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IKK Plays an Essential Role in the Phosphorylation of RelA/p65 on Serine 536 Induced by Lipopolysaccharide¹

Fan Yang^*, Eric Tang, Kunliang Guan^, and Cun-Yu Wang^2,*,

^* Laboratory of Molecular Signaling and Apoptosis, Department of Biologic and Materials Sciences, Department of Biological Chemistry, and Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109

	Abstract

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Activation of the IB kinase (IKK) complex by LPS induces phosphorylation and degradation of IB, leading to the nuclear translocation of NF-B. Although it is essential for NF-B activation, emerging evidence has indicated that the nuclear translocation of NF-B is not sufficient to activate NF-B-dependent transcription. Here, we reported that LPS induced the phosphorylation of the p65 trans-activation domain on serine 536 in monocytes/macrophages. Using mouse embryonic fibroblasts lacking either IKK or IKK, we found that IKK played an essential role in LPS-induced p65 phosphorylation on serine 536, while IKK was partially required for the p65 phosphorylation. The LPS-induced p65 phosphorylation on serine 536 was independent of the phosphatidylinositol 3'-kinase/Akt signaling pathway. Furthermore, we found that the phosphorylation on serine 536 increased the p65 transcription activity. In summary, our results demonstrate that IKK plays an essential role in the LPS-induced p65 phosphorylation on serine 536, which may represent a mechanism to regulate the NF-B transcription activity by LPS.

	Introduction

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Lipopolysaccharides, a major component of the outer membrane of Gram-negative bacteria, is a primary inducer of chronic inflammatory diseases and septic shock (1). LPS stimulates host cells to produce a variety of proinflammatory cytokines and mediators, such as IL-1, TNF, and PGs, through activation of the transcription factor NF-B (2). NF-B is initially identified and named for its role in the control of Ig -chain gene expression in B lymphocytes. The prototypical form of NF-B is a heterodimer consisting of a DNA binding subunit (p50) and a trans-activation subunit (RelA/p65) and is ubiquitously expressed. In most unstimulated cells, NF-B is retained in the cytoplasm by IB family proteins. Upon stimulation by LPS or the proinflammatory cytokines TNF and IL-1, the IB kinase (IKK)³ complex is activated, resulting in the phosphorylation of IBs on two conserved N-terminal serine residues. The phosphorylated IBs are ubiquitinated and subsequently degraded by the 26S proteasome pathway, resulting in the nuclear translocation of NF-B (3, 4, 5). The IKK complex consists of two catalytic subunits, IKK and IKK, and a scaffold molecule, NF-B essential modulator (or IKK). Gene depletion studies demonstrate that IKK, but not IKK, plays an essential role in NF-B activation mediated by LPS, TNF, or IL-1 (3, 6, 7, 8, 9, 10, 11).

Although the phosphorylation of IB by IKK and the subsequent nuclear translocation of NF-B are the key steps for NF-B activation, recent evidence has indicated that the phosphorylation of NF-B subunit p65 modulates NF-B transcription activity (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21). Zhong et al. (14) found that LPS stimulated protein kinase A-dependent phosphorylation of p65 on serine 276 and subsequently recruited the transcription coactivators CREB binding protein (CBP)/p300 to potentiate NF-B transcription. Recently, additional studies from them demonstrated that in resting cells homodimers of either p65 or p50 interacted with the histone deacetylase HDAC1 and suppressed NF-B transcription. The phosphorylated p65 that is associated with CBP entered the nucleus and activated gene transcription by displacing p50-HDAC1 complexes (15). Wang et al. (16) found that TNF induced phosphorylation of the p65 trans-activation domain on serine 529 by casein kinase II. This phosphorylation also increased p65 transcription activity. Although it is unknown whether phosphorylation of p65 has any effect on the NF-B transcriptional activity, Sakurai et al. (17) reported that overexpression of IKK or IKK induced the phosphorylation of p65 on serine 536 in vivo and in vitro. Studies from Madrid et al. (18, 19) and Sizemore et al. (20) found that the cell survival kinase Akt induced NF-B transcription activity by stimulating the NF-B trans-activation domain, suggesting that NF-B plays an important role in the Akt-mediated cell survival. Sizemore et al. (21) further reported that both IKK and IKK were required for phosphatidylinositol 3'-kinase (PI3K)/Akt-mediated phosphorylation of the p65 trans-activation domain in response to IL-1 and TNF, although the phosphorylation sites remain to be identified.

Since LPS is also a potent activator of IKK, in this study (3) we performed experiments to determine whether LPS also induced phosphorylation of the p65 trans-activation domain in addition to the Rel homology domain identified by Zhong et al. (14). We found that LPS rapidly induced phosphorylation of the p65 trans-activation domain on serine 536 in macrophages/monocytes in an IKK-dependent manner. Using IKK^-/- and IKK^-/- mouse embryonic fibroblasts (MEFs), we found that IKK was essential for the LPS-induced p65 phosphorylation on serine 536, whereas loss of IKK only partially impaired LPS-induced p65 phosphorylation. In contrast, although phosphorylation of p65 on serine 536 was decreased, IKK was not essential for TNF-induced phosphorylation of p65 on serine 536. Furthermore, we found that the phosphorylation on serine 536 increased p65 transcription activity. These results suggest that IKK plays an essential role in LPS-induced p65 phosphorylation on serine 536 and provide new insight into the regulation of NF-B activation by LPS.

	Materials and Methods

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Cell culture and reagents

Wild-type, IKK^-/-, and IKK^-/- MEFs; p65^-/- MEFs; human THP.1 monocytes; and mouse RAW 264.7 macrophages were maintained in DMEM (Invitrogen, San Diego, CA) supplemented with 10% FCS, 100 µg/ml penicillin G, and 100 µg/ml streptomycin. The PI3K inhibitor LY294002 was purchased from Calbiochem (La Jolla, CA). Escherichia coli LPS was obtained from Sigma-Aldrich (St. Louis, MO), and TNF was purchased from Promega (Madison, WI).

Western blot analysis

Cells were treated with LPS (500 ng/ml) or TNF (20 ng/ml) for the indicated time period, harvested, and washed once with PBS. Cells were pelleted and lysed with lysis buffer containing 1% Nonidet P-40, 5% sodium deoxycholate, 1 mM PMSF, 100 mM sodium orthovanadate, and 1/100 protease inhibitor cocktails (Sigma-Aldrich). The protein concentration was determined according to the manufacturer’s protocol (Bio-Rad, Hercules, CA). Whole-cell lysates were subjected to 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membranes were blotted with 5% dry milk overnight at 4°C and probed with the primary Abs. The immunocomplexes were visualized with HRP-coupled goat anti-rabbit or anti-mouse IgG (Promega) using the ECL reagents (Amersham Pharmacia Biotech, Arlington Heights, IL). The nuclear extracts were prepared as described previously (22, 23, 24). The primary Abs were obtained from the following sources: anti-histone H1, anti-IKK, anti-IKK, and anti-IB polyclonal Abs from Santa Cruz Biotechnology (Santa Cruz, CA); and anti-phospho-p65 (serine 536) polyclonal Abs and anti-phospho-IB (serine 32) polyclonal and mAbs from Cell Signaling (Beverly, MA).

Plasmids, transient transfection, and NF-B luciferase reporter assay

The pCMV2-Flag-tagged p65 (p65) and p65-S529A containing an alanine substitution on serine 529 were provided by Dr. A. Baldwin (University of North Carolina, Chapel Hill, NC). The wild-type p65 expression vector was used as a template to make the p65-S536A mutant. The p65-S529 expression vector was used as a template to make the p65-S2A mutant containing an alanine substitution on both serine 529 and 536. The mutagenesis for both p65-S536A and p65-S2A was performed according to the manufacturer’s instructions (Stratagene, La Jolla. CA). The p65^-/- MEFs were cotransfected with 2x NF-B luciferase reporter plasmids with p65 or mutant p65 expression vectors using Lipofectamine reagents according to the manufacturer’s instructions (Invitrogen). Cells were cotransfected with the pRL-TK Renilla luciferase reporter for normalizing transfection efficiency. Luciferase activities were measured with a dual luciferase system (Promega).

Retroviral infection and stable cell lines

The full-length p65 or p65-S536A cDNA was subcloned into the retroviral vector pBabe-puromycin. Retroviruses were generated by transfecting the vectors into 293T cells, and retrovirus-containing supernatants were collected and stored at -70°C. The p65^-/- MEFs were infected with retroviruses in the presence of 6 µg/ml polybrene (Sigma-Aldrich). Forty-eight hours after infection, cells were selected with puromycin (1.5 µg/ml) for 1 wk. The resistant cells were pooled and examined by Western blot analysis.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
LPS induced phosphorylation of the p65 trans-activation domain on serine 536 in monocytes/macrophages

In addition to the stimulation of IB phosphorylation, LPS has been found to induce phosphorylation of the p65 Rel homology domain, an event that promotes the interaction of p65 with the transcription coactivator CBP/p300 and enhances NF-B transcription (14, 15). To determine whether LPS stimulated phosphorylation of the p65 trans-activation domain, we used a newly available anti-phospho-p65 Ab that specifically detects the phosphorylated form of p65 on serine 536 on the trans-activation domain. Mouse Raw 264.7 macrophage-like cells were treated with LPS isolated from E. coli (500 ng/ml) for various time periods, and whole-cell extracts were subjected to Western blot analysis. As shown in Fig. 1A, LPS rapidly induced p65 phosphorylation on serine 536. Since IB phosphorylation on serine 32 and 36 is solely mediated by IKK (2, 3, 4, 5, 6, 25, 26), we also determined LPS-induced IKK activation by examining IB phosphorylation. As shown in Fig. 1B, LPS stimulated IKK activity, which was concurrent with the phosphorylation of p65. While the level of p65 protein remained unchanged (Fig. 1A), phosphorylated IB was degraded by the proteasome pathway (Fig. 1B). To determine whether the phosphorylated p65 was translocated to nucleus, cells were fractionated, and nuclear proteins were isolated. As shown in Fig. 1C, phosphorylated p65 was detected in the nuclear extracts following LPS stimulation. Similarly, LPS stimulated p65 phosphorylation on serine 536 in human THP.1 monocytes, which was concurrent with IKK activation induced by LPS (Fig. 1, D and E). We also tested whether LPS from other pathogens induced p65 phosphorylation. LPS from Porphyromonas gingivalis, a common pathogen of chronic oral inflammatory diseases, has been found to induce chronic oral inflammation and inflammatory bone resorption (1). We found that, like LPS from E. coli, P. gingivalis LPS stimulated phosphorylation of p65 on serine 536 and activated IKK in macrophages (data not shown).

View larger version (35K): [in this window] [in a new window]   FIGURE 1. LPS from E. coli induced the phosphorylation of p65 trans-activation domain on serine 536 in monocytes/macrophages. A, LPS-induced p65 phosphorylation in mouse Raw 264.7 macrophage-like cells. Cells were treated with LPS from E. coli (500 ng/ml) for the indicated time periods. The whole-cell extracts were prepared as described in Materials and Methods. Fifty-microgram aliquots of extracts were probed with anti-phospho-p65 (536) polyclonal Abs (1/1000; upper panel). For loading control, the blots were stripped and reprobed with anti-p65 polyclonal Abs (1/5000; lower panel). B, LPS-induced phosphorylation and degradation of IB. Thirty-microgram aliquots of extracts from A were probed with anti-phospho-IB Abs (1/1000; upper panel) and anti-IB Abs (1/500; lower panel). C, The phosphorylated p65 was detected in the nucleus. Cells were treated with LPS for the indicated time points. The nuclear proteins were isolated, and 20-µg aliquots of extracts were probed with anti-phospho-p65 Abs. For loading control, the blots were stripped and reprobed with anti-histone H1 Abs. D, LPS-induced p65 phosphorylation in human THP.1 monocytes. Human THP.1 monocytes were treated with LPS (500 ng/ml), and Western blot analysis was performed as described in A. E, LPS-induced phosphorylation and degradation of IB in human THP.1 monocytes. Each experiment was performed three times.

IKK was essential for LPS-induced p65 phosphorylation on serine 536

Studies by Sakurai et al. (17) found that the activation of the IKK complex by overexpressing IKK or IKK induces p65 phosphorylation on serine 536. Since the IKK complex contains two related catalytic kinases, IKK and IKK, we were interested to determine the functional role of IKK and IKK in LPS-induced p65 phosphorylation on serine 536 using IKK^-/- and IKK^-/- MEFs. As in macrophages and monocytes, LPS rapidly induced p65 phosphorylation on serine 536 and stimulated IKK activity in wild-type MEFs (Fig. 2, A and B, lanes 1–4). Compared with wild-type cells, LPS-induced p65 phosphorylation on serine 536 in IKK^-/- MEFs was partially reduced (Fig. 2A, lanes 5–8). LPS-induced IB phosphorylation was also decreased in IKK^-/- MEFs (Fig. 2B, lanes 5–8). Strikingly, LPS was unable to induce p65 phosphorylation on serine 536 in IKK^-/- MEFs (Fig. 2A, lanes 9–12), indicating that IKK played an essential role in LPS-induced p65 phosphorylation. As shown in Fig. 2B (lanes 9–12), LPS also failed to induce the phosphorylation and degradation of IB in IKK^-/- cells. Wild-type MEFs expressing both IKK and IKK (Fig. 2C, lanes 1–4), IKK^-/- MEFs lacking IKK (Fig. 2C, lanes 5–8), and IKK^-/- MEFs lacking IKK (Fig. 2C, lanes 9–12) were confirmed by Western blot analysis. Collectively, our results demonstrate that IKK plays an essential role in LPS-induced p65 phosphorylation on serine 536.

View larger version (73K): [in this window] [in a new window]   FIGURE 2. IKK plays an essential role in LPS-induced p65 phosphorylation on serine 536. A, IKK, but not IKK, was essential for LPS-induced p65 phosphorylation. Wild-type, IKK-/-, and IKK MEFs were treated with LPS (500 ng/ml) for the indicated times, and whole-cell lyses were prepared. Fifty-microgram aliquots of extracts were probed with anti-phospho-p65 (536) Abs. For loading control, blots were stripped and reprobed with anti-p65 Abs. WT, wild type. B, IKK was essential for LPS-induced phosphorylation and degradation of IB. Thirty-microgram aliquots of cell extracts from A were probed with anti-phospho-IB Abs and anti-IB Abs, respectively. C, Confirmation of wild-type, IKK-/-, and IKK-/- MEFs. Fifty-microgram aliquots of cell extracts from A were probed with anti-IKK (1/1000) and anti-IKK (1/1000) Abs, respectively. Each experiment was repeated three times.

IKK, but not IKK, was important in TNF-induced p65 phosphorylation on serine 536

Since TNF has been implicated in stimulation of phosphorylation of the p65 trans-activation domain (16), we examined TNF-induced p65 phosphorylation using IKK^-/- and IKK^-/- MEFs. Cells were treated with TNF (20 ng/ml), and whole-cell extracts were examined by Western blot analysis. As shown in Fig. 3A (lanes 1–4), TNF rapidly induced p65 phosphorylation on serine 536 in wild-type MEFs. Compared with wild-type MEFs, the p65 phosphorylation stimulated by TNF was slightly reduced in IKK^-/- MEFs (Fig. 3A, lanes 5–8), indicating that IKK was not essential for TNF-induced p65 phosphorylation. Although it was dramatically impaired, TNF was still capable of inducing p65 phosphorylation on serine 536 in IKK^-/- cells (Fig. 3A, lanes 9–12). This suggests that IKK is important, but not essential, for TNF-induced p65 phosphorylation on serine 536. Consistent with previous studies, TNF-induced IB phosphorylation and degradation were severely impaired in IKK^-/- MEFs (Fig. 3B, lanes 9 and 10), but not in wild-type MEFs or IKK^-/- MEFs (lanes 1–8). Collectively, these results suggest that IKK plays a more critical role than IKK in TNF-induced p65 phosphorylation on serine 536.

View larger version (48K): [in this window] [in a new window]   FIGURE 3. IKK plays an important role in TNF-induced p65 phosphorylation on serine 536. A, TNF-induced p65 phosphorylation decreased in IKK-/- MEFs. Wild-type, IKK-/-, and IKK-/- MEFs were treated with TNF (20 ng/ml) for the indicated periods, and whole-cell extracts were prepared. Fifty-microgram aliquots of extracts were probed with anti-phospho-p65 (536) Abs (upper panel). For the loading control, the membranes were stripped and reprobed with anti-65 Abs (lower panel). WT, wild type. B, TNF-induced phosphorylation and degradation of IB. Fifty-milligram aliquots of extracts were probed with anti-phospho-IB (upper panel) and anti-IB Abs (lower panel), respectively. Each experiment was repeated three times.

Several studies reported that the PI3K/Akt signaling pathway activated NF-B by targeting the p65 trans-activation domain. Recently, Sizemore et al. (21) reported that IKK is required for both IB and p65 phosphorylation, and IKK is required solely for phosphorylation of the p65 trans-activation domain induced by Akt. Nevertheless, the phosphorylation site(s) was unknown in their studies. Therefore, we wanted to determine whether the TNF- or LPS-induced phosphorylation of p65 on serine 536 was dependent on the PI3K/Akt signaling pathway. To inhibit Akt activation, the specific chemical inhibitor LY294002 was used. Cells were pretreated with LY294002 and then treated with TNF (20 ng/ml). As shown in Fig. 4 (top panel, lanes 1–5), TNF-induced p65 phosphorylation on serine 536 was not inhibited by LY294002 in wild-type MEF cells. Also, LY294002 did not have any effect on TNF-induced p65 phosphorylation in IKK^-/- or IKK^-/- MEFs (lanes 6–15), indicating that neither IKK- nor IKK-induced p65 phosphorylation was dependent on PI3K/Akt activation. As a positive control, pretreatment of LY294002 suppressed basal and TNF-induced AKT phosphorylation, as detected with anti-phospho-Akt Ab (Fig. 4, bottom panel). Additionally, LY294002 did not inhibit the phosphorylation and degradation of IB induced by TNF (data not shown).

View larger version (48K): [in this window] [in a new window]   FIGURE 4. TNF-induced p65 phosphorylation is independent of the PI3K/Akt signaling pathway. Wild-type, IKK-/-, and IKK-/- MEFs were pretreated with LY294002 (10 µM) for 30 min and then stimulated with TNF (20 ng/ml) for the indicated time periods. Cells were harvested, and whole-cell extracts were prepared. Fifty-microgram aliquots of extracts were probed with anti-phospho-p65 (536) Abs (top panel). For loading control, the membrane was stripped and reprobed with anti-p65 Abs (middle panel). Akt phosphorylation was probed with anti-phospho-Akt Abs (1/1000) (bottom panel). WT, wild-type MEFs; IKK-/-, IKK-/- MEFs; IKK-/-, IKK-/- MEFs. Each experiment was performed three times.

View larger version (64K): [in this window] [in a new window]   FIGURE 5. LPS-induced phosphorylation on serine 536 is independent of the PI3K/Akt signaling pathway. Both wild-type and IKK-/- MEFs were pretreated with LY294002 (10 µM) and then stimulated with LPS (500 ng/ml) for the indicated times. Fifty-microgram aliquots of extracts were probed with anti-phospho-p65 Abs (top panel). For the loading control, the membrane was stripped and reprobed with anti-p65 Abs (second panel). Fifty-microgram aliquots of extracts were probed with anti-phospho-IB Abs (third panel) and anti-IB Abs (bottom panel), respectively. Each experiment was repeated three times.

The p65 phosphorylation on serine 536 by IKK enhanced p65 transcription activity

Next, we were interested to determine whether the phosphorylation on serine 536 induced by LPS or TNF modulated p65 transcription activity using the NF-B-dependent luciferase reporter assay. To minimize the side effects due to the overexpression of p65 proteins, transfection experiments were performed in p65-deficient (p65^-/-) MEFs. As shown in Fig. 6A, the NF-B-dependent luciferase reporter was increased 5-fold by transfection of the wild-type p65 expression vector (p65). In comparison, the fold activation of NF-B reporter induced by mutant p65-S536A (alanine 536) or p65-S2A (alanine 529 and 536) was significantly compromised (35% reduction), perhaps suggesting that the basal phosphorylation of serine 536 due to p65 overexpression had effects on p65 transcription activity. As a control, an alanine substitution on serine 529 (p65-S529A), which was the phosphorylation site for casein kinase II (16), had no effect on p65-induced NF-B activity. As shown in Fig. 6B (lanes 1–5), we confirmed that p65, p65-S529A, p65-S536A, and p65-S2A were equivalently expressed in p65^-/- MEFs, indicating that the reduction of NF-B activation by mutant p65 was not due to the level of protein expression. Since our results indicated that IKK was essential for LPS-induced p65 phosphorylation on serine 536, we tested whether IKK could directly enhance p65 transcription activity that was dependent on serine 536. As shown in Fig. 6A, transfection of IKK significantly potentiated the transcriptional activity of wild-type p65. An alanine mutation on serine 529 (p65-S529A) had no effect on the potentiation of p65 transcription activity mediated by IKK, indicating that serine 529 was not critical for IKK stimulation. In contrast, an alanine mutation at serine 536 (p65-S536A) or at both serine 529 and 536 (p65S2A) severely reduced the potentiation of p65 transcription activity induced by IKK, indicating that phosphorylation on serine 536 was a key site for IKK-potentiated p65 transcriptional activity. As shown in Fig. 6B (lanes 6–10), wild-type p65 and p65 mutants were expressed at similar levels. Also, we determined whether the phosphorylation on serine 536 by IKK modulated p65 transcription activity in human THP.1 monocytes. As shown in Fig. 6C, transfection of IKK also significantly enhanced the transcriptional activity of wild-type p65, but not that of p65-S536A. Transfection of IKK moderately increased the transcriptional activity of wild-type p65, but not that of p65-S536A. Collectively, these results indicate that the phosphorylation of the p65 trans-activation domain by IKK on serine 536 increases p65 transcription activity. Serine 536, but not serine 529, is a key site for IKK-stimulated enhancement of NF-B transcription.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. Phosphorylation on serine 536 by IKK increases p65 transcription activity. A, IKK enhanced the transcriptional activity of p65 through phosphorylation on serine 536 in MEFs. The p65-/- MEFs were cotransfected with 2x B luciferase reporter, the indicated p65 expression vectors, and IKK expression vector or control empty vector by Lipofectamine. Cells were cotransfected with pRK-Renilla for the internal normalization. Twenty-four hours after transfection, cells were lysed, and luciferase activities were measured with a dual-luciferase system. The fold activation was determined by comparison with basal luciferase activity when cells were cotransfected with 2x B luciferase reporter and empty vector. The assay was performed in duplicate, and the activation value represents the mean ± SD from three independent experiments. *, p < 0.01. B, The expression of wild-type p65 and p65 mutants. One hundred-microgram aliquots of extracts from A were probed with mAbs against Flag epitope (1/1000). C, IKK enhanced the transcriptional activity of p65 through the phosphorylation on serine 536 in human THP.1 monocytes. Cell transfection was performed as described in A. The assay were performed in duplicate, and the activation value represents the mean ± SD from three independent experiments. *, p < 0.01.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. The phosphorylation of p65 on serine 536 induced by LPS plays a critical role in NF-B transcriptional activity. A, The reconstitution of p65-/- MEFs with wild-type p65 or p65-S536A mutant. The p65-/- MEFs were transduced with the retroviruses expressing p65, p65-S536A, or the control vector and selected with puromycin (1.5 µg/ml) for 1 wk. The resistant clones were pooled and probed with polyclonal Abs against p65. For loading control, the membrane was stripped and reprobed with mAbs against -tubulin. B, LPS-induced NF-B activity was impaired by p65-S536A mutant. The p65-WT, p65-S536A, or control cells were transfected with 2x B luciferase reporter using Lipofectamine reagents. Cells were cotransfected with pRK-Renilla for the internal normalization. Twenty-four hours after transfection, cells were treated with LPS for 16 h, and luciferase assay was performed as described in Fig. 6A. The fold activation was determined by comparison with basal luciferase activity of p65-WT cells. The assay was performed in duplicate, and the activation value represents the mean ± SD. *, p < 0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Madrid et al. (18, 19) have found that Akt stimulated NF-B activity by targeting the trans-activation domain of NF-B, which was dependent on IKK. Studies by Sizemore et al. (20, 21) demonstrated that PI3K/Akt played an important role in the phosphorylation and trans-activation of p65 in response to IL-1 and TNF and that Akt-mediated NF-B activation required both IKK and IKK. In this regard, our results support the idea that phosphorylation of the p65 trans-activation domain by IKK plays an important role in NF-B transcription. Furthermore, using IKK-deficient MEFs, we found that IKK, but not IKK, played an essential role in LPS-induced p65 phosphorylation on serine 536. Our results have significantly extended studies by Madrid et al. (18, 19) and Sizemore et al. (20, 21). However, studies by Sizemore et al. (21) suggested that the PI3K/Akt signaling pathway played a critical role in TNF- and IL-1-induced p65 phosphorylation on the trans-activation domain. In contrast, our results demonstrated that phosphorylation of the p65 trans-activation domain on serine 536 induced by TNF or LPS was independent of the PI3K/Akt signaling. We also found that PI3K/Akt inhibitor could not inhibit LPS- and TNF-mediated transcription activity (unpublished observation). Currently, we cannot provide an explanation for this difference.

IKK, but not IKK, has been found to play an essential role in both TNF- and LPS-stimulated NF-B activation (3, 6, 7, 8, 9, 10). Interestingly, we found that LPS-induced p65 phosphorylation in IKK^-/- MEFs was partially reduced and totally abolished in IKK^-/- MEFs, indicating that IKK also plays an essential role in LPS-induced phosphorylation of p65 on serine 536. However, compared with LPS stimulation, the effects of TNF-induced p65 phosphorylation on serine 536 were less severe in both IKK^-/- and IKK^-/- MEFs. These results might reflect the difference between the two stimuli. LPS induces NF-B activation, which is dependent on TNF receptor-associated factor 6 (TRAF6) (2, 3). TRAF6, together with the ubiquitin-conjugating enzyme Ubc13/Uev1A, catalyzes the formation of a Lys⁶³ (K63)-linked polyubiquitin chain that stimulates TGF-activated kinase 1 and the subsequent activation of IKK (28). Although it is not fully understood how IKK is activated by TNF, according to the studies by Devin et al. (29, 30), TNF activates IKK by stimulating the relocalization of IKK and IKK to the TNF receptor 1 complex via TRAF2. Therefore, it is likely that the activation of the IKK complex induced by different mechanisms may play a distinct role in the differential regulation of p65 phosphorylation on serine 536 by IKK or IKK.

Zhong et al. (14, 15) reported that LPS-induced p65 phosphorylation on serine 276 stimulated p65 transcription activity by promoting its interaction with the transcription coactivator CBP/p300. Our studies presented here identified a new phosphorylation site of p65 induced by LPS that was located on serine 536 in the p65 trans-activation domain. Importantly, the phosphorylation on serine 536 potentiated p65 transcription activity. Currently, we do not know how the phosphorylation on serine 536 regulates p65 transcription activity. We failed to detect any effect of the phosphorylation of serine 536 on the interaction between p65 and the transcription coactivators CBP/p300 using a coimmunoprecipitation assay. It is possible that LPS-induced p65 phosphorylation on serine 536 may modify the interaction of p65 with other transcription factors and/or chromatin remodeling factors. In the future it will be of interest to identify these factors to fully understand the mechanisms underlying NF-B activation.

	Acknowledgments

 
We thank Dr. Inder Verma for providing IKK^-/- and IKK^-/- MEFs, and Drs. Dan Wang and Al Baldwin for plasmids.

	Footnotes

 
¹ This work was supported by National Institute of Dental and Craniofacial Research Grants R01DE13335 and R01DE13848 (to C.-Y.W.).

² Address correspondence and reprint requests to Dr. Cun-Yu Wang, Laboratory of Molecular Signaling and Apoptosis, Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, MI 48109-1078. E-mail: cunywang{at}umich.edu

³ Abbreviations used in this paper: IKK, IB kinase; MEF, mouse embryonic fibroblast; PI3K, phosphatidylinositol 3'-kinase; TRAF, TNF receptor-associated factor; CBP, CREB binding protein.

Received for publication August 28, 2002. Accepted for publication March 25, 2003.

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