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MUSCLE CELL BIOLOGY AND CELL MOTILITY
-induced persistent activation of NF-
B through phosphorylation of IB
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
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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B requires the phosphorylation and degradation of its associated inhibitory proteins, IB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1
to induce persistent activation of NF-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-B activation. In cultured rat VSMCs, IL-1 activated ERK and induced degradation of both IB
and IB, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-B p65. RSK1, a downstream kinase of ERK, was associated with an IB/NF-B complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1 decreased IB in the RSK1/IB/NF-B complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1-induced IB decrease without influencing ether ERK phosphorylation or the earlier IB degradation. By using recombinant wild-type and mutant IB proteins, both active ERK2 and RSK1 were found to directly phosphorylate IB, but only active RSK1 phosphorylated IB 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 IB and contributes to the persistent activation of NF-B by IL-1. extracellular signal-regulated kinase; in vitro phosphorylation assay; recombinant proteins; small interference RNA; vascular smooth muscle cell
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-B can occur transiently or persistently, which may result in different gene expression patterns. In the vessel wall, NF-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-B-dependent genes in part by regulating the duration of NF-B activation (13, 15).
In most cells without inflammatory stimulation, NF-B is sequestered in the cytoplasm by inhibitory proteins IB (, , etc.). Stimulation of cells with cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF)- leads to the phosphorylation and ubiquitination-dependent proteasomal degradation of IB, accompanied by NF-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). IB and IB have different kinetics for degradation and synthesis in response to cytokine stimulation. IB is degraded rapidly after cytokine stimulation and is then rapidly resynthesized because of the presence of NF-B-binding motifs in the promoter of the IB gene (17). This autoregulatory loop limits NF-B activation in a transient manner (17, 27). IB is slowly but persistently decreased after cytokine stimulation, a process that contributes to persistent NF-B activation (27). Two IB kinases, IKK and IKK (also known as IKK1 and IKK2), are capable of phosphorylating IB and inducing NF-B activation (16, 21, 30). Both IKK and IKK phosphorylate Ser32 and Ser36 of IB but phosphorylate IB weakly (24, 30). The mechanism causing the phosphorylation and degradation of IB and the subsequent persistent activation of NF-B is not fully understood.
Extracellular signal-regulated kinase (ERK) regulates several NF-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-B without influencing the early (transient) activation of NF-B induced by IL-1 (11, 13, 14). Inhibition of ERK activation did not affect IL-1-induced IB degradation but attenuated IL-1-induced IB degradation, suggesting that an IB-mediated mechanism is involved in the ERK-dependent persistent activation of NF-B (11, 14). This observation provided an important clue for the signaling mechanism that controls the duration of NF-B activation. Ribosomal S6 kinase (RSK)1 is a downstream kinase of ERK that is able to phosphorylate IB on Ser32 (7). It is unknown whether RSK1 could also phosphorylate IB and eventually contribute to the persistent activation of NF-B. In the present study, we have demonstrated that RSK1, but not ERK, directly associates with the IB/NF-B complex and targets IB 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 IB phosphorylation and degradation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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(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 IKK 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-IB (Ser19/23), as well as monoclonal antibodies against phospho-p44/42 MAPK (Thr202/Tyr204) and phospho-IB (Ser32/36), were obtained from Cell Signaling Technologies. Monoclonal antibody against RSK was obtained from Transduction Laboratories. NF-B consensus oligonucleotide agarose conjugate, recombinant IB, and polyclonal antibodies against RSK1, IB, NF-B p50, and NF-B p65 were obtained from Santa Cruz Biotechnology. NF-B consensus oligonucleotide was obtained from Promega. [
-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-1 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 -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 -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 IB/NF-B complex, we performed immunoprecipitation with either monoclonal antibody against RSK or polyclonal antibody against NF-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 IB/NF-B complex.
IB/NF-B complex was prepared from whole cell lysates with an NF-B consensus oligonucleotide agarose conjugate. Rat VSMCs were either untreated or treated with IL-1 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 IB/NF-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 IB.
Full-length cDNA encoding wild-type rat IB 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-IB 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 TG 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--D-thiogalactopyranoside was added to a final concentration of 1 mM to induce expression of wild-type and mutant IB proteins. The His-IB 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 IB antibody.
In vitro phosphorylation assay.
To determine whether ERK or RSK1 would phosphorylate IB, 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 [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 IB/NF-B complex (eluted from the oligonucleotide agarose conjugate) or purified IB, 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 IB 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 IB, followed by Western blot analysis of IB and RSK1, accordingly, to confirm equal loading of the proteins. To determine the ability of recombinant kinases to phosphorylate Ser19/23 of IB or Ser32/36 of IB, we also performed the reaction in the above kinase buffer with ATP and then subjected the membranes to Western blot analysis with phospho-IB (Ser19/23) or phospho-IB (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-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-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-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).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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activates ERK and induces nuclear translocation of both RSK1 and NF-B p65.
In rat VSMCs, IL-1-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-1-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-1 inducible and was insensitive to inhibition of ERK activation. IL-1 treatment with or without MEK inhibitors did not change cellular total RSK1 levels.
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induced translocation of both p65 and RSK1 to the nucleus as detected at 1 and 6 h after IL-1 addition. Inhibition of ERK activation by U0126 reduced IL-1-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-B p65 in cultured rat VSMCs treated with IL-1 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-1 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-1-induced RSK phosphorylation and nuclear accumulation during the 6-h treatment. U0126 did not influence IL-1-induced earlier p65 translocation (at 1 h) but reduced prolonged p65 translocation (at 6 h). These results suggest that upon IL-1 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-B and has less effect on the earlier transient activation of NF-B.
Knockdown of RSK1 attenuates IL-1-induced decrease in IB.
To examine whether ERK could mediate IL-1-induced IB 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-1-induced phosphorylation of ERK or temporal changes in IB levels. However, the decrease in RSK1 expression attenuated the IL-1-induced decrease in IB, which was more pronounced at 6 h than at 30 min after IL-1 addition. SiRNA-Ctl did not affect these cell responses to IL-1. A densitometric analysis of the Western blots showed a significant change in IB at 6 h (Fig. 2B). These results suggest that RSK1 could be a major component in the ERK signaling pathway in regulating IL-1-induced IB degradation.
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B/NF-B complex.
Because IL-1-induced IB degradation is influenced by changing ERK activity and RSK1 levels, we examined whether ERK or its downstream kinase RSK1 might physically interact with the IB/NF-B complex. It has been reported that both IB and IB can bind to the NF-B p65/p50 heterodimer but that only IB binding efficiently blocks p65/p50 DNA-binding activity, whereas IB binding does not (6, 26). Therefore, it should be feasible to pull down the IB/NF-B complex from whole cell lysates with an oligonucleotide agarose conjugate that contains an NF-B-binding sequence. To test this possibility, nuclear extracts from VSMCs treated with IL-1 for 16 h were incubated with recombinant IB or IB for 30 min before EMSA was performed. The p65/p50 heterodimer and p50/p50 homodimer were identified using antibodies against NF-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, IB completely blocked the DNA-binding activity of p65/p50, whereas IB at the same amount showed no effect. Neither IB nor IB affected p50/p50 DNA-binding activity. Based on this fact, NF-B oligonucleotide agarose conjugate was used to prepare an IB/NF-B complex from cell lysates. VSMCs treated with IL-1 for 6 h had a decreased IB level, determined in the whole cell lysates, under conditions where there were no changes in the levels of NF-B p65, ERK1/2, and RSK1 (Fig. 3B, left). Western blot analysis of the NF-B complex that was pulled down with the NF-B oligonucleotide agarose conjugate (Fig. 3B, right) showed an increased p65 binding in the cell lysates from IL-1-treated cells compared with that from control cells, which may relate to an increased dissociation of NF-B from IB following IL-1 treatment. The relative amount of IB was less in the complex from IL-1-treated cells, consistent with its decrease in the whole cell lysates. IB was not detectable in the complex (data not shown). Because in untreated control cells NF-B DNA-binding activity in the nuclear extract was almost undetectable by EMSA (see Fig. 3A), the p65 pulled down with NF-B oligonucleotide agarose conjugate from control whole cell lysates should predominantly represent NF-B from the IB/NF-B complex in the cytoplasm. The complex bound to NF-B oligonucleotide contained not only NF-B p65 and IB 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 IB/NF-B complex. Interestingly, similarly to p65, RSK1 was increased in the NF-B oligonucleotide agarose conjugate-bound complex from IL-1-treated cells, suggesting the possibility that RSK1 interacts with p65 but not with IB.
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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 IB in the RSK1/IB/NF-B complex, we treated rat VSMCs with IL-1 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 IB, respectively (Fig. 3D). The results show that changing ERK activity did not influence the interaction of p65 and RSK1, but the amount of IB in the complex decreased after IL-1 treatment, and the decrease was attenuated by U0126. In a separate experiment, rat VSMCs were treated for 6 h with IL-1, or IL-1 plus PD-98059, and the cell lysates were used in immunoprecipitation experiments with a rabbit polyclonal antibody against NF-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-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 IB on different sites.
To examine whether activated ERK might phosphorylate IB, we incubated the NF-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 [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 IB (band 1), NF-B p65 (band 2), and RSK1 (band 3), respectively. To demonstrate whether ERK or RSK1 or both could directly phosphorylate IB, we used purified recombinant IB 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 IB. The specific phosphorylation of IB 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 IB). However, phosphorylation by p-ERK2 of IB with Ser19Ala or Ser23Ala single mutation or Ser19/23Ala double mutation was similar to that of wild-type IB (Fig. 4C), indicating that p-ERK2 phosphorylates IB on site(s) other than Ser19 and Ser23.
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B/NF-B complex suggested that RSK1 could be the kinase downstream of ERK that directly phosphorylates IB. 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 IB, and phosphorylation by RSK1 was dramatically decreased in the Ser19Ala or Ser23Ala IB mutants. Figure 5A, middle and bottom, shows the equal amounts of IB 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 IB 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 IB by RSK1 might not be limited to Ser19 and Ser23.
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B Ser19/23 was further demonstrated by in vitro phosphorylation assay and immunoblotting with anti-phospho-IB (Ser19/23) antibody and compared with that of IKK and ERK2. After incubation of the recombinant IB proteins with recombinant active RSK1, IKK, or ERK2, it was clearly demonstrated that both active RSK1 and IKK phosphorylated wild-type IB, whereas ERK2 did not (Fig. 6A). The antibody also detected phosphorylation at Ser23 alone as seen in the IB with Ser19Ala mutation, although it was weak compared with that of wild-type IB. No signal was detectable in the Ser23Ala mutant or the Ser19/23Ala double mutant, as well as in the wild-type IB incubated with no kinase. The kinases were also tested in a phosphorylation assay with recombinant IB as the substrate (Fig. 6B). Similarly, both RSK1 and IKK phosphorylated IB, 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 IB by RSK1 or IKK in a concentration-dependent manner. The phosphorylation was increased with kinase concentrations from 6.25 to 50 ng per assay with 100 ng of IB each in the reaction mixture.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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B/NF-B complex and that following its phosphorylation and activation by ERK, RSK1 is able to directly phosphorylate IB 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-1-induced degradation of IB but not that of IB, offers an explanation as to how ERK signaling can regulate cytokine-mediated persistent activation of NF-B and subsequent gene expression.
Phosphorylation of IB and IB by IKK/ may not completely explain the differential regulation of transient and persistent activation of NF-B (24, 28). For example, TNF- caused the degradation of both IB and IB in some cell types such as NIH3T3 and rat VSMCs (14, 28), whereas only IB degradation was affected by this cytokine in several other cell types such as 70Z/3, Jurkat, and EL-4 cells (27, 28). Furthermore, TNF- does not induce IB degradation in the 70Z/3 cells, whereas IL-1 or lipopolysaccharides initiate complete IB degradation despite the activation of IKK and degradation of IB by all the above stimuli (17, 27, 28). There is still no clear understanding of the different mechanisms causing the phosphorylation and degradation of IB and the subsequent persistent activation of NF-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-1-induced expression of iNOS, which was associated with a decrease in prolonged NF-B activation (14, 15). Furthermore, growth factors such as PDGF and EGF, which do not activate NF-B when added alone, enhanced IL-1-induced persistent activation of NF-B via an ERK-dependent mechanism (13). Inhibition of ERK activation attenuated IL-1-induced IB degradation without affecting IL-1-induced IB degradation, suggesting that the ERK-dependent persistent activation of NF-B is via an IB-mediated mechanism (11, 14). In addition, treatment of Jurkat cells with TNF- induced IKK activation and IB degradation but did not induce IB degradation (27, 28). It is notable that in these cells, TNF- did not activate RSK (7).
The facts that active ERK2 did not phosphorylate IB Ser19 and Ser23 and that ERK did not directly interact with IB/NF-B suggest that ERK may regulate IL-1-induced IB phosphorylation and degradation through its downstream kinases. In this study, we have shown that IL-1 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-1, partially prevented IL-1-induced IB degradation. The ability of RSK1 to directly phosphorylate IB Ser19 and Ser23 was demonstrated by in vitro phosphorylation assay with recombinant wild-type or mutant IB as substrates, detected by either [32P]ATP labeling or immunoblotting with anti-phospho-IB (Ser19/23). The association of RSK1 with the IB/NF-B complex, demonstrated with both the DNA-bound NF-B complex and by immunoprecipitation, was not different with or without IL-1 treatment and was not influenced by MEK inhibitors, indicating that the association was not dependent on RSK1 phosphorylation. IL-1 decreased IB in the complex but did not obviously change the association of RSK1 with p65, suggesting that RSK1 normally interacts with p65, permitting IB dissociation from the complex upon its phosphorylation and degradation. The dissociation of IB was partially dependent on the activation of ERK, because it was attenuated by MEK inhibitors.
RSK1 has been reported to phosphorylate IB (7). However, in rat VSMCs, we found that inhibition of ERK signaling shows only a minor effect on IL-1-induced IB phosphorylation and degradation (11, 14). In the present study, decrease of RSK1 levels by siRNA also shows no obvious effect on IL-1-induced IB degradation. This could be because the IKK/ activity induced by IL-1 are potent enough to maximally phosphorylate IB. This finding also suggests that the ERK cascade does not act upstream of IKK/ or on IKK/. Furthermore, although RSK1 is able to phosphorylate both IB and IB, activation of RSK1 alone is insufficient to trigger NF-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-B, despite their potent ability to activate the ERK signaling pathway (13). As noted in our previous study, the IL-1-induced prolonged NF-B DNA-binding activity and IB degradation were not completely prevented by inhibition of ERK activation (11, 13, 14), suggesting that the activity of IKK on IB phosphorylation, although weak, is not inhibited by the MEK inhibitors and that the ERK cascade may act in conjunction with IKK to phosphorylate IB. In addition, it is possible that RSK1 may target newly synthesized IB in the nucleus, where the phosphorylated RSK1 is accumulated. This may explain why once NF-B activation is triggered by cytokines such as IL-1, PDGF or EGF can enhance the persistent activation of NF-B through an ERK-dependent mechanism (13). A possible mechanism for the involvement of ERK/RSK1 in the persistent activation of NF-B is summarized schematically in Fig. 7.
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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-B activation may define the patterns of expression of NF-B-dependent genes. A clear example exists in cultured rat VSMCs, in which transient or persistent activation of NF-B leads to different combinations of NF-B-inducible gene products (14). The present study describes a novel mechanism for regulation of the duration of NF-B activation by ERK and RSK1. |
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FOOTNOTES |
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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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REFERENCES |
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P < 0.05 vs. control (no IL-1
