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IKKß phosphorylates p65 at S468 in transactivaton domain 2

Robert F. Schwabe^*,1 and Hiroaki Sakurai

^* Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York, USA; and
Department of Pathogenic Biochemistry, Toyama Medical and Pharmaceutical University, Toyama, Japan

¹ Correspondence: E-mail: rfs2102{at}columbia.edu

SPECIFIC AIMS

1. To investigate the presence of IKK sites in transactivation domain 2 of the NF-B subunit p65. 2. To characterize the physiological relevance these p65 IKK site(s).

PRINCIPAL FINDINGS

1. IKK phosphorylates TAD1 and TAD2 of p65
We tested the ability of recombinant human (rh) IKK or rhIKKß to phosphorylate GST-p65 substrates in which either 1) the known IKK site at S536 was mutated, 2) TAD1 was deleted, or 3) TAD1 and TAD2 were deleted. In the S536A GST-p65 (354-551) substrate, we found two phosphorylation bands indicating the existence of IKK sites besides the known site at S536. We observed one phosphorylation band in the GST-p65 (354-520) substrate incubated with rhIKKß indicating the existence of one IKK site within TAD2 whereas IKKß-induced phosphorylation was almost absent in the substrates that did not contain TAD1 and TAD2. In all substrates tested, IKKß was a far more potent p65 kinase than IKK.

2. IKKß phosphorylates S468 in vitro
In order to map the IKK site within TAD2, we mutated all serines within TAD2 (S437, S457, S468, S472) in the GST-p65 (354-520) substrate and performed in vitro kinase assays in the presence of rhIKKß. In the GST-p65 S468A (354-520) substrate, but not in GST-p65 (354-520, 437A, 457A, or 472A) substrates, phosphorylation almost completely disappeared, demonstrating that S468 is the only IKK site within TAD2 (Fig. 1 B). Accordingly, one of the phosphorylation bands observed in the GST-p65 (354-551, S536A) also disappeared when we introduced the S468A mutation (Fig. 1A ). The other phosphorylation band corresponds to a site which lies outside of TAD2 (R.F.S., unpublished observation). Next we determined the relative role of IKK and IKKß in S468 phosphorylation using a S468 phospho-specific antibody which recognizes S468 and not to T464 or S472 (Fig. 1C ). We observed a strong S468 phosphorylation in the GST-p65 (354-551) substrate that had been incubated with rhIKKß but almost completely absent phosphorylation in GST-p65 (354-551) that had been incubated with rhIKK (Fig. 1D ).

View larger version (63K): [in this window] [in a new window]   Figure 1. IKKß phosphorylates S468 of in TAD2. A) Radioactive kinase assays were performed using rhIKKß (0.5 µg/mL) and GST-p65 (354-551, S536A) substrates in which S437, S457, S468, or S472 were mutated to alanine. The GST-p65 (354-551, SS468,536AA) shows only one phosphorylation band. B) Radioactive kinase assays were performed as described above using GST-p65 (354-520) substrate in which S437, S457, S468, and S472 were mutated to alanine. The GST-p65 (354-520, S468A) substrate is not phosphorylated. C) A nonradioactive kinase assay was performed using rhIKKß and GST-p65 (354-551) substrates in which T464, S468, or S472 were mutated to alanine. The phospho-S468 specific antibody did not bind to the GST-p65 (354-551, S468A) substrate (upper panel), but to all other substrates. D) A nonradioactive kinase assay was performed using rhIKK or rhIKKß and GST-p65 (354-551) followed by western blot analysis using phospho-S468 specific antibodies.

3. IKKß phosphorylates p65 in vivo
To determine whether IKK-induced p65 phosphorylation at S468 represents a physiological event in vivo, we stimulated HeLa cells with TNF (10 ng/mL) and IL-1ß (5 ng/mL). TNF and IL-1ß-induced S468 phosphorylation was detected with a peak after 7.5 min and a rapid decline in phosphorylation thereafter. After pretreatment with the phosphatase inhibitor calyculin A (10 nM), we observed a stronger phosphorylation band and a longer lasting phosphorylation after TNF and IL-1ß, indicating that protein phosphatase 1/2a is involved in the rapid dephosphorylation of S468. Strong TNF- and IL-1ß-inducible phosphorylation was also observed in human T cell, breast cancer, hepatoma, and neuroblastoma cell lines and primary human hepatic stellate cells. In primary human peripheral blood mononuclear cells, PMA induced robust S468 phosphorylation.

4. IKKß phosphorylates p65 at S468 and S536 while it is bound to IB
Dominant negative IKKß, but not dominant negative IKK, blocked TNF- or IL-1ß-induced S468 phosphorylation, whereas HeLa cells transduced with an IBsr that is resistant to IKK phosphorylation showed an increased phosphorylation of S468. Immunoprecipitation experiments demonstrated that S468- and S536-phosphorylated p65 and IB co-immunoprecipiated. These results suggest that p65 is phosphorylated while bound to IB and that p65 and IB may compete for IKK-mediated phosphorylation. To exclude the involvement of other kinases in this process, we tested TNF and IL-1ß-inducible S468 phosphorylation in cells which had been pretreated with pharmacological inhibitors of IKK, p38, Erk, PI-3 kinase, and GSK-3. The IKK inhibitor PS-1145 completely blocked TNF and IL-1ß induced S468 phosphorylation whereas inhibitors of p38, Erk, JNK, PI-3 kinase, and GSK-3 did not influence S468 phosphorylation after TNF or IL-1ß.

5. S468 phosphorylation is not required for TNF- and IL-1ß-induced NF-B activation
To determine the role S468 in NFB-dependent gene transcription, we created p65S468A- and p65wt-reconstituted p65 –/– MEFs which showed similar p65 expression and S536 phosphorylation, but differed in TNF- or IL-1ß-induced S468 phosphorylation (Fig. 2 A). In contrast to mock-transfected p65 –/– MEFs, both p65wt- and the p65S468A-transfected p65 –/– MEFs displayed a strong increase in NF-B-dependent luciferase activity and RANTES mRNA levels and a more pronounced increase of MCP-1 mRNA levels after rmTNF or rmIL-1ß (Fig. 2B-D ). We found that the p65S468A-transfected clones had on average a 59% and 53% higher induction of NF-B-driven luciferase activity after rmTNF and IL-1ß, respectively, and a 100% and 48% higher induction of RANTES mRNA (Student’s t test, P <0.05) than the p65wt-transfected clones (Fig. 2B-C ). The induction of MCP-1 mRNA was also higher in p65S468A-expressing clones but did not reach statistical significance (Fig. 2D ). After rmTNFa stimulation, p65wt- and p65S468-transfected cells displayed a similar rate of p65 nuclear translocation with strong nuclear staining 15 and 30 min after stimulation that gradually decreased to baseline within 120 min indicating that S468 phosphorylation is not required for p65 nuclear translocation (Fig. 2E ).

View larger version (55K): [in this window] [in a new window]   Figure 2. S468 is not required for NF-B-dependent transcription or p65 nuclear translocation after TNF or IL-1ß. A) p65 –/– MEFs were reconstituted with human p65wt or human p65S468A. Western blot analysis for p65 shows similar p65 expression in the p65wt and p65S468A clones (upper panel). Western blot for S468 and S536 phosphorylation shows absent S468 phosphorylation after 15 min of rmTNF (30 ng/mL) or rmIL-1ß (5 ng/mL) in p65S468A MEFs but not in the p65wt MEFs (lower panel). B) p65wt or p65S468A reconstituted p65 –/– MEFs were coinfected with adenoviruses containing 3xb-driven luciferase (moi 50) and CMV-driven ß-galactosidase (moi 50) followed by treatment with rmTNF or rmIL-1ß for 6 h. Luciferase activity was normalized to ß-galactosidase activity expressed as fold-induction compared with untreated control. This assay is the average of 3 independent experiments using 5 separate p65wt and 5 p65S468A clones in each experiment. C–D) p65 wt or p65S468A-reconstituted p65 –/– MEFs were treated with rmTNF or rmIL-1ß for 4h. Levels of RANTES mRNA (C) and MCP-1 mRNA (D) were measured by real time PCR and normalized to 18s levels. Each condition was performed in duplicate in 5 different clones and is expressed as average (±SD) E) To analyze p65 nuclear translocation, p65wt, or p65S468A reconstituted p65 –/– MEFs were treated with rmTNF followed by p65 immunofluorescent staining and DAPI nuclear counterstain. *P < 0.05 (compared withrmTNF- and rmIL-1ß-treated p65wt cells).

CONCLUSIONS AND SIGNIFICANCE

It has been suggested that phosphorylation of NF-B subunits represents a second mechanism by which NF-B transcriptional activity is regulated. Schmitz et al. postulated that phosphorylation of p65 TAD1 or TAD2 up-regulates NF-B-dependent transcription. We have shown that IKK is a p65 kinase that phosphorylates S536 in TAD1, implying that the IKK complex has dual functions in the NF-B pathway (i.e., phosphorylation of 1) IB and 2) p65). In the current study, we demonstrate that IKK phosphorylates p65 in TAD2 at S468. We show that IKK-mediated S468 phosphorylation is a physiological event that occurs after TNF and IL-1ß stimulation in cell lines from the liver, breast, brain, cervix, T and B cell lines as well as in primary cells. IKKß is a far more efficient S468 p65 kinase than IKK both in vitro and in vivo. We observed that IBsr enhanced S468 and S536 phosphorylation. Moreover, phosphorylated p65 and IB co-immunoprecipitated. This demonstrates that p65 phosphorylation occurs in a complex formed by IB, p65, and IKK. It is likely that IB recruits IKK and brings it in proximity with p65 to allow p65 phosphorylation. This result is similar to previous findings showing that the proteasome inhibitors ALLN and MG132, which block IB degradation, enhanced S536 phosphorylation.

We and others have reported that GSK-3ß phosphorylates p65 and that S468 is a GSK-3 site. We observed that IKKß is a far more efficient S468 kinase in vitro (data not shown) and found no reduction in S468 phosphorylation in cells pretreated with GSK-3 inhibitors, whereas IKK inhibition almost completely abrogates S468 phosphorylation. Although it is possible that GSK-3ß phosphorylates p65 at S468 in a cell type specific manner, we think that the previous studies also suggest an involvement of IKK in S468 phosphorylation in vivo: 1) high doses of calyculin A used in previous studies led to IB phosphorylation at S32 and S36 indicating concomitant IKK activation; and 2) even high doses of the GSK-3 inhibitor LiCl did not completely block S468 phosphorylation. It is possible that under some conditions, S468 phosphorylation is mediated by a multi-kinase complex as shown for S536 phosphorylation or that IKKß and GSK-3ß compete for S468. Moreover, our study excludes a role for JNK, p38, Erk, or PI-3kinase/Akt in TNF- or IL-1ß-induced S468 phosphorylation.

Although phosphorylation of the TADs of p65 is believed to up-regulate p65 transactivation, some reports do not find a role for S536 in up-regulating NF-B-dependent transcription. Our study shows that phosphorylation of S468 is not required for NF-B-dependent transcription. On the contrary, we found that p65S468A-reconstituted p65 –/– MEFs showed a slightly higher degree of NF-B-dependent luciferase activity and higher mRNA levels of the endogenous NF-B-dependent gene RANTES after TNF and IL-1ß than p65wt reconstituted p65 –/– MEFs. This result is similar to a previous study showing that p65S536A-reconstituted p65 –/– MEFs have no defect in the NF-B pathway and shows an 2-fold higher level of IL-6 secretion than wt cells. Therefore it is possible that IKK-mediated serine phosphorylation in the p65 TADs actually represses NF-B activation. The effects of the S468A mutation on NF-B-dependent transcription were relatively weak with 1.5- to 2-fold induction of NF-B-driven luciferase and RANTES mRNA induction. This may be caused by the rapid dephosphorylation of p65. However, phosphatase inhibition did not uncover stronger effects of S468 mutation on the NF-B pathway (data not shown). Another likely scenario is that the multiple IKK sites within the p65 TADs have additive or redundant functions and that it is required to mutate all residues to reveal the functional role of IKK-mediated p65 phosphorylation. Alignment of the TA’₁ and TAD1 sequences shows that S468 in TA’₁ corresponds to S536 in TAD1. These data not only suggest that TAD1 and TAD2 share a sterical configuration that exposes S468 and S536 to IKK-mediated phosphorylation, but also that S468 and S536 may fulfill similar functions with respect to transactivation. Other possible functions of S468 may be the regulation of genes with atypical B sites or regulatory functions outside of the NF-B pathway such as the feedback to other signaling pathways. Further studies are required to unravel the relationship between the 3 IKK sites in p65 TADs and to investigate whether they serve distinct functions or whether they cooperate to regulate NF-B activation.

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3736fje;

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