IκB Kinases Phosphorylate NF-κB p65 Subunit on Serine 536 in the Transactivation Domain*

Recent investigations have elucidated the cytokine-induced NF-κB activation pathway. IκB kinase (IKK) phosphorylates inhibitors of NF-κB (IκBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-κB. We have examined the possibility that IKK can phosphorylate the p65 NF-κB subunit as well as IκB in the cytokine-induced NF-κB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-α in a time dependent manner similar to IκB 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 IKKβ 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-κB-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-κB·IκB complex.

The transcription factor nuclear factor-B (NF-B) 1 plays a pivotal role in inflammatory and immune responses (1)(2)(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 NH 2 -terminal Ser residues. The phosphorylation targets them for rapid degradation through a ubiquitin-prote-asome 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)(23)(24), MAPK/ extracellular signal-regulated kinase kinase kinases (MEKK1-3) (25)(26)(27)(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
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 g/ml streptomycin at 37°C in 5% CO 2 . 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 [ 32 P]orthophosphate, HeLa cells were washed twice with phosphatefree Dulbecco's modified Eagle's medium (Life Technologies, Inc.) and subsequently incubated with 1 mCi/ml [ 32 P]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 GSTfused 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 epitopetagged p65 using LipofectAMINE reagents (Life Technologies, Inc.). For * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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 32 P 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).
p65 Phosphorylating Activity in HeLa Cells-To characterize the p65 kinase activity in vitro, we generated NH 2 -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.
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 NH 2terminal 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.
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 ϫ Histagged 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. DISCUSSION 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)(36)(37)(38)(39)(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 IKKs⅐NF-B⅐IB complex will elucidate the spatial localization of these components and the role of dimerization of IKKs in the phosphorylation of the NF-B⅐IB 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)(42)(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 NH 2terminal 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.