J Biol Chem, Vol. 274, Issue 43, 30353-30356, October 22, 1999

COMMUNICATION
IB Kinases Phosphorylate NF-B p65 Subunit on Serine 536 in the Transactivation Domain^*

Hiroaki Sakurai, Hiroaki Chiba, Hidetaka Miyoshi, Takahisa Sugita, and Wataru Toriumi

From the Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505, Japan

	ABSTRACT

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Recent investigations have elucidated the cytokine-induced NF-B activation pathway. IB kinase (IKK) phosphorylates inhibitors of NF-B (IBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-B. We have examined the possibility that IKK can phosphorylate the p65 NF-B subunit as well as IB in the cytokine-induced NF-B activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF- in a time dependent manner similar to IB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKK efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-B-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-B·IB complex.

	INTRODUCTION

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The transcription factor nuclear factor-B (NF-B)¹ plays a pivotal role in inflammatory and immune responses (1-3). NF-B is composed of a heterodimer of p65 and p50 subunits in most cell types and is sequestered in the cytoplasm by its inhibitor proteins, the IBs (4-8). Several NF-B-activating agents, including pro-inflammatory cytokines, phorbol esters, and bacterial lipopolysaccaride, induce the phosphorylation of IBs at two NH₂-terminal Ser residues. The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, thereby releasing NF-B to enter the nucleus for gene expression (9-15).

Recent investigations have focused on the phosphorylation of IBs and clearly elucidated the molecular mechanisms of the phosphorylation. In brief, two closely related kinases, designated IB kinase (IKK)  and IKK, have been identified as components of the multiprotein IKK complex (500-900 kDa) that directly phosphorylates the critical Ser residues of IBs (16-20). IKK and IKK together form a heterodimer through their COOH-terminal leucine zipper motifs, and the functional IKK complex contains both IKK subunits. NF-B-inducing kinase (NIK), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, interacts with and activates the IKK complex (21). Other MAP3Ks, including transforming growth factor- activated kinase 1 (TAK1) (22-24), MAPK/extracellular signal-regulated kinase kinase kinases (MEKK1-3) (25-28), and Cot/Tpl2 (29), have been shown to be involved in the IKK activation pathways, indicating the important roles of MAP3K family kinases in the IKK activation by diverse extracellular stimuli.

The activity of several inducible transcription factors, including cAMP response element-binding protein (CREB) (30) and c-Jun (31), has been shown to be regulated by phosphorylation. It has been shown that the p65 NF-B subunit is also phosphorylated during the phosphorylation and degradation of IBs. However, the cytokine-inducible phosphorylating activity of p65 remains to be characterized. Here we show that IKKs are possible p65 kinases in the TNF--induced NF-B activation, and the phosphorylation site is Ser-536 in the COOH-terminal transactivation domain.

	MATERIALS AND METHODS

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Cell Cultures-- HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in 5% CO₂. Cells were treated with 20 ng/ml TNF- (R & D Systems) for the indicated time period. Where indicated, cells were treated with a proteasome inhibitor, N-acetyl-leucyl-leucyl-norleucinal (Nacalai Tesque).

Nuclear Translocation of p65 and Degradation of IB-- After stimulating with TNF-, cytoplasmic and nuclear extracts were prepared as described previously (23). The nuclear translocation of p65 and the degradation of IB were determined by Western blotting with an anti-p65 antibody (C-20; Santa Cruz Biotechnology) and an anti-IB antibody (C-21; Santa Cruz Biotechnology) using nuclear and cytoplasmic extracts, respectively.

In Vivo Phosphorylation of p65-- For metabolic labeling with [³²P]orthophosphate, HeLa cells were washed twice with phosphate-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and subsequently incubated with 1 mCi/ml [³²P]orthophosphate for 3 h. After stimulating the cells with TNF- for a given period, we immunoprecipitated p65 or HA-p65 with the anti-p65 antibody or anti-HA antibody as described previously. The precipitated proteins were separated by 7.5% SDS-PAGE and autoradiographed.

Generation of GST-fused p65-- The cDNA encoding full-length p65 was obtained from HeLa cells by reverse transcription-polymerase chain reaction. Several deletion cDNAs were inserted into pGEX-5X-3 bacterial expression vector (Amersham Pharmacia Biotech). The GST-fused p65 proteins were expressed in Escherichia coli and purified with glutathione-Sepharose (Amersham Pharmacia Biotech). Point mutations were made by using a QuikChange site-directed mutagenesis kit (Stratagene) and all of the mutations were verified by DNA sequencing analysis.

Transfection and Baculovirus Expression-- HeLa cells were transfected with expression vectors for Xpress (XP) epitope-tagged IKKs, FLAG epitope-tagged TAK1, HA epitope-tagged TAB1, and HA epitope-tagged p65 using LipofectAMINE reagents (Life Technologies, Inc.). For expression of IKK, NIK, and TAK1/TAB1 as 6 × His-tagged proteins in the Baculovirus system, Sf21 insect cells were infected with recombinant viruses generated by co-transfection with the BaculoGold DNA and transfer vectors (pAcHLT-NIK, pAcHLT-IKK, or pAcUW51-TAK1/TAB1) (PharMingen). The recombinant kinases were purified by nickel column chromatography (Amersham Pharmacia Biotech).

In Vitro Phosphorylation of p65-- TNF--stimulated whole cell lysates, cytoplasmic extracts, immunoprecipitated IKKs, or Baculovirus-expressed recombinant IKK were incubated with 1 µg of GST-fused p65 in kinase buffer (20 mM HEPES (pH 7.6), 20 mM MgCl₂, 2 mM dithiothreitol, 20 µM ATP, 20 mM -glycerophosphate, 20 mM disodium p-nitrophenyl phosphate, 0.1 mM sodium orthovanadate, 3 µCi [-³²P]ATP) at 30 °C for 30 min. The phosphorylated GST-p65 was separated by 10% SDS-PAGE and autoradiographed.

	RESULTS

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TNF--induced Phosphorylation of p65 in Vivo-- Treatment of HeLa cells with TNF- induced the degradation of IB and the subsequent nuclear translocation of p65 NF-B within 5 min after the treatment (Fig. 1A). A phosphorylated form of IB was detected at 2-5 min, indicating an inducible kinase activity of the endogenous IKK complex (Fig. 1A). Interestingly, an in vivo ³²P metabolic labeling immunoprecipitation analysis showed that p65 was also phosphorylated at the time of the IB phosphorylation (Fig. 1B). To establish whether the phosphorylation of p65 occurred in the cytoplasm or in the nucleus, the cells were treated with a proteasome inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN). The treatment with ALLN caused an accumulation of the phosphorylated form of IB, resulting in an impaired nuclear translocation of p65 (Fig. 1C). In contrast, the phosphorylated p65 could be detected even in the presence of ALLN, indicating that the phosphorylation occurred in the cytoplasm prior to the nuclear translocation (Fig. 1D).

View larger version (53K): [in this window] [in a new window]   Fig. 1.   Phosphorylation of p65 in vivo. A, HeLa cells were stimulated with recombinant TNF- (20 ng/ml) for the indicated time period. Nuclear or cytoplasmic extracts were evaluated by Western blotting with p65 and IB antibodies, respectively. P-IB indicates a phosphorylated form of IB. B, cells were labeled with [32P]orthophosphate for 3 h and were either left untreated or were treated with TNF-. After harvest, whole cell lysates were immunoprecipitated with the anti-p65 antibody (upper panel). P-p65 indicates a phosphorylated form of p65. The p65 expression level in lysates was determined by Western blotting with the p65 antibody (lower panel). C and D, cells were pretreated with ALLN (100 µM) for 15 min. After stimulating with TNF- for 5 min, nuclear translocation of p65 and phosphorylation and degradation of IB (C), and phosphorylation of p65 in whole cell lysates (D), were determined by the procedure described in A and B, respectively.

p65 Phosphorylating Activity in HeLa Cells-- To characterize the p65 kinase activity in vitro, we generated NH₂-terminal (from amino acid 1 to 305) and COOH-terminal (from amino acid 354 to 551) p65 proteins fused with GST. An inducible kinase activity was detected in whole cell lysates of TNF--stimulated HeLa cells when the COOH-terminal p65 was used as a substrate (Fig. 2A). Zhong et al. (32) reported that Ser-276 of p65 was phosphorylated by PKA; however, the TNF--induced p65 kinase did not phosphorylate GST-p65-(1-305) containing the Ser residue (Fig. 2A). Interestingly, the in vitro p65 phosphorylating activity was induced in a time-dependent manner similar to the phosphorylation of p65 in vivo (Fig. 1B). In addition, the activity was extracted into the cytoplasmic fraction (Fig. 2B), suggesting that the p65 phosphorylating activity was efficiently extracted from TNF--treated HeLa cells.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Phosphorylation of p65 in vitro. A, HeLa cells were treated with TNF- for the indicated time period. Whole cell lysates were prepared and then in vitro kinase assays (KA) were performed with GST-p65-(1-305) or GST-p65-(354-551) as a substrate. The GST-fused p65 proteins used in the kinase reaction were stained with CBB. B, GST-p65-(354-551) was phosphorylated with cytoplasmic (Cyto.) or nuclear (Nucl.) extracts prepared from untreated or TNF--stimulated cells.

Determination of the Phosphorylation Site-- We next determined the phosphorylation site in the COOH-terminal p65 using the TNF--treated cytoplasmic extracts as a kinase source. GST-p65-(354-521), a mutant lacking the 30 NH₂-terminal amino acids (TA1 domain), failed to be phosphorylated by the activity (Fig. 3A). Wang et al. (33) recently reported that TNF- induced p65 phosphorylation at Ser-529 in this domain. In contrast, our in vitro kinase assays using Ser to Ala substitution mutants indicate that the phosphorylation site is Ser-536 (Fig. 3B). In contrast to Ser-529, the target Ser residue is conserved in human, mouse, chicken, and Xenopus p65 subunits (Fig. 3C), suggesting a role for the phosphorylation in the transactivation of NF-B.

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Determination of the phosphorylation site on p65. Several deletion (A) and Ser to Ala substitution (B) mutants of GST-p65 were phosphorylated with cytoplasmic extracts (Cyto. ext.) from TNF--treated HeLa cells by in vitro kinase assays (KA). C, structure of the p65 subunit and sequences of the last 30 COOH-terminal amino acids (TA1 domain) of human, mouse, chicken, and Xenopus p65. p65 consists of a DNA-binding and dimerization domain (RHD), nuclear localization signal (NLS), and transactivation domains (TA1 and TA2). The phosphorylation site in human p65 is conserved in mouse, chicken, and Xenopus p65 subunits.

IKK-mediated Phosphorylation of p65 in Vitro-- We previously reported that the endogenous IKK kinase activity was induced by TNF- in a time-dependent manner similar to the p65 phosphorylating activity (23). We therefore investigated whether the p65 kinase is a component of the IKK complex. The endogenous IKK complex was examined for kinase activity against GST-p65-(354-551) and GST-IB-(1-54) (Fig. 4A). Activity could be detected for GST-p65-(354-551), which was comparable with the IKK activity for GST-IB-(1-54). Moreover, the p65 phosphorylating activity was competed by an excess amount of GST-IB-(1-54), but not GST-c-Jun-(1-79) (Fig. 4B). We further examined whether two IKK subunits can phosphorylate the p65 subunit by using an overexpression experiment (Fig. 4C). HeLa cells were transfected with expression vectors for Xpress-epitope tagged IKKs (XP-IKKs) with or without expression vectors for FLAG-epitope tagged TAK1 (FLAG-TAK1) and the TAK1 activator, hemagglutinin-epitope-tagged TAB1 (HA-TAB1). An anti-XP immunocomplex kinase assay showed that TAK1-activated IKKs phosphorylated p65 (Fig. 4C). These results suggest that the p65 kinase is a component of the IKK complex.

View larger version (56K): [in this window] [in a new window]   Fig. 4.   Phosphorylation of p65 by IKKs in vitro. A, HeLa cells were treated with TNF- for the indicated time period. The endogenous IKK complex was immunoprecipitated (IP) with anti-IKK antibody and was examined kinase activities (KA) for GST-p65-(354-551) or GST-IB-(1-54). To monitor the expression of IKK, whole cell lysates were immunoblotted (WB) with the anti-IKK antibody. B, GST-p65-(354-551) (1 µg) was phosphorylated with the anti-IKK immunoprecipitates in the absence or presence of an excess amount (10 µg) of GST-IB-(1-54) or GST-c-Jun-(1-79). C, HeLa cells (1 × 106/60 mm dish) were transfected with expression vectors (1 µg each) for XP-IKKs, FLAG-TAK1, and HA-TAB1. The total amount of DNA was adjusted with an empty vector at 3 µg. Anti-XP-immunoprecipitates were evaluated in an in vitro phosphorylation assay with GST-p65-(354-551). The expression levels of the tagged proteins were monitored by Western blotting (WB) with anti-XP, anti-FLAG, and anti-HA antibodies. D, recombinant TAK1/TAB1, NIK, and IKK were incubated with wild type (WT) or mutant (SA) GST-p65-(354-551). Auto indicates autophosphorylation activities.

Some protein kinases, such as TAK1, NIK, IKK, and IKK, have been shown to be components of the IKK complex. We generated recombinant TAK1, NIK, and IKK as 6 × His-tagged proteins by using the Baculovirus expression system and examined their abilities to phosphorylate GST-p65-(354-551) (Fig. 4D). Only IKK could phosphorylate GST-p65, whereas TAK1 and NIK showed autophosphorylation activities. The site of phosphorylation by IKK was also Ser-536. These results indicate that the p65 phosphorylation may be mediated by IKKs in vitro.

IKK-mediated Phosphorylation of p65 in Vivo-- We next investigated whether p65 is a substrate for IKKs in vivo by co-transfection and metabolic labeling analyses. HA-p65 was transiently co-expressed in HeLa cells together with XP-IKK, XP-IKK, and FLAG-NIK. Cell lysates were immunoprecipitated with an anti-HA antibody and analyzed by SDS-PAGE (Fig. 5A). The phosphorylation of HA-p65 was detected when XP-IKKs were activated by the co-expression with Flag-NIK. In addition, the TNF--induced phosphorylation of p65 occurred at Ser 536, as demonstrated by the reduced phosphorylation of HA-p65 (S536A). Taken together, these results indicate that the p65 NF-B subunit is phosphorylated by IKKs in the cytokine-induced NF-B activation pathway.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Phosphorylation of p65 by IKKs in vivo. A, HeLa cells were transfected with expression vectors for HA-p65, XP-IKKs, and FLAG-NIK. Twenty-one h after the transfection, cells were labeled with [32P]orthophosphate for 3 h. Whole cell lysates were immunoprecipitated (IP) with anti-HA antibody. Loading control of the immunoprecipitated HA-p65 was determined by Western blotting (WB) with the anti-p65 antibody. Expression levels of XP-IKKs and FLAG-NIK in cell lysates were analyzed by Western blotting with anti-XP and anti-FLAG antibodies, respectively. B, HeLa cells (2 × 105 cells/12-well plate) were transfected with expression vectors for wild type HA-p65 or HA-p65 (S536A) (0.2 µg each), and FLAG-IB (0.5 µg). Twenty-one h after the transfection, cells were labeled with [32P]orthophosphate for 3 h and treated with TNF- for 5 min. Phosphorylation of HA-p65 was examined by an immunoprecipitation with the anti-HA antibody.

	DISCUSSION

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NF-B p65 has been shown to be phosphorylated along with phosphorylation of IB. In contrast to the IB phosphorylation, the p65 phosphorylation has not been well characterized. Here we show that TNF-induced phosphorylation of p65 is mediated by IKKs prior to the nuclear translocation. It is reasonable that p65 and IB are phosphorylated by the same protein kinases, since they associate in the cytoplasm and are phosphorylated in a similar time-dependent fashion in response to TNF-. Mercurio et al. (34) recently reported that p65, but not other Rel family members c-Rel and p52, was phosphorylated by a recombinant constitutive active mutant of IKK in vitro with a specificity constant similar to that for IB, suggesting a physiological role of the phosphorylation. There are two IKK subunits, and they form a homodimer or heterodimer in the IKK complex; however, the physiological role of the dimerization is still unclear. It is possible that one component of the dimer phosphorylates IB and the other phosphorylates p65. Recently, IKK- and IKK-deficient mice have been developed (35-40). The IKK complex derived from these mice may form a homodimer of the counterpart IKK subunit and showed intact kinase activities for p65 in vitro (37, 38). In contrast, IKK complex derived from IKK- deficient mice, but not IKK-, has impaired phosphorylation of IBs in vitro, indicating that the IKK homodimer might recognize p65, but not IBs, as a substrate. Future analysis of the three-dimensional structure of IKKs·NF-B·IB complex will elucidate the spatial localization of these components and the role of dimerization of IKKs in the phosphorylation of the NF-B·IB complex. In addition, the development of an IKK/IKK double knockout mouse will provide more information on the p65 phosphorylation.

The transcriptional activation domain (TAD) of p65 has been characterized by using fusion protein with the DNA-binding domain of the yeast GAL4 transcription factor (41-43). The COOH-terminal 30 amino acids (TA1 domain) comprise the most important transactivation domain, which is predicted to be -helix. In addition, there are seven Ser residues in the TA1 domain of human p65 and they locate on one face of the presumptive -helix, suggesting transcriptional regulation by phosphorylation. In fact, Wang et al. (33) recently reported that p65 was phosphorylated at Ser-529 in the TA1 domain by an undefined protein kinase in response to TNF-. Here we demonstrated the IKK-mediated phosphorylation of p65 at Ser-536 in the TA1 domain. In addition, Zhong et al. (32) reported that protein kinase A phosphorylated p65 at Ser-276 in the NH₂-terminal Rel homology domain (RHD), which promoted an interaction of p65 with the transcriptional co-activator CBP/p300. Furthermore, MAPK cascades that are sensitive to the MAPK kinase (MEK1, MEK2) inhibitor PD98059 and the p38 MAPK inhibitor SB203580 were shown to enhance the TNF--induced transactivation of the p65 subunit (44). Thus, these observations indicate that the NF-B transactivation may be regulated by multiple phosphorylations in TAD and RHD.

In summary, we demonstrated the IKK-mediated phosphorylation of p65 in the cytokine-induced NF-B activation pathway. Previous studies on the characterization of p65 TAD employed the GAL4 system. However, this system does not reflect inducible phosphorylations in the cytoplasm, because the fusion proteins translocated into the nucleus in a stimulus-independent manner. Therefore, the development of a new transactivation assay system evaluating the IKK-mediated phosphorylation is necessary for future characterization of the physiological role of the phosphorylation.

	ACKNOWLEDGEMENTS

We are grateful to Drs. M. Hibi and K. Hasegawa for materials. We are also grateful to Drs. K. Matsumoto and N. Yanaka for helpful discussions on the manuscript. We thank E. Yamada for DNA sequencing.

	FOOTNOTES

^* 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.

To whom correspondence should be addressed: Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505, Japan. Tel: 81-6-6300-2571; Fax: 81-6-6300-2593; E-mail: hsakurai@tanabe.co.jp.

	ABBREVIATIONS

The abbreviations used are: NF-B, nuclear factor-B; IKK, IB kinase; NIK, NF-B-inducing kinase; MAP3K, mitogen-activated protein kinase kinase kinase; TNF, tumor necrosis factor; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; ALLN, N-acetyl-leucyl-leucyl-norleucinal; XP, Xpress; TAD, transcriptional activation domain; RHD, Rel homology domain; TAK1, transforming growth factor- activated kinase 1; TAB1, TAK1-binding protein 1.

	REFERENCES

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