membranes were subjected to autoradiography to detect 32P-labeling of I{kappa}Bbeta, followed by Western blot analysis of I{kappa}Bbeta and RSK1, accordingly, to confirm equal loading of the proteins. To determine the ability of recombinant kinases to phosphorylate Ser19/23 of I{kappa}Bbeta or Ser32/36 of I{kappa}B{alpha}, we also performed the reaction in the above kinase buffer with ATP and then subjected the membranes to Western blot analysis with phospho-I{kappa}Bbeta (Ser19/23) or phospho-I{kappa}B{alpha} (Ser32/36) antibodies.

Electrophoretic mobility shift assay. Nuclear extracts were prepared, and DNA-binding activity was assessed by electrophoretic mobility shift assay (EMSA), using NF-{kappa}B consensus oligonucleotide as described previously (12).

Immunofluorescence staining. VSMCs were cultured on four-well Lab-Tek II chamber slides (Nalge Nunc International) under the same conditions described above. After treatment, the cells were washed with cold PBS, fixed for 8 min in methanol at –20°C, and air-dried at room temperature. To perform double staining of NF-{kappa}B p65 and phosphor-RSK (Thr359/Ser363), we incubated the slides with 1% BSA in PBS at room temperature for 20 min, followed by incubating overnight at 4°C with primary antibodies [7.5 µg/ml goat anti-NF-{kappa}B p65 polyclonal antibody (Santa Cruz Biotechnology) and 2 µg/ml rabbit anti-phospho-RSK (Thr359/Ser363) polyclonal antibody (Cell Signaling)] in PBS with 1% BSA, washing three times with PBS, incubating for 1 h with FITC-conjugated donkey anti-rabbit antibody and tetramethylrhodamine isothiocyanate-conjugated donkey anti-goat antibody (Jackson Laboratories; each 1:100 dilution) in PBS with 1% BSA, washing three times with PBS, and finally, mounting with aqueous mounting medium. The images observed under a fluorescence microscope were recorded on a linked computer using Openlab software (version 2.2.5; Improvision).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IL-1beta activates ERK and induces nuclear translocation of both RSK1 and NF-{kappa}B p65. In rat VSMCs, IL-1beta-induced activation of both ERK and RSK1, as shown by Western blot analysis of the phosphorylation of ERK and RSK (Fig. 1A). It is known that multiple sites of RSK can be phosphorylated during activation. Thr359 and Ser363 are targeted by ERK, whereas Ser380 is autophosphorylated by the RSK C-terminal kinase domain when it is activated by ERK. IL-1beta-induced RSK phosphorylation at Ser380 and Thr359/Ser363 (the upper bands shown in the blots in Fig. 1A) was consistent with the phosphorylation of ERK, and was inhibited by the addition of PD-98059 or U0126, MEK inhibitors that prevent ERK activation. The lower bands in Fig. 1A detected in all lanes by anti-phospho-RSK (Thr359/Ser363) antibody were probably nonspecific, because it was not IL-1beta inducible and was insensitive to inhibition of ERK activation. IL-1beta treatment with or without MEK inhibitors did not change cellular total RSK1 levels.


Figure 1
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  		Fig. 1. IL-1beta activates ERK and induces nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-{kappa}B p65. Rat vascular smooth muscle cells (VSMCs) were treated with IL-1beta (3 ng/ml) for indicated times in the absence or presence of MEK inhibitor PD-98059 (20 µM) or U0126 (10 µM) that was added 1 h before IL-1beta. A: Western blot analysis (20 µg whole cell lysate proteins/lane) shows that IL-1beta-induced phosphorylation of RSK at Thr359/Ser363 (T359/S363) and Ser 380 (S380) is consistent with the phosphorylation of ERK1/2 and is inhibited by the presence of MEK inhibitors. The bar graph shows the densitometric analysis of IL-1beta-induced phosphorylation of ERK and RSK, with the levels detected in controls (first lane) as 1-fold arbitrary unit. Values are means (SD) from 3 separate experiments. *P < 0.01; {dagger}P < 0.05 vs. control (no IL-1beta), by Student's t-test. B: Western blot analysis of nuclear extracts (10 µg proteins/lane) shows that U0126 inhibits IL-1beta-induced nuclear translocation of RSK1 at both 1 and 6 h but inhibits the nuclear translocation of NF-{kappa}B p65 only at 6 h. The bar graph shows the densitometric analysis of Western blotting of RSK1 and p65, with the levels detected in controls (first lane) as 1-fold arbitrary unit. Values are means (SD) from 3 separate experiments. *P < 0.05 vs. control (no IL-1beta); {dagger}P < 0.05 vs. IL-1beta alone, by 1-way ANOVA. C: immunofluorescent staining shows the nuclear accumulation of phospho-RSK (T359/S363) (green) and nuclear translocation of NF-{kappa}B p65 (red) at 1 and 6 h after IL-1beta addition. Note that U0126 inhibits IL-1beta-induced RSK phosphorylation at both 1 and 6 h but inhibits p65 translocation only at 6 h.

 
Western blot analysis of nuclear extracts (Fig. 1B) showed that IL-1beta induced translocation of both p65 and RSK1 to the nucleus as detected at 1 and 6 h after IL-1beta addition. Inhibition of ERK activation by U0126 reduced IL-1beta-induced RSK1 nuclear translocation at both 1 and 6 h but only attenuated p65 translocation at 6 h. Furthermore, immunofluorescent double staining was used to show the cellular location of p-RSK (Thr359/Ser363) and NF-{kappa}B p65 in cultured rat VSMCs treated with IL-1beta in the absence or presence of U0126 (Fig. 1C). In control cells (without treatment), weak signals for p-RSK (Thr359/Ser363) were observed in the nuclei, and p65 was predominantly stained in the cytoplasm. Upon IL-1beta stimulation, nuclear accumulation of p-RSK (Thr359/Ser363) was seen at both 1 and 6 h, which was associated with nuclear translocation of p65. U0126 inhibited IL-1beta-induced RSK phosphorylation and nuclear accumulation during the 6-h treatment. U0126 did not influence IL-1beta-induced earlier p65 translocation (at 1 h) but reduced prolonged p65 translocation (at 6 h). These results suggest that upon IL-1beta stimulation, activated RSK is accumulated in the nucleus in response to ERK activation, and they support the hypothesis that ERK is integrated into the signaling for the persistent activation of NF-{kappa}B and has less effect on the earlier transient activation of NF-{kappa}B.

Knockdown of RSK1 attenuates IL-1beta-induced decrease in I{kappa}Bbeta. To examine whether ERK could mediate IL-1beta-induced I{kappa}Bbeta degradation via RSK1, we used siRNA to silence Rsk1 gene expression in rat VSMCs. Cellular total RSK1 protein levels, estimated by densitometric analysis of the Western blots, were 31 ± 10 and 99 ± 10% in the cells at 72 h after the transfection with siRNA-Rsk1 and siRNA-Ctl, respectively, compared with the levels in nontransfected cells (100 ± 8%). As shown in Fig. 2A, the decrease of RSK1 expression caused by siRNA-Rsk1 did not influence the IL-1beta-induced phosphorylation of ERK or temporal changes in I{kappa}B{alpha} levels. However, the decrease in RSK1 expression attenuated the IL-1beta-induced decrease in I{kappa}Bbeta, which was more pronounced at 6 h than at 30 min after IL-1beta addition. SiRNA-Ctl did not affect these cell responses to IL-1beta. A densitometric analysis of the Western blots showed a significant change in I{kappa}Bbeta at 6 h (Fig. 2B). These results suggest that RSK1 could be a major component in the ERK signaling pathway in regulating IL-1beta-induced I{kappa}Bbeta degradation.


Figure 2
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  		Fig. 2. Knockdown of RSK1 by small interference RNA (siRNA) attenuates IL-1beta-induced I{kappa}Bbeta degradation. Rat VSMCs were nontransfected or transfected with siRNA-Ctl (nonsilencing control siRNA) or siRNA-Rsk1 (targeting rat Rps6ka1) for 72 h. The cells were then either untreated or treated with IL-1beta (3 ng/ml) for 30 min or 6 h. Whole cell lysates were prepared and subjected to Western blot analysis (10 µg proteins/lane). A: Western blot analysis shows that siRNA-Rsk1 reduced RSK1 expression in the cells, which did not influence IL-1beta-induced phosphorylation of ERK and temporal changes of I{kappa}B{alpha} but attenuated IL-1beta-induced I{kappa}Bbeta decrease. Transfection and IL-1beta treatment did not change total ERK and beta-actin levels. B: densitometric analysis of the Western blotting for I{kappa}Bbeta, with the levels detected in controls (first lane) as 1-fold arbitrary unit. Values are means (SD), n = 4, from 3 separate experiments. *P < 0.001 vs. respective control [IL-1beta (–)]; {dagger}P < 0.01 vs. no siRNA or SiRNA-Ctl [IL-1beta (6 h)], by 1-way ANOVA.

 
RSK1 interacts with I{kappa}Bbeta/NF-{kappa}B complex. Because IL-1beta-induced I{kappa}Bbeta degradation is influenced by changing ERK activity and RSK1 levels, we examined whether ERK or its downstream kinase RSK1 might physically interact with the I{kappa}Bbeta/NF-{kappa}B complex. It has been reported that both I{kappa}B{alpha} and I{kappa}Bbeta can bind to the NF-{kappa}B p65/p50 heterodimer but that only I{kappa}B{alpha} binding efficiently blocks p65/p50 DNA-binding activity, whereas I{kappa}Bbeta binding does not (6, 26). Therefore, it should be feasible to pull down the I{kappa}Bbeta/NF-{kappa}B complex from whole cell lysates with an oligonucleotide agarose conjugate that contains an NF-{kappa}B-binding sequence. To test this possibility, nuclear extracts from VSMCs treated with IL-1beta for 16 h were incubated with recombinant I{kappa}B{alpha} or I{kappa}Bbeta for 30 min before EMSA was performed. The p65/p50 heterodimer and p50/p50 homodimer were identified using antibodies against NF-{kappa}B subunits p65 and p50, respectively (Fig. 3A, right). Either anti-p65 or anti-p50, or both, attenuated the upper band. The lower band was only attenuated by anti-p50. As shown in Fig. 3A, left, I{kappa}B{alpha} completely blocked the DNA-binding activity of p65/p50, whereas I{kappa}Bbeta at the same amount showed no effect. Neither I{kappa}B{alpha} nor I{kappa}Bbeta affected p50/p50 DNA-binding activity. Based on this fact, NF-{kappa}B oligonucleotide agarose conjugate was used to prepare an I{kappa}Bbeta/NF-{kappa}B complex from cell lysates. VSMCs treated with IL-1beta for 6 h had a decreased I{kappa}Bbeta level, determined in the whole cell lysates, under conditions where there were no changes in the levels of NF-{kappa}B p65, ERK1/2, and RSK1 (Fig. 3B, left). Western blot analysis of the NF-{kappa}B complex that was pulled down with the NF-{kappa}B oligonucleotide agarose conjugate (Fig. 3B, right) showed an increased p65 binding in the cell lysates from IL-1beta-treated cells compared with that from control cells, which may relate to an increased dissociation of NF-{kappa}B from I{kappa}B{alpha} following IL-1beta treatment. The relative amount of I{kappa}Bbeta was less in the complex from IL-1beta-treated cells, consistent with its decrease in the whole cell lysates. I{kappa}B{alpha} was not detectable in the complex (data not shown). Because in untreated control cells NF-{kappa}B DNA-binding activity in the nuclear extract was almost undetectable by EMSA (see Fig. 3A), the p65 pulled down with NF-{kappa}B oligonucleotide agarose conjugate from control whole cell lysates should predominantly represent NF-{kappa}B from the I{kappa}Bbeta/NF-{kappa}B complex in the cytoplasm. The complex bound to NF-{kappa}B oligonucleotide contained not only NF-{kappa}B p65 and I{kappa}Bbeta but also RSK1, as shown by immunoblotting with RSK1-specific antibody, whereas ERK was not detectable in the complex, suggesting a physical association of RSK1, but not ERK, with the I{kappa}Bbeta/NF-{kappa}B complex. Interestingly, similarly to p65, RSK1 was increased in the NF-{kappa}B oligonucleotide agarose conjugate-bound complex from IL-1beta-treated cells, suggesting the possibility that RSK1 interacts with p65 but not with I{kappa}Bbeta.


Figure 3
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  		Fig. 3. RSK1 physically associates with NF-{kappa}B p65, and activation of ERK/RSK1 enhances the dissociation of I{kappa}Bbeta from RSK1/I{kappa}Bbeta/NF-{kappa}B complex. A: I{kappa}B{alpha}, but not I{kappa}Bbeta, prevents NF-{kappa}B p65/p50 DNA-binding activity. Nuclear extracts from rat VSMCs treated with IL-1beta for 16 h were incubated with recombinant I{kappa}B{alpha} or I{kappa}Bbeta for 30 min before EMSA (left). Ctl, control (without IL-1beta treatment); WT, wild type. The p65/p50 and p50/p50 were identified by incubation of nuclear extracts (from rat VSMCs treated with IL-1beta for 16 h) with p65 and p50 subunit-specific antibodies ({alpha}) before EMSA (right). B: I{kappa}Bbeta/NF-{kappa}B complex contains RSK1. Rat VSMCs were either untreated or treated with IL-1beta for 6 h. I{kappa}Bbeta/NF-{kappa}B complex was pulled down from whole cell lysates with NF-{kappa}B oligonucleotide agarose conjugate. Cell lysates (20 µg proteins) and NF-{kappa}B oligonucleotide (DNA)-bound complex (from 20 µg cell lysate proteins) were resolved by 10% SDS-PAGE and analyzed by Western blotting. Bar graphs show the densitometric analysis of DNA-bound proteins with those from control as 1-fold arbitrary unit. Values are means (SD); n = 2. Statistical analysis was performed using Student's t-test. C: control cell lysates (50 µg proteins) were immunoprecipitated (IP) with mouse monoclonal antibody (mAb) against RSK or normal mouse IgG. The immunoprecipitates were analyzed by SDS-PAGE and immunoblotting (IB) with rabbit polyclonal antibodies (pAb) against RSK1 or p65. Whole cell lysate (20 µg proteins) was loaded as a non-IP control. D and E: RSK1 associates with NF-{kappa}B p65, which is independent of RSK1 phosphorylation status. Rat VSMCs were either untreated or treated for 6 h with IL-1beta (3 ng/ml) or IL-1beta plus U0126 (10 µM) or PD-98059 (20 µM) that was added 1 h before IL-1beta. Cell lysates were used for IP with antibodies as indicated or normal rabbit IgG (E, with the same cell lysate as used in first lane). The immunoprecipitates were analyzed by SDS-PAGE and immunoblotting. gAb, goat polyclonal antibody. Note the IL-1beta-induced decrease of I{kappa}Bbeta in the immunoprecipitated complex (D) was partially prevented by U0126.

 
The interaction of RSK1 and p65 was further confirmed by immunoprecipitation. With the use of a monoclonal antibody to pull down RSK1 from untreated control cell lysates, both RSK1 and p65 were detectable in the immunoprecipitate (Fig. 3C), although compared with the total amounts of RSK1 and p65 in the whole cell lysates, the RSK1-associated p65 was only a small proportion (~7%), as estimated by relative intensities of the immunoblots. The normal mouse IgG that was used as a nonspecific control did not pull down RSK1, although it did show a slight association with a trace amount of p65. To examine whether activation of ERK could enhance the interaction of p65 and RSK1 and change the amount of I{kappa}Bbeta in the RSK1/I{kappa}Bbeta/NF-{kappa}B complex, we treated rat VSMCs with IL-1beta for 6 h in the absence or presence of U0126. Whole cell lysates were then subjected to immunoprecipitation with monoclonal antibody against RSK, followed by Western blotting with polyclonal antibodies against RSK1, p65, and I{kappa}Bbeta, respectively (Fig. 3D). The results show that changing ERK activity did not influence the interaction of p65 and RSK1, but the amount of I{kappa}Bbeta in the complex decreased after IL-1beta treatment, and the decrease was attenuated by U0126. In a separate experiment, rat VSMCs were treated for 6 h with IL-1beta, or IL-1beta plus PD-98059, and the cell lysates were used in immunoprecipitation experiments with a rabbit polyclonal antibody against NF-{kappa}B p65. A goat polyclonal antibody against p65 and a mouse monoclonal antibody against RSK were then used for immunoblotting. As shown in Fig. 3E, RSK was coimmunoprecipitated with the p65-specific antibody. Neither p65 nor RSK were detectable in the nonspecific precipitation by normal rabbit IgG (Fig. 3E). There was no obvious difference in coimmunoprecipitation of RSK and NF-{kappa}B p65 among the different treatments. Thus Fig. 3, D and E, both indicate the association of RSK1 with p65, which is independent of the phosphorylation status of RSK1.

ERK and RSK1 phosphorylate I{kappa}Bbeta on different sites. To examine whether activated ERK might phosphorylate I{kappa}Bbeta, we incubated the NF-{kappa}B oligonucleotide-bound complex from control cell lysates for 30 min with either ERK2 (inactive) or p-ERK2 (active) in an in vitro phosphorylation assay system containing [{gamma}32P]ATP. Active ERK2 phosphorylated (as shown by 32P labeling) several components of the complex (Fig. 4A, left). Western blot analysis (Fig. 4A, middle and right) demonstrated that three of the 32P-labeled proteins matched the molecular masses of I{kappa}Bbeta (band 1), NF-{kappa}B p65 (band 2), and RSK1 (band 3), respectively. To demonstrate whether ERK or RSK1 or both could directly phosphorylate I{kappa}Bbeta, we used purified recombinant I{kappa}Bbeta proteins to detect in vitro phosphorylation of Ser19 and/or Ser23, two sites related known to mediate its subsequent ubiquitination and degradation. The results shown in Fig. 4B indicate that p-ERK2 directly phosphorylates wild-type I{kappa}Bbeta. The specific phosphorylation of I{kappa}Bbeta by p-ERK2 was identified by 32P labeling in lane 2 compared with lane 1 (no addition of p-ERK2) and lane 3 (no addition of I{kappa}Bbeta). However, phosphorylation by p-ERK2 of I{kappa}Bbeta with Ser19Ala or Ser23Ala single mutation or Ser19/23Ala double mutation was similar to that of wild-type I{kappa}Bbeta (Fig. 4C), indicating that p-ERK2 phosphorylates I{kappa}Bbeta on site(s) other than Ser19 and Ser23.


Figure 4
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  		Fig. 4. ERK phosphorylates I{kappa}Bbeta on the sites other than S19 and S23. A: ERK phosphorylates several components of NF-{kappa}B oligonucleotide-bound complex including RSK1, p65, and I{kappa}Bbeta. NF-{kappa}B oligonucleotide-bound complex from 20 µg of control cell lysate proteins was incubated with 5 ng of recombinant active ERK2 (p-ERK2) or inactive ERK2 in a phosphorylation assay buffer containing [{gamma}32P]ATP for 30 min at 30°C. The proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by sequential autoradiography (left), immunoblotting with anti-p65 antibody (middle), and, after cutting the membrane between bands 1 and 2, further immunoblotting with anti-RSK1 and anti-I{kappa}Bbeta antibodies (right). B and C: purified recombinant I{kappa}Bbeta proteins (WT and mutants, 50 ng) were incubated with 5 ng of p-ERK2 or ERK2 in the same phosphorylation assay buffer as in A for 30 min at 30°C. After being resolved by SDS-PAGE and transferred to membrane, the phosphorylated I{kappa}Bbeta and total I{kappa}Bbeta amounts were detected by autoradiography and immunoblotting, respectively.

 
The association of RSK1 with the I{kappa}Bbeta/NF-{kappa}B complex suggested that RSK1 could be the kinase downstream of ERK that directly phosphorylates I{kappa}Bbeta. To test this possibility, we treated VSMCs for 10 min with PDGF, which potently activates the ERK pathway, and RSK1 was immunoprecipitated from the cell lysates and used for the in vitro phosphorylation assay. As shown in Fig. 5A, top, the immunoprecipitated RSK1 directly phosphorylated wild-type I{kappa}Bbeta, and phosphorylation by RSK1 was dramatically decreased in the Ser19Ala or Ser23Ala I{kappa}Bbeta mutants. Figure 5A, middle and bottom, shows the equal amounts of I{kappa}Bbeta and RSK1, respectively, added to the reaction. A similar result was obtained by using activated recombinant RSK1 (Fig. 5B, left), suggesting that the phosphorylation of I{kappa}Bbeta by immunoprecipitated RSK1 shown in Fig. 5A was predominantly caused by RSK1, even if there might be contamination by other kinases in the immunoprecipitate. However, double mutation of Ser19/23Ala did not completely eliminate RSK1-mediated phosphorylation (Fig. 5B, right), suggesting that the phosphorylation of I{kappa}Bbeta by RSK1 might not be limited to Ser19 and Ser23.


Figure 5
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  		Fig. 5. RSK1 phosphorylates I{kappa}Bbeta on S19 and S23. A: purified recombinant I{kappa}Bbeta proteins (WT and S19A or S23A mutants, 50 ng each) were incubated with active RSK1 in the phosphorylation assay buffer containing [{gamma}32P]ATP for 30 min at 30°C. After being resolved by 10% SDS-PAGE and transferred to membrane, the phosphorylated I{kappa}Bbeta and total I{kappa}Bbeta and RSK1 amounts were detected by autoradiography (top) and immunoblotting (middle and bottom), respectively. Active RSK1 was immunoprecipitated from cell lysates after cells were treated with PDGF for 10 min. HC, heavy chain. B: the same phosphorylation assay was performed as in A, but the recombinant I{kappa}Bbeta proteins (including S19/23A mutant) were incubated with recombinant active RSK1 (20 ng each). The gel was dried and directly exposed to film. Bar graphs in A and B show the fold changes of relative intensities [means (SD), n = 3]. S19A, S23A, or S19/23A mutation of I{kappa}Bbeta markedly reduced its phosphorylation by active RSK1 compared with WT I{kappa}Bbeta.

 
The ability of RSK1 to phosphorylate I{kappa}Bbeta Ser19/23 was further demonstrated by in vitro phosphorylation assay and immunoblotting with anti-phospho-I{kappa}Bbeta (Ser19/23) antibody and compared with that of IKKbeta and ERK2. After incubation of the recombinant I{kappa}Bbeta proteins with recombinant active RSK1, IKKbeta, or ERK2, it was clearly demonstrated that both active RSK1 and IKKbeta phosphorylated wild-type I{kappa}Bbeta, whereas ERK2 did not (Fig. 6A). The antibody also detected phosphorylation at Ser23 alone as seen in the I{kappa}Bbeta with Ser19Ala mutation, although it was weak compared with that of wild-type I{kappa}Bbeta. No signal was detectable in the Ser23Ala mutant or the Ser19/23Ala double mutant, as well as in the wild-type I{kappa}Bbeta incubated with no kinase. The kinases were also tested in a phosphorylation assay with recombinant I{kappa}B{alpha} as the substrate (Fig. 6B). Similarly, both RSK1 and IKKbeta phosphorylated I{kappa}B{alpha}, but ERK2 did not. Figure 6B, far right, is a positive control showing ERK2 activity with recombinant Elk1 as the substrate. Figure 6C shows the phosphorylation of wild-type I{kappa}Bbeta by RSK1 or IKKbeta in a concentration-dependent manner. The phosphorylation was increased with kinase concentrations from 6.25 to 50 ng per assay with 100 ng of I{kappa}Bbeta each in the reaction mixture.


Figure 6
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  		Fig. 6. RSK1 and IKKbeta, but not ERK2, phosphorylate I{kappa}Bbeta S19/23 and I{kappa}B{alpha} S32/36 detected by antibodies against phospho-I{kappa}Bbeta and phospho-I{kappa}B{alpha}. A: recombinant I{kappa}Bbeta (WT and mutants, 100 ng each) were incubated with active RSK1 (50 ng), IKKbeta (50 ng), or ERK2 (10 ng) in the phosphorylation assay buffer containing 100 µM ATP but without [{gamma}32P]ATP for 1 h at 30°C. S19/23 phosphorylation was detected by Western blotting with phospho-I{kappa}Bbeta (Ser19/23) antibody. I{kappa}Bbeta equal loading was analyzed with I{kappa}Bbeta antibody. B: recombinant I{kappa}B{alpha} (WT) was incubated with or without active RSK1, IKKbeta, or ERK2 under the same conditions as described in A. S32/36 phosphorylation was detected by Western blotting with phospho-I{kappa}B{alpha} (Ser32/36) antibody. I{kappa}B{alpha} equal loading was analyzed with I{kappa}B{alpha} antibody. The recombinant Elk1 (50 ng) was incubated with or without active ERK2 (10 ng) followed by Western blotting with phospho-Elk1 (Ser383) antibody (far right). C: WT I{kappa}Bbeta (100 ng) was incubated with 6.25, 12.5, 25, and 50 ng of active RSK1 or IKKbeta under the same conditions as described in A. p-I{kappa}Bbeta (Ser19/23) was detected by Western blotting. Relative intensities are charted, with 6.25 ng of IKKbeta-caused phosphorylation as 1-fold arbitrary unit.

 

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In the present study, we showed that RSK1 is associated with the I{kappa}Bbeta/NF-{kappa}B complex and that following its phosphorylation and activation by ERK, RSK1 is able to directly phosphorylate I{kappa}Bbeta on Ser19 and Ser23, two phosphorylation sites associated with subsequent ubiquitination and degradation. This finding, together with our previous observations that inhibition of ERK selectively attenuates IL-1beta-induced degradation of I{kappa}Bbeta but not that of I{kappa}B{alpha}, offers an explanation as to how ERK signaling can regulate cytokine-mediated persistent activation of NF-{kappa}B and subsequent gene expression.

Phosphorylation of I{kappa}B{alpha} and I{kappa}Bbeta by IKK{alpha}/beta may not completely explain the differential regulation of transient and persistent activation of NF-{kappa}B (24, 28). For example, TNF-{alpha} caused the degradation of both I{kappa}B{alpha} and I{kappa}Bbeta in some cell types such as NIH3T3 and rat VSMCs (14, 28), whereas only I{kappa}B{alpha} degradation was affected by this cytokine in several other cell types such as 70Z/3, Jurkat, and EL-4 cells (27, 28). Furthermore, TNF-{alpha} does not induce I{kappa}Bbeta degradation in the 70Z/3 cells, whereas IL-1beta or lipopolysaccharides initiate complete I{kappa}Bbeta degradation despite the activation of IKK and degradation of I{kappa}B{alpha} by all the above stimuli (17, 27, 28). There is still no clear understanding of the different mechanisms causing the phosphorylation and degradation of I{kappa}Bbeta and the subsequent persistent activation of NF-{kappa}B underlying these differences in cellular responses to cytokines. We hypothesize that besides IKK, other signaling pathways are involved. Several studies have suggested that the ERK signaling cascade is one such pathway. In cultured rat VSMCs, for example, we showed that inhibition of ERK activation, either by selective MEK inhibitors such as U0126 and PD-98059 or by overexpression of a dominant negative MEK-1, inhibited IL-1beta-induced expression of iNOS, which was associated with a decrease in prolonged NF-{kappa}B activation (14, 15). Furthermore, growth factors such as PDGF and EGF, which do not activate NF-{kappa}B when added alone, enhanced IL-1beta-induced persistent activation of NF-{kappa}B via an ERK-dependent mechanism (13). Inhibition of ERK activation attenuated IL-1beta-induced I{kappa}Bbeta degradation without affecting IL-1beta-induced I{kappa}B{alpha} degradation, suggesting that the ERK-dependent persistent activation of NF-{kappa}B is via an I{kappa}Bbeta-mediated mechanism (11, 14). In addition, treatment of Jurkat cells with TNF-{alpha} induced IKK activation and I{kappa}B{alpha} degradation but did not induce I{kappa}Bbeta degradation (27, 28). It is notable that in these cells, TNF-{alpha} did not activate RSK (7).

The facts that active ERK2 did not phosphorylate I{kappa}Bbeta Ser19 and Ser23 and that ERK did not directly interact with I{kappa}Bbeta/NF-{kappa}B suggest that ERK may regulate IL-1beta-induced I{kappa}Bbeta phosphorylation and degradation through its downstream kinases. In this study, we have shown that IL-1beta induced RSK1 phosphorylation at Thr359/363 and Ser380, which required ERK activation and were prevented by MEK inhibitors. Importantly, knockdown of RSK1 levels in the cells, which did not affect ERK activation by IL-1beta, partially prevented IL-1beta-induced I{kappa}Bbeta degradation. The ability of RSK1 to directly phosphorylate I{kappa}Bbeta Ser19 and Ser23 was demonstrated by in vitro phosphorylation assay with recombinant wild-type or mutant I{kappa}Bbeta as substrates, detected by either [{gamma}32P]ATP labeling or immunoblotting with anti-phospho-I{kappa}Bbeta (Ser19/23). The association of RSK1 with the I{kappa}Bbeta/NF-{kappa}B complex, demonstrated with both the DNA-bound NF-{kappa}B complex and by immunoprecipitation, was not different with or without IL-1beta treatment and was not influenced by MEK inhibitors, indicating that the association was not dependent on RSK1 phosphorylation. IL-1beta decreased I{kappa}Bbeta in the complex but did not obviously change the association of RSK1 with p65, suggesting that RSK1 normally interacts with p65, permitting I{kappa}Bbeta dissociation from the complex upon its phosphorylation and degradation. The dissociation of I{kappa}Bbeta was partially dependent on the activation of ERK, because it was attenuated by MEK inhibitors.

RSK1 has been reported to phosphorylate I{kappa}B{alpha} (7). However, in rat VSMCs, we found that inhibition of ERK signaling shows only a minor effect on IL-1beta-induced I{kappa}B{alpha} phosphorylation and degradation (11, 14). In the present study, decrease of RSK1 levels by siRNA also shows no obvious effect on IL-1beta-induced I{kappa}B{alpha} degradation. This could be because the IKK{alpha}/beta activity induced by IL-1beta are potent enough to maximally phosphorylate I{kappa}B{alpha}. This finding also suggests that the ERK cascade does not act upstream of IKK{alpha}/beta or on IKK{alpha}/beta. Furthermore, although RSK1 is able to phosphorylate both I{kappa}B{alpha} and I{kappa}Bbeta, activation of RSK1 alone is insufficient to trigger NF-{kappa}B activation and requires ERK pathway-independent mechanism(s). At least in rat VSMCs, treatment of the rat VSMCs with growth factors such as PDGF and EGF alone cannot activate NF-{kappa}B, despite their potent ability to activate the ERK signaling pathway (13). As noted in our previous study, the IL-1beta-induced prolonged NF-{kappa}B DNA-binding activity and I{kappa}Bbeta degradation were not completely prevented by inhibition of ERK activation (11, 13, 14), suggesting that the activity of IKK on I{kappa}Bbeta phosphorylation, although weak, is not inhibited by the MEK inhibitors and that the ERK cascade may act in conjunction with IKK to phosphorylate I{kappa}Bbeta. In addition, it is possible that RSK1 may target newly synthesized I{kappa}Bbeta in the nucleus, where the phosphorylated RSK1 is accumulated. This may explain why once NF-{kappa}B activation is triggered by cytokines such as IL-1beta, PDGF or EGF can enhance the persistent activation of NF-{kappa}B through an ERK-dependent mechanism (13). A possible mechanism for the involvement of ERK/RSK1 in the persistent activation of NF-{kappa}B is summarized schematically in Fig. 7.


Figure 7
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  		Fig. 7. ERK/RSK1 and NF-{kappa}B activation. Upon cytokine stimulation, activated IKK{alpha}/beta principally phosphorylates I{kappa}B{alpha} but poorly phosphorylates I{kappa}Bbeta. Phosphorylated I{kappa}B{alpha} then undergoes ubiquitination-dependent proteasomal degradation, which results in transient NF-{kappa}B activation. RSK1 physically associates with I{kappa}B/NF-{kappa}B complex. Activation of ERK leads to activation of RSK1. It is postulated that with IKK{alpha}/beta activation, active RSK1 will act (in either the cytoplasm or the nucleus, or both) in concert with IKK{alpha}/beta to phosphorylate I{kappa}Bbeta on S19 and S23 and cause its degradation, as well as subsequently enhancing persistent NF-{kappa}B activation. RSK1 also phosphorylates I{kappa}B{alpha}, but its activity may not be necessary when IKK{alpha}/beta is activated, because the latter activity is potent enough to induce maximum phosphorylation and degradation of I{kappa}B{alpha}. The duration of NF-{kappa}B activation may determine different gene expression patterns. iNOS, inducible nitric oxide synthase; P, phosphorylation.

 
Temporal modulation of NF-{kappa}B activation has an important role in regulating diverse pathophysiological processes, including the initiation and resolution of inflammation, the replication and clearance of virus, the apoptosis and survival of injured cells or tumor cells, and the differentiation of specialized cells. The duration of NF-{kappa}B activation may define the patterns of expression of NF-{kappa}B-dependent genes. A clear example exists in cultured rat VSMCs, in which transient or persistent activation of NF-{kappa}B leads to different combinations of NF-{kappa}B-inducible gene products (14). The present study describes a novel mechanism for regulation of the duration of NF-{kappa}B activation by ERK and RSK1.


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This study was supported by American Heart Association Grant 0435205N and National Institutes of Health Grants R01 HL-55620 and R01 AG-27080.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Jiang, Vascular Biology Unit, Whitaker Cardiovascular Institute, Dept. of Medicine, Boston Univ. School of Medicine, 650 Albany St., X704, Boston, MA 02118 (e-mail: bjiang{at}bu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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