1

Constitutive and IL-1-inducible phosphorylation of p65 NF-κB at serine 536 is mediated

by multiple protein kinases including IKKα, IKKβ, IKKε, TBK1 and an unknown

kinase and couples p65 to TAFII31-mediated IL-8 transcription

Holger Buss‡**, Anneke Dörrie‡**, M. Lienhard Schmitz♣, Elke Hoffmann‡, Klaus

Resch‡ and Michael Kracht‡ ¶

‡Institute of Pharmacology, Medical School Hannover, Carl-Neuberg Strasse 1, D-30625,

Hannover, Germany

♣ Department of Chemistry and Biochemistry, University of Bern, Freiestr. 3, 3012 Bern,

Switzerland

*This work was supported by grants from the Deutsche Forschungsgemeinschaft KR1143/4-

1, KR-1143/4-2, SFB566/B06 (to M.K.) and Schm 1417/3-1, 3-2, 3-3 to (L.S.)

** H.B. and A.D. contributed equally to this work

Key words: IL-1, p65 NFκB, IKKα, IKKβ, TBK1, IKKε, TAFII31, AES

¶Corresponding author:

Michael Kracht Institute of Pharmacology

Medical School Hannover Carl-Neuberg-Strasse 1

D-30625 Hannover, Germany Phone:0049-511-532-2800

Fax: 0049-511-532-4081 E-mail:Kracht.Michael@MH-Hannover.de

JBC Papers in Press. Published on October 15, 2004 as Manuscript M409825200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARY

Phosphorylation of NFκB p65(RelA) serine 536 is physiologically induced in response to a

variety of proinflammatory stimuli but the responsible pathways have not been conclusively

unravelled and the function of this phosphorylation is largely elusive. In contrast to previous

studies we found no evidence for a role of JNK, p38, ERK or PI3 kinase in IL-1- or TNF-

induced ser536 phosphorylation, as revealed by pharmacological inhibitors. Neither RNAi

directed at IKKα/β - the best characterized ser536 kinases so far -, nor the IKKβ inhibitor SC-

514 or dominant negative mutants of both IKKs we were able to suppress ser536

phosphorylation. A GFP-p65 fusion protein was phosphorylated at ser536 in the absence of

IKK activation, suggesting the existence of IKKα/β-independent ser536 kinases.

Chromatographic fractionation of cell extracts allowed the identification of two distinct

enzymatic activities phosphorylating ser536. Peak one represents an unknown kinase, while

peak two contained IKKα, IKKβ, IKKε and TBK1. Overexpressed IKKε and TBK1

phosphorylate ser536 in vivo and in vitro. Reconstitution of mutant p65 proteins in p65-

deficient fibroblasts that either mimicked phosphorylation (S536D) or preserved a predicted

hydrogen-bond between ser536 and asp533 (S536N) revealed that phosphorylation of S536

favours IL-8 transcription mediated by TAFII31, a component of TFIID. In the absence of

phosphorylation, the hydrogen-bond favours binding of the corepressor AES to the p65 TAD.

Collectively, our results provide evidence for at least five kinases that converge on ser536 of

p65 and a novel function for this phosphorylation site in the recruitment of components of the

basal transcriptional machinery to the IL-8 promoter.

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Introduction

The transcription factor NF-κB regulates the expression of a large number of genes

with important functions in the immune response, inflammation, cellular stress reactions,

carcinogenesis and apoptosis. In resting cells NF-κB is trapped in the cytoplasm by its

interaction with the inhibitor IκB. A central step in activation of NF-κB is the stimulus-

induced phosphorylation of IκB by IκB kinases (IKK) α and β. Both IKKs, IκB and NF-κB

subunits form a large signalling complex (1). Phosphorylation of IκB results in targeting of

IκB to the proteasome followed by release and nuclear translocation of NF-κB. Recent

evidence suggests that NF-κB activity is determined by additional mechanisms.

Cells lacking the protein kinases GSK3β (2), TBK1/NAK (3;4), IKKε (5), NIK (6)

and PKCζ (7) show a normal IκB degradation pathway, but impaired activation of NF-κB-

dependent gene expression.

Furthermore, biochemical and genetic experiments in cells deficient for IKKα or

IKKβ strongly suggest that direct phosphorylation and other modifications of NF-κB are

essential for NF-κB function (8-10). The complexity of this regulation is exemplified by

recent studies that identified four serine residues in the p65 NF-κB subunit that are inducibly

phosphorylated by TNF or LPS and that are targeted by distinct signalling pathways.

Phosphorylation of serine 276 of p65 occurs by PKAc in response to LPS (11;12), or

by MSK1 in response to TNF (13). Phosphorylation at ser276 promotes its interaction with

the coactivator CBP – a histone acetylase - and is required for TNF-induced IL-6 expression

(14;15). Phosphorylation in the REL homology domain (RHD) of p65 at ser311 is required

for efficient p65 transactivation mediated by PKCζ (7;16;17).

Serine 529 of p65 is phosphorylated by casein kinase II (18;19) and by the TAX-

activated IKK complex in vitro (20). In experiments using reconstituted p65 -/- cells, ser529

contributes to p65 transactivation (20). However, others have found no role for ser529 in p65

activity in response to TNF treatment or IKKα/IKKβ overexpression (15;21;22).

Regulated phosphorylation of serine 536 of the C-terminal transactivation domain

(TAD) of p65 was originally found by Sakurai et al who searched for kinase activities in

extracts from TNF-stimulated cells that would phosphorylate various truncated recombinant

p65 proteins in vitro (22). This group also suggested IKKβ as a p65 TAD kinase (22). Since

then, several groups have confirmed that both IKKs directly phosphorylate the C-terminal

TAD of p65 (20;23-25).

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Recombinant or overexpressed IKKs phosphorylate ser536 in vitro, with IKKβ being

somewhat more efficient, as shown by mutational analysis of GST-p65 fusion proteins

(22;24), or by mass spectrometry analysis of p65 peptides (25)

Ser536 is physiologically induced in response to a variety of proinflammatory stimuli

(21;24;26;27) but the responsible pathways and the function of this phosphorylation have not

been conclusively unravelled. Sakurai et al. identified TRAF2, TRAF5, TAK1 and IKKα/β as

important mediators for TNFα-induced serine 536 phosphorylation (24), while – as shown by

us - induction of this phosphorylation by T cell costimulation depends on Cot (Tpl2), RIP,

PKCθ, NIK and IKKβ (27). A further study showed that the LPS- and TNF-induced

phosphorylation of endogenous p65 at ser536 is unaffected in IKKα -/- fibroblasts. Only the

LPS-induced but not the TNF-induced phosphorylation is lost in IKKβ -/- cells (21).

In the light of these contrasting findings and the apparent complexity of p65 regulation

by phosphorylation we have set up experiments to analyse more conclusively the kinases that

contribute to ser536 phosphorylation of p65 and its function in response to one of the major

activators of p65, interleukin-1. We report here that besides IKKα and IKKβ, the IKK-related

kinases TBK1 and IKKε also phosphorylate ser536. We also provide evidence for a novel

ser536 kinase. Further experiments into the mechanism by which ser536 contributes to p65

function suggest that phosphorylation disrupts a hydrogen-bond between ser536 and asp533.

As a result, p65 has a lower affinity for the corepressor AES and can interact efficiently with

TAFII31, a component of the basal transcription factor machinery, to induce transcription of

IL-8, a known p65 target gene.We thus identify a hitherto unknown mechanism of p65-

mediated gene regulation.

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EXPERIMENTAL PROCEDURES

Cells and Materials

HeLa cells stably expressing the tet transactivator protein and HEK293 cells stably expressing

the IL-1 receptor (HEK293IL-R) were kind gifts of H. Bujard and K.Matsumoto, respectively.

KB cells were from the American Type Culture Collection, Rockville, MD. p65 -/- cells were

a kind gift of H. Nakano (Tokyo, Japan). All cells were cultured in Dulbecco’s modified

Eagle's medium, complemented with 10% fetal calf serum, 2mM L-glutamine, 1mM sodium

pyruvate, 100U/ml penicillin, 100µg/ml streptomycin. Antibodies against the following

proteins or peptides were used in this study: IκBα (9242), phospho IκBα (9241),

phosphoser536 NF-κB (3031), all from Cell Signaling Technology; p65 NF-κB (C-20), IKKε

(H-116), TBK1 (M-375), IKKα (H-744), IKKβ (T-20), ERK2 (C-14) all from Santa Cruz,

GFP (Boehringer), FLAG (M2, Kodak). Horseradish peroxidase-coupled secondary

antibodies were from Sigma. ProteinA/G sepharose was from AmershamPharmacia. Human

recombinant IL-1α was a kind gift of J.Saklatvala, London. PD98059 was from Alexis,

SP600125 was from Tocris, SB203580 and MG132 were from Calbiochem, SC-514 was a

kind gift of Pharmacia Corp. Other reagents were from Sigma-Aldrich or Fisher and were of

analytical grade or better.

Plasmids and Transfections

The expression plasmids pcDNA3-FLAG-IKKα and pcDNA3-FLAG-IKKβ were kind gifts

of David Wallach, Israel. pMTNHA-TAFII31 (28) was from X.Wu, Houston, USA,

pGEX2TK-TAFII31 (29) was from A.A.Ladias, Boston, USA, pcDNA3.1/HiSA-AES and

pGEX-5X-2-AES (30) were from T.Okamoto, Nagoya, Japan, pEGFP-p65 was from Rainer

de Martin, Vienna, Austria. Expression vectors for FLAG-IKKαKN, FLAG-IKKβKN, IKKε,

IKKεKN, TBK1, TBK1KN, IκBα and NF-κB (3) luc have been published (27) The

expression plasmids for the p65 TAD, pGEX-p65 (354-551) and versions mutated in

ser536ala, ser529Ala, ser529/ala +ser536/ala were kind gifts of H.Sakurai, Toyama,Japan.

pMT7-p65 NF-κB and the IL-8 promoter-luciferase reporter plasmid pUHC13-3-IL-8pr

(nucleotides 1348-1527 of the IL-8 gene) have been described (31). pSV-β-gal coding for

SV40 promoter driven β-galactosidase was from Promega. GST-fusion proteins were

expressed in bacteria and purified on GSH-sepharose using standard procedures.

HEK293IL-1R cells were transiently transfected by the calcium phosphate method and

determination of luciferase reporter gene activity were performed as described (31). Equal

amounts of plasmid DNA within each experiment were obtained by adding empty vector. For

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determination of promoter activity, cells (seeded at 5 x 105 per well of 6-well plates) were

transfected with 0.25 µg of the IL-8-promoter luciferase reporter plasmids and 0.5 µg of pSV-

β-gal. β-galactosidase activity was determined (using reagents from Clontech) to allow

normalization of luciferase activity in different transfections. p65 -/- cells cells were seeded in

6-well plates and transfected using Rotifect (Roth) according to the manufacturer’s

instructions.

Site-directed mutagenesis

All mutations were perfomed by quick change site-directed mutagenesis kit (Stratagene)

according to the manufacturer’s protocol and verified by DNA sequencing on an ABI Prism

310 instrument.

siRNA experiments

Cells were seeded in 24 well plates at 6,5x104 cells/ml. At 70% confluency cells were washed

2x in serum-free medium and transfected with a total of 200nM of a mixture of four double-

stranded RNA oligonucleotides directed against human IKKα and IKKβ (Smart Pool,

Dharmakon) using 3µl Oligofectamine per well. After 72h cells were treated as indicated,

lysed and protein expression determined by western blot. The sequences of the luciferase

siRNA oligonucleotides used as a control were as follows (5’-

GGCCUUGUGAACAGAUCAGdTdT-3’, 5’- CUGAUCUGUUCACAAGGCCdTdT-3’)

Anionexchange chromatography

Four T175 cm2 flasks of HeLa cells were stimulated or left untreated as indicated in the

figure legend. Cells were washed in icecold PBS and resuspended in 20 mM Tris, pH 8.5, 20

mM ß-glycerophosphate, 20 mM NaF, 0.1 mM Na3VO4, 0.5mM EGTA, 0.5mM EDTA, 0.1%

NP-40, 2mM DTT, 10µM E64, 2.5µg/ml leupeptin, 1mM PMSF, 1µM pepstatin and 400nM

okadaic acid. Cells were broken mechanically by passaging them three times through a

26-gauge needle. Then the lysate was cleared at 15.000xg for 20min at 4°C. Supernatants

were stored in liquid nitrogen or directly used for chromatography. 4,75mg of lysate was

diluted into 2.5ml of buffer A (20 mM Tris, pH 8.5, 20 mM ß-glycerophosphate, 20 mM NaF,

0.1 mM Na3VO4, 0.5mM EGTA, 0.5mM EDTA, 0.05% NP-40, 2mM DTT) and loaded onto

a 1ml ResQ column run by an Äktaprime system (Amersham Pharmacia). The column was

equilibrated in buffer A and proteins were eluted with a linear salt gradient (0-0.75 M NaCI in

16 ml). 1ml fractions were collected and stored frozen at –80°C until further use.

Preparation of cell extracts

For the preparation of whole cell extracts cells were lysed directly in SDS-sample buffer.

DNA was sheared by brief sonification and soluble proteins were recovered after

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centrifugation of lysates at 15.000xg for 15min at 4°C. For in vitro kinase assays, cells were

lysed in 10 mM Tris, pH 7.05, 30 mM NaPPi, 150 mM NaCl, 1% Triton X-100, 2 mM

Na3VO4, 50 mM NaF, 20 mM ß-glycerophosphate and freshly added 0.5 mM PMSF, 0.5

µg/ml Leupeptin, 0.5 µg/ml pepstatin, 400 nM okadaic acid). After 10 min on ice, lysates

were clarified by centrifugation at 10,000 x g for 15 min at 4 °C.

Nuclear and cytosolic extracts were prepared as described previously (31). Protein

concentration of cell extracts was determined by the method of Bradford, and samples were

stored at -80 °C.

Western blotting

Cell extract proteins were separated on 10% SDS-PAGE and electrophoretically transferred to

PVDF membranes (Immobilon, Millipore). After blocking with 5% dried milk in Tris-

buffered saline overnight, membranes were incubated for 4-24 h with primary antibodies,

washed in Tris-buffered saline and incubated for 2-4 h with the peroxidase-coupled secondary

antibody. Proteins were detected by using the Amersham enhanced chemiluminescence

system.

In vitro kinase assays

For immune complex kinase assays 500 µg of cell extract protein was diluted in 500 µl of ice-

cold immunoprecipitation (IP) buffer (10 mM Tris, pH 7.05, 30 mM NaPPi, 150 mM NaCl,

1% Triton X-100, 2 mM Na3VO4, 50 mM NaF, 20 mM ß-glycerophosphate, 2mM DTT and

400µM PMSF). Samples were incubated for 3 h with 1 µg of antibodies against IKKα, or

FLAG (to precipitate FLAG- IKKε or FLAG-TBK1) followed by the addition of 20 µl of a

50% suspension of protein A or G--Sepharose beads and incubation for 1-2 h at 4 °C. Beads

were spun down, washed 3x in 1 ml of IP buffer A and resuspended in 10 µl of the same

buffer.

Then 1 µg of recombinant protein substrates (GST-p65354-551 or mutants thereof) in 10 µl

H2O and 10 µl of kinase buffer (150 mM Tris, pH 7.4, 30 mM MgCl2, 60 µM ATP, 4 µCi [γ-32P]ATP) were added. After 30 min at room temperature SDS-PAGE sample buffer was

added, and proteins were eluted from the beads by boiling for 5 min. After centrifugation at

10,000 x g for 5 min, supernatants were separated on 10% or 12.5% SDS-PAGE.

Phosphorylated proteins were visualized by autoradiography.

Alternatively, 10µl of ResQ fractions were used in the kinase reactions. For the kinase assay

shown in Fig.4A, 10µl of cell lysate (50µg of protein) was added to the kinase reaction and

the in vitro phosphorylated GST-p65 fusion protein purified on GSH-sepharose prior to SDS

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PAGE and autoradiography as described in (32). For the kinase assays shown in Fig.6A, 10µl

(50µg) of cytosolic or nuclear proteins were mixed with 10µl of (150 mM Tris, pH 7.4, 30

mM MgCl2, 600 µM ATP) and 10µl (1µg) of GST-p65(354-551) fusion proteins for 15 min at

30°C. Reaction mixtures were separated by SDS PAGE and phosphorylation of p65 detected

by immunoblotting with the anti phosphoser536 antibody.

EMSA

EMSA was performed as described in (31) using an oligonucleotide (5’-

tgacagagGGGACTTTCCagaga-3’) containing a NF-κB consensus site (shown in capital

letters).

GST-pull down assays

p65, or AES proteins were in vitro transcribed and translated using the TNT rabbit

reticulocyte Kit from Promega. Approximately 500ng of purified GST, GST-AES or GST-

TAFII31 (as judged from Coomassie-stained gels) were immobilized on GSH Sepharose,

resuspended in 70µl of binding buffer (20mM Tris, pH7.4, 0.3M NaCl, 1% (w/v) BSA, 0.1%

NP-40, 2mM DTT, 1mM PMSF) and incubated with 3.5 µl of in vitro translated p65 or AES

proteins for 60min at 30°C. Beads were spun down, washed twice in binding buffer without

BSA and bound proteins eluted in SDS-sample buffer for 5min at 95°C. Bound proteins and

1/10 of the in vitro translated proteins (used as input control) and were separated on SDS

PAGE and proteins analysed by autoradiography or western blotting.

Chromatin immunopecipitation (ChIP)

Proteins bound to DNA were crosslinked in vivo by replacement of the medium with warm

PBS including 1% formaldehyde. After 1 minute, this solution was replaced by warm PBS

including 0.125 M glycine to stop cross-linking. Cells were washed, collected and lysed in

ChIP-RIPA buffer (10mM Tris, pH 7.5, 150mM NaCl, 1% NP-40, 1% desoxycholate, 0.1%

SDS, 1mM EDTA, and freshly added 1% aprotinin). Lysates were cleared by sonification (4x

one minute on ice) followed by centrifugation at 15.000xg at 4°C for 20min. 4-10µl of

antibodies were added to 250-500µl of lysates and the mixture rotated at 4°C overnight. Then

40µl of a ProteinA/G mixture, preequilibrated in ChIP-RIPA buffer was added for 1h at 4°C.

Beads were washed two times in ChIP-RIPA buffer, once in high salt buffer (10mM Tris, pH

7.5, 2M NaCl, 1% NP-40, 0.5% desoxycholate, 1mM EDTA), once in ChIP-RIPA buffer,

once in TE buffer (10mM Tris, pH 7.5, 1mM EDTA) and finally resuspended in 55µl of

elution buffer (10mM Tris, pH 7.5, 1mM EDTA, 1% SDS). Samples were vigorously mixed

for 15min at 30°C and than centrifuged. 50µl of supernant was diluted to 200µl with TE

buffer including RNAse A (50µg/ml). Similarly 50µl of the initial lysate (input samples) were

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diluted to 200µl with TE buffer including 1% SDS and 50µg/ml RNAse A. After 30min at

37°C, proteinase K was added (0.5mg/ml) and both, input and immunoprecipitates were

incubated for at least 6h at 37°C followed by at least 6h at 65°C. Then, DNA was purified

using Qiaquick spin columns (Qiagen). DNA was eluted with TE buffer and stored at –20°C

until further use. PCR was performed on input and immunoprecipitated DNA using 2.5 U of

hotstart taq polymerase (Qiagen), 1µM of sense and antisense primer, 2.5-3 µl of template

DNA, 0.2mM of dNTPs in a total volume of 30µl. PCR cycles were as follows: 15min 95°C,

34-36 cycles of 94°C (20 secondes), 55-60°C (20 seconds), 72°C (20 seconds), followed by a

final extension reaction at 72°C for 7 minutes. PCR products were separated by agarose gel

electrophoresis, visualized by ethidium bromide staining and fluorescence intensities of bands

were quantified using a Biometra TI5 system (Biometra) and the BioDocAnalyse software,

version 1.0 (Biometra). ChIP primers for the IL-8 gene (accession number M28130) were as

follows: IL-8 promoter: sense (nucleotides (nt) 1303-1325): 5’-aagaaaactttcgtcatactccg-3’,

antisense (nt 1450-1473): 5’-tggctttttatatcatcaccctac -3’.

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RESULTS

Serine 536 phosphorylation of p65 NF-κB occurs independent from JNK, p38, ERK and

PI3 kinase pathways but is induced by proteasome inhibition via the IKK complex

Increasing evidence suggests that the three major MAPK pathways, i.e. JNK, p38 and ERK

are interconnected with NF-κB activation (13;33;34). In the light of the previous implication

of p38 and PI3K/AKT in the IKK-mediated phosphorylation of p65 at ser536 we used

pharmacological inhibitors to assess the impact of known pathways on endogenous p65

ser536 phosphorylation triggered by various stimuli.

Neither the low level of constitutive ser536 phosphorylation, nor the induced phosphorylation

activated by PMA, TNF-or IL-1 was affected by the pathway-specific inhibitors PD98059,

SP600125 or SB203580 (Fig. 1). At the concentrations used these three inhibitors specifically

inhibit their target kinases and affect inflammatory gene expression (data not shown).

Wortmannin at high concentrations (1µM) blocked the PMA-induced, but not the IL-1- or

TNF-induced ser536 phosphorylation, suggesting that PI3K/AKT is only operational in

specific pathways. Interestingly, the proteasome inhibitor MG132 which blocks NF-κB

activation by preventing IκB proteasomal degradation, enhanced basal ser536

phosphorylation, but did not affect the inducible ser536 phosphorylation. Further experiments

showed that this effect was dose- and time-dependent. 30 min after its addition, MG132

caused the induction of ser536 phosphorylation even in a concentration as low as 1µM. In

parallel the substance induced phosphorylation of IκBα (Fig.2A, B). The IKK complex

purified from cells treated with MG132 or IL-1 phosphorylated the TAD of p65 in vitro and

MG132 activated endogenous IKK to a similar extent as IL-1 (Fig.2C). Collectively, the data

presented in Fig.1 suggest that with the exception of PMA-stimulation, phosphorylation of

p65 at ser536 is independent from MAPK and PI3 kinase pathways. Hence, the usage of

pharmacological inhibitors suggested that IL-1-induced 536 phosphorylation was mediated by

kinases that are contained in the IKK complex and are unrelated to PI3 kinase and the three

MAPK pathways.

Detection of IKK-independent ser536 phosphorylation

Both IKKs, in particular IKKβ are the best characterized ser536 kinases activated in response

to TNF or LPS. To further analyse the relative contribution of IKKs for the constitutive and

inducible ser536 phosphorylation in the IL-1 signalling pathway, we performed a series of

different experiments interfering with IKK activation by various approaches. Parallel

suppression of endogenous IKKα and IKKβ by transfection of siRNA strongly reduced the

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amount of endogenous IKKα and IKKβ compared to luciferase siRNA and other controls.

However, this did not affect basal or IL-1-induced ser536 phosphorylation (Fig.3A). IKKβ is

the IL-1-induced IKK that mediates cytokine-induced gene expression (35;36). A novel IKKβ

inihibitor, SC514 effectively inhibited the IL-1-induced IκB phosphorylation at doses (50 and

100µM) that were previously published (25). This inhibitor did not affect constitutive ser536

phosphorylation, but partially impaired the IL-1-induced ser536 phosphorylation (Fig.3B),

suggesting the existence of further ser536 kinases in addition to IKKβ.

To study phosphorylation of p65 at ser536 independent from p65 associated with the IKK-

complex we used an ectopically expressed GFP-p65 fusion protein. Overexpressed GFP-p65

activates a cotransfected NF-κB reporter gene (data not shown) and induces expression of

endogenous IκBα, a known NF-κB target gene (Fig.3D, lane 2). At the exposure time chosen,

endogenous constitutively expressed IκBα was not yet visible in the vector control lane

(Fig.3D, lane 1), indicating the strong induction of IκBα by GFP-p65. This indicated that at

least a fraction of the overexpressed GFP-p65 fusion protein escaped trapping by endogenous

IκB, translocated to the nucleus and activated gene expression. This fully functional GFP-p65

protein was strongly phosphorylated at ser536 in vivo in the absence of any detectable IKK

activation as assessed by phosphorylation of endogenous IκB (Fig.3D, lane 2). These results

suggest the existence of a constitutively active protein kinase that accounts for ser536

phosphorylation of p65 proteins independent from IKKs. Neither wildtype (wt) IKKs nor

dominant negative (d.n.) IKKs blocked the constitutive GFP-p65 phosphorylation at ser536.

Only ovexpressed IκBα completely suppressed constitutive ser536 phosphorylation of GFP-

p65 (Fig.3D, lane 5). IκBα also completely inhibited GFP-p65 induced gene expression of a

cotransfected NF-κB reporter gene (data not shown) and of endogenous IκBα, suggesting that

it efficiently bound to cytosolic p65, blocked its access to the IKK-independent ser536 kinase

and also prevented translocation of the ser536-phosphorylated form of p65 to the nucleus

(Fig.3D, lane 5).

Collectively, the data shown in Fig.3 suggest that kinases in addition to and independent from

both IKKs account for most of the constitutive ser536 phosphorylation of p65 and may

contribute substantially to IL-1-induced IKK-mediated ser536 kinase activity. The data also

indicate that in unstimulated cells access of these novel kinases to p65 might be limited to free

p65 not trapped in p65/IκB complexes. Therefore, overexpression of IκB inhibits basal ser536

phosphorylation (Fig.3D, lane 5).

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Chromatographic separation of cellular kinases reveales three ser536 kinases in addition

to IKKs: IKKε, TBK1 and one unknown kinase

To get further evidence for ser536 kinases in addition to IKKs we fractionated extracts from

untreated and IL-1-stimulated cells by anionexchange chromatography (Fig.4A, B). In vitro

kinase assays using proteins from the individual fractions performed with the GST-p65 TAD

fusion protein revealed two broad peaks of p65 kinase activities that could be detected in

unstimulated cells and whose activity was stimulated by IL-1 (Fig.4B). Peak 2, but not peak1,

contained IKKα and IKKβ (Fig.4B). Peak two also contained TBK1 and IKKε, two novel

IKK-related protein kinases (Fig.4C). Notably, the majority of the IKKε and TBK1 antigen

(fractions 10-11) eluted slightly earlier than the amount of antigen associated with p65 kinase

activity (fraction 12-14). This phenomen has also been observed for other kinases such as

ERK (37) and suggests that enzymatically active IKKε and TBK1 carry more negatively

charges, e.g. phosphorylated residues. Both p65 kinase acitivities of peak 1 and 2

phosphorylated the p65 TAD on ser536, but not on ser529, as assessed by in vitro kinase

assays using alanine mutations of these phosphorylation sites (Fig.4D).

IKKε and TBK1 phosphorylate endogenous p65 at ser536 in vivo

To assess if IKKε and TBK1 induce phosphorylation of p65 on ser536 in intact cells we

coexpressed both kinases as well as their inactive forms together with GFP-p65 in HEK293

cells. Interestingly, wild type IKKε induced a strong phosphorylation of ser536 of p65

(Fig.5A, lane 3). IKKε wt also enhanced constitutive and IL-1-induced GFP-p65 activity,

while the kinase inactive form of IKKε strongly blocked constitutive and IL-1-induced ser536

phosphorylation of GFP-p65 as well as transactivation mediated by GFP-p65 (Fig.5, lanes

4+10). IKKε also induced a slower migrating form of p65, suggesting that it may

phosphorylate further residues in p65 in addition to ser536 (Fig.5A, lane 3 + 9). We did not

detect any endogenous IKKε in HEK293 cells, and we also noticed that the kinase inactive

form of IKKε was much more stable than the wt form (Fig.5A). In contrast, endogenous

TBK1 was readily detectable (Fig.5A). Overexpression of TBK1 induced ser536

phosphorylation but did neither enhance nor suppress GFP-p65 activity (Fig.5A, lanes 5+11).

Furthermore, the kinase inactive form did not inhibit ser536 phosphorylation or GFP-p65

activity (Fig.5A, lanes 6+12). Both, flag-tagged immunoprecipitated IKKε and TBK1

phosphorylated the p65 TAD in vitro at serine 536, suggesting that they are direct ser536

kinases (Fig.5B). In aggreement with its efficient intracellular phosphorylation of ser536,

IKKε was much more effective in phosphorylating ser536 in vitro. Overexpressed IKKε and

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TBK1 from HEK293 cells were not stimulated by IL-1, suggesting that they may account

mostly for the constitutive IKK- independent ser536 kinase activity (Fig.5B) or that they may

be induced by other stimuli.

Establishment of a function for phospho ser536 in IL-8 transcription -

ser536 phosphorylated p65 binds to the IL-8 promoter

So far, no p65 target genes have been identified that require phosphorylation on ser536 for

activation. We detected the ser536 protein kinases in the cytoplasm and in the nucleus by in

vitro kinase assays (Fig.6A). Endogenous p65 phosphorylated at ser536 rapidly accumulates

in the nucleus between 10 to 30 min after IL-1 stimulation (Fig.6B). Furthermore, IL-1

induces two nuclear protein complexes in vitro that bind to an NF-κB probe in EMSA, both

of which can be supershifted with antibodies to p65, while the slower migrating complex is

supershifted with the ser536 antibody (Fig.6C). Following this kinetics, p65 phosphorylated at

ser536 is also recruited within 10min to the endogenous IL-8 promoter in vivo as assessed by

chromatin immunoprecipitation (Fig.6D). We therefore set up experiments to analyse the

potential mechanism by which phosphorylation at ser536 contributes to p65-mediated IL-8

transcription.

Identification of active and inactive ser536 mutants

The acidic transactivation domain of p53 contains an FXXΦΦ motif that is conserved in p65,

VP16 and other transcriptional activators. Upon activation, in p53 this domain has been

shown to fold into a structured α-helix that enables contact with TAFII31, a human TFIID

TATA box-binding protein associated factor (Fig.7A, (38;39). Inspection of the primary

structure of p65 revealed two FXXΦΦ motifs in p65 in a region that was previously also

predicted to fold into an α-helix (40). Ser536 is contained in the first FXXΦΦ motif which

suggested that ser536 might couple p65 to the basal transcriptional machinery, in particular

TAFII31. Molecular modelling of the stretch of amino acids around ser536 revealed a

hydrogen-bond between ser536 and asp533 in wild type p65. Exchange of ser536 to either

alanine (S536A), or, aspartic acid (S536D), disrupted this hydrogen bond, whereas exchange

to asparagine (S536N) preserved the hydrogen bond (Fig.7B). We therefore generated three

different mutations in order to abolish (S536A mutant) or to mimic phosphorylation (S536D

mutant), or, to mimic hydrogen-bonding in the absence of phosphorylation (S536N mutant) in

intact cells. The wildtype and mutant proteins showed similar expression levels and DNA-

binding capacity (Fig.7C). Likewise all three mutants were expressed in p65-deficient Mefs to

levels comparable to the wt p65 protein (Fig.7D). As in the experiments shown before, wt p65

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was constitutively phosphorylated in p65 -/- Mefs and this was significantly stimulated by IL-

1. Interestingly, the anti phospho ser536 antibody strongly reacted which the S536D mutant,

very weakly with the S536A mutant and not with the S536N mutant, confirming that it

specifically recognizes the negative charge either resulting from endogenous phosphorylation

or aspartic acid side chains (Fig.7D). Expression of wt p65 activated an IL-8 promoter

reporter gene in these cells by about ten-fold, while the S536D and the S536A mutant

increased IL-8 transcription by more than twenty-fold. IL-1 stimulated wt p65-dependent IL-8

transcription and this effect was stronger in cells expressing either the S536D or the S536A

mutant. Interestingly, the S536N mutant showed the lowest transcriptional potency and it was

almost resistant to induction by IL-1 (Fig.7E).

Ser536 participates in binding of TAFII31 and AES to p65

Our results suggest that the reduced function of the S536N mutant may be explained by the

lack of phosphorylation at ser536 and by the preserved hydrogen-bond. It is thus possible that

the hydrogen-bond prevents interaction with a transcriptional coactivator by stabilizing an

interaction with a repressor that binds to the same region in p65 NF-κB. Such a mechanism

has been described for p53, in which the hydrogen-bond between thr18 and asp21 favours

binding of the repressor Mdm2 and negatively affects recruitment of TAFII31 to the FXXΦΦ

motif of p53 (41). To address the question whether a similar mechanism occurs for p65, we

tested binding of p65 proteins to a GST-TAFII31 fusion protein. Pull-down experiments

revealed similar binding of p65 wt and the S536D mutant to TAFII31, while the S536N

mutant bound slightly less strongly (Fig.8A). The groucho-like protein AES has recently been

shown to bind to the C-terminal TAD of p65 and to act as a repressor (30). 35S-labelled AES

mixed with an equal amount of 35S-labelled p65 reduced by about 50% the amount of wt p65

and of the S536N mutant, but not that of the S536D mutant, that could be captured

subsequently by immobilized GST-TAFII31 in vitro (Fig.8B). This competition experiment

suggests that efficient formation of p65-AES complexes is determined by ser536

modifications and can negatively affect binding of p65 to TAFII31. Hence, phosphorylation

on ser536 followed by destruction of the hydrogen bond may serve to induce binding to

TAFII31, while in the absence of phosphorylation the hydrogen-bond promotes preferential

binding of the region around ser536 to AES and represses p65 activity.

To test the functional consequences of TAFII31/p65 interactions for gene expression, both

proteins were coexpressed in p65 -/- Mefs. p65-mediated transactivation of a IL-8 reporter

gene was strongly triggered upon coexpression of TAFII31 (Fig.9A, lane 5), while AES had

no effect (Fig.9A, lane 6). Upon stimulation by IL-1 TAFII31 potentiated the p65-mediated

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IL-8 transcription (Fig.9A, lane 11), while AES suppressed p65-mediated IL-8 transcription

by at least two-fold (Fig.9A, lane 12). Neither, TAFII31 nor AES significantly affected IL-8

transcription in the absence of p65 (Fig.9A, lanes 3,4, 8,9) suggesting that their gene-

regulatory effects are predominantly mediated through p65. These data show that shifting the

balance of TAFII31 and AES by overexpressing one of the partners shifts IL-8 transcription

towards induction or repression, respectively. The TAFII31 effect is stronger when p65 is

phosphorylated, i.e. in the IL-1-stimulated situation (Fig.9A, lane 11). In agreement with this

assumption, the activity of the phosphomimetic S536D mutant can be further induced by

coexpressed TAFII31 and is not suppressed by AES, while the S536N mutant is resistant to

coexpressed TAFII31 and significantly suppressed by coexpressed AES (Fig.9B). In fact, in

cotransfection experiments in IL-1-stimulated cells overexpression of TAFII31 overrides the

AES-mediated repression (data not shown), suggesting that endogenous TAFII31 might be a

limiting component that competes with endogenous AES.

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DISCUSSION

Cytoplasmic retention of NF-κB by IκB is the major mechanism that prevents spontaneous

NF-κB activity. Hence, the IKKα and IKKβ-mediated phosphorylation of IκB followed by its

proteasomal degradation is believed to be essential and sufficient for full NF-κB activation as

it permits the subsequent nuclear entry of free NF-κB (9). However, recent evidence has

shown that post-translational modifications contribute significantly to, or, may even be

sufficient for full NF-κB activation (9). A number of protein kinases have been shown to

phosphorylate the strongly transactivating subunit p65 at at least five distinct amino acids,

ser276 (14), ser311(17), ser468 (42), ser529 (19) and ser536 (22).

In this study we have investigated in detail the IL-1-mediated ser536 phosphorylation and

have tested the possibility that kinases in addition to IKK contribute to ser 536

phosphorylation. In contrasts to previous findings from overexpression systems which

implied the PI3K and p38 MAPK pathways in phosphorylation of the p65 TAD (23;23;43-45)

we found no evidence for MAPK or PI3 kinase activation in ser536 phosphorylation by using

pharmacological inhibitors at concentrations that specifically inhibit PI3K, JNK, p38 or ERK.

For PI3 kinase our data are in aggreement with Yang et al., who showed recently that

LY294002, a PI3 kinase inhibitor did not affect ser536 phosphorylation in response to LPS or

TNF ((21). Furthermore, like Yang et al. we did not find an effect of PI3 kinase inhibitors on

IL-1-induced NF-κB activation (data not shown). Thus our data do not support a role for

PI3K/AKT in ser536 phosphorylation in response to TNF or IL-1. On the other hand, ser536

phosphorylation induced by phorbol ester is impaired by LY294002, showing that the PI3

kinase pathway may be operational in response to specific stimuli. Of the compounds used

only the proteasome inhibitor MG132 unexpectedly induced phosphorylation of ser536

phosphorylation in unstimulated cells, but did not affect the IL-1-induced ser536

phosphorylation. MG132 also activated IKK kinase activity. This finding may suggest that

MG132 promotes the formation of IKK:p65: IκBα complexes (1) in which IKK comes into

close proximity with its substrate p65. The exact mechanism of the MG132-mediated ser536

phosphorylation awaits further investigation, but together with the additional evidence

presented in this study (see below), we suggest that p65 exists in at least three complexes in

cells: (i), the majority is bound to IκB and is thereby trapped in the cytoplasm and not

accessible to ser536 kinases, (ii), a low amount exists as free dimer which is phosphorylated

by ser536 kinases (and other kinases) and is responsible for basal NF-κB activity, (iii), some

p65 can also associate with IKK and apparently it is this fraction which accumulates by

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MG132 treatment. The relative composition of these complexes is highly dynamic and can

therefore change by extracellular stimuli (1;27), by overexpression of individual components

(as shown in this study for GFP-p65 or IκBα), or, by disturbing their natural protein turnover

by proteasome blockade, e.g. by MG132.

Both IKKs have been shown to phosphorylate ser536 by using cells deficient for either IKKα

or IKKβ, or, by assessing the ser536 kinase acitivities associated with immunoprecipitated or

recombinant IKKs (21-24). To study the relevance of IKKs for IL-1-mediated ser536

phosphorylation of endogenous p65, we used a number of strategies to suppress or inhibit

endogenous IKKα and β. Neither by RNAi directed at both IKKs simultaenously, nor by the

novel IKKβ inhibitor SC-514 or by dominant negative mutants of IKKα and IKKβ we were

able to suppress ser536 phosphorylation efficiently. Additionally, in all experiments shown in

this study there was some constitutive ser536 phosphorylation detectable by the ser536

antibody which is specific for the phosphorylated form of p65. Experiments using

overexpressed p65 indicated that a GFP-p65 fusion could be phosphorylated in the absence of

IKK activation and this effect was lost by trapping of GFP-p65 by coexpression of IκBα.

These observations led us to screen for IKK-independent ser536 protein kinases. By

chromatographic fractionation of cell extracts we identified two distinct enzymatic activities

that were detectable in unstimulated cells and that were both stimulated by IL-1. Peak two

contained IKKα, IKKβ, IKKε and TBK1 while peak one represents an unknown kinase.

Further experiments confirmed that IKKε and TBK1 phosphorylate ser536 in vivo and in

vitro. These experiments also revealed that the activity of IKKε and TBK1 is not regulated by

IL-1. This contrasts to TNF, which activates TBK1 in complex with a regulatory subunit

called NAP-1 (46).

Ectopically expressed IKKε and TBK1 do not increase the constitutive ser536

phosphorylation of endogenous p65 (data not shown). The potency of IKKε and of TBK1 to

phosphorylate ser536 in vivo becomes only apparent when the amount of p65 is

concomitantly increased by overexpression, an observation which is in line with the existence

of different p65 complexes as described above.

An important conclusion from our data therefore is, that IKKε and TBK1 are likely candidates

that account for p65 ser536 phosphorylation in those situations where p65 is not bound to

IKK or IκB. In the absence of external stimulation, p65 shuttles between the cytoplasm and

the nucleus (9;47), a phenomen that may account for a detecable, but low amount of p65 in

the nucleus of unstimulated cells (Fig.6) and that may explain the low level of constitutive

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NF-κB activity that is found in many cell types. Our data would support a model, in which

IKKε and TBK1 phosphorylate free p65 that is found in unstimulated cells, while the IKKs

have their major role in phosphorylating ser536 of p65 that becomes associated with the IKK

complex in response to IL-1. IKKε and TBK1 may also play a role in phosphorylating p65 in

response to DNA-damaging agents that activate NF-κB in the absence of IKKα and IKKβ

(48). While we clearly show that IKKε and TBK1 can phosphorylate ser536, their

contribution to p65 transactivation via this site still needs to be established and may only

become apparent when analysing individual NF-κB target genes.

We also discovered an IL-1-stimulated protein kinase that was distinct from IKKs, TBK1 and

IKKε. We have not identified this activity yet. While this manuscript was in preparation,

Bohuslav et al reported that RSK1 phosphorylates ser536 in an p53-dependent manner (49).

Ribosomal S6 kinases (RSK) elute from Mono Q at around 200-300mM NaCl at pH 7.0 to pH

7.8 (50), and RSK1 activation depends on ERK (49). Peak I ser536 kinase elutes at around

30-170 mM at pH 8.5 from Q resins and we did not observe any effect of PD98059 on

constitutive or IL-1-inducible ser536 phosphorylation. Therefore, peak I kinase is a novel,

unknown ser536 kinase that remains to be identified.

Collectively our results show that ser536 phosphorylation is regulated by at least five

different protein kinases, IKKα, IKKβ, IKKε , TBK1 and an unknown kinase.

Despite its wide occurrence, the function of serine 536 phosphorylation for p65 activity is

largely elusive. This contrasts with phosphorylation of ser276, which is required for full p65-

induced transactivation (11;13-15). Only a few and controversial findings regarding ser536

function exist. Expression of a p65 serine 536 to alanine mutant in a p65-/- background

revealed that serine 536 is dispensable for TNF-mediated IL-6 gene induction (Okazaki et al.,

2003), but on the other hand it is required for TNF- or LPS-induced induced activation of a

NF-κB reporter gene in p65 -/- cells (20;21). Furthermore, mutation of ser536 to alanine did

not affect basal or TNF-induced activation of a GAL4-p65 fusion protein (13). The role of

serine 536 phosphorylation in coupling p65 to coactivators, corepressors, or components of

the basal transcriptional machinery has not been investigated to date. We found that p65

phosphorylated on ser536 binds to the promoter of IL-8, a human gene whose transcriptional

regulation in response to IL-1 we have studied previously in detail (51). Transfection of an

IL-8 promoter construct in p65 deficient fibroblasts confirmed the importance of p65 for IL-8

transcription, as in its absence the IL-8 promoter was largely inactive. We therefore

reconstituted the p65 -/- Mefs with ser536 mutants to study the functional relevance of this

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site in IL-8 transcription. In the light of the previously reported lack of effect of the alanine

mutants of ser536 (15) we generated two additional mutants that either mimicked

phosphorylation or preserved a predicted hydrogen-bond between ser536 and asp533. The

alanine and the phosphomimetic mutant enhanced IL-8 transcription compared to the wild

type protein, while the S536N mutant had the lowest activity and was largely resistent to IL-

1-stimulation.

This phenomen was reminiscent for a mechanism by which thr18 regulates p53

transactivation. The acidic transactivation domains of p53 and p65 share low sequence

homology and have unfolded structures in the absence of their targets. However, both contain

a so-called FXXΦΦ motif that – as shown for p53 – folds into an α-helical structure upon

binding to TAFII31, a component of TFIID (52). By this mechanism the acidic residues

establish initial longe-range contact with basic residues in TAFII31 to allow binding. Induced

transition into the α-helical structure then exposes the hydrophobic residues within the

FXXΦΦ motif which now contact non polar residues in TAFII31. By this mechanism

multiple weak interactions synergize to modulate transactivation in a cooperative manner. In

addition to the FXXΦΦ motif of p65 that has been located previously between amino acids

542-546 a second region positioned around ser536 fulfilled the criteria for a FXXΦΦ motif.

We therefore tested the hypothesis that ser536 plays a role in TAFII31 binding. In vitro, p65

wild type and the S536D mutant bound to TAFII31similarly, while the S536N mutant bound

less strongly. Likewise, in p53, phosphomimetic mutation of thr18 within the FXXΦΦ motif

does not increase TAFII31 binding in vitro, but disrupts the hydrogen bond to the

neighbouring asp21 and thereby, lowers the affinity for the corepressor Mdm-2. This raised

the question if ser536 phosphorylation may similarly affect binding to a repressor of p65. We

have previously identified the NRF protein as a corepressor of basal IL-8 transcription (31).

However, in IL-1-stimulated cells NRF switches its function and serves as a coactivator by an

unknown mechanism. In contrast, the groucho-related protein amino-terminal enhancer of

split (AES) inhibits both, basal and TNF-induced IL-6 transcription and has been found in

yeast two hybrid screens to bind to the C-terminal region (aa 477-522) of p65 (30).Thus AES

binds in close vicinity to the FXXΦΦ motif around ser536. Due to the strong transcriptional

activity of the p65 TAD in yeast two hybrid screens, aa 523-551 of p65 could not be tested for

AES binding (30), but we confirmed the interaction of p65 with GST-AES in pull down

experiments (data not shown). Bacterially expressed GST-AES bound indistinguishable to wt

p65, the S536D and the S536N mutant in vitro (data not shown). However, if in vitro

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synthezised AES was preincubated with equal amounts of p65 proteins, it became apparent

that AES negatively influenced the ability of wild type p65 and of the S536N mutant, but not

that of the S536D mutant, to interact with GST-TAFII31. Based on these results we

considered the possibility that in analogy to p53, the hydrogen bond between ser536 and

asp533 may serve to modulate the balance of TAFII31 and AES binding to p65. In p65

deficient cells, coexpression of TAFII31 enhanced p65-mediated IL-8 transcription and this is

strongly stimulated by IL-1, presumably by phosphorylation. Coexpression of AES has little

effect on basal p65-mediated IL-8 transcription but suppresses IL-1-induced IL-8

transcription. Upon treatment with IL-1 neither TAFII31 or AES can activate IL-8

transcription significantly in the absence of p65, suggesting that their gene regulatory effects

are mediated via p65. Evidence for the involvement of ser536 in these effects is derived from

coexpression of either the S536D or the S536N mutant with TAFII31 or AES. The S536D

mutant is largely resistant to AES-mediated repression while the S536N mutant is efficiently

suppressed by AES. The S536N mutant is also unresponsive to TAFII31-mediated

enhancement of IL-8 transcription.

Collectively our data show that at least five kinases converge on ser536 of p65 NF-κB.

Together with the observations that ERK-RSK pathways also phosphorylate ser536 during

oncogenic transformation this site may be the target of many different kinases (49;53). We

further provide evidence that this serine residue is an important determinant of chemokine

transcription by promoting efficient coupling of p65 to the basal transcriptional machinery.

Mutations of ser536 do not render the p65 protein inactive, suggesting that this

phosphorylation site plays a modulatory rather than an essential role and acts in concert with

other p65 phosphorylations. Targeting the ser536 protein kinases, therefore, may be an

effective means of controlling excessive NF-κB activity in inflammation or proliferative

diseases while preserving residual activity required for cell survival.

Acknowledgements:

We thank Takashi Okamoto, Hiroyasu Nakano, Hiroaki Sakurai, Rainer deMartin, Xiangwei

Wu, John Ladias and David Wallach for the gift of valuable reagents.

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LEGENDS

Fig.1. Serine 536 phosphorylation of p65 NF-κB in response to IL-1 or TNF occurs

independent of JNK, p38, ERK and PI3 kinase pathways but is induced by proteasome

inhibition

HeLa cells were treated with PD98059 (50µM), SP600125 (20µM), SB203580 (2µM),

Wortmannin (1µM) or MG132 (10µM) for 30min and then stimulated for the indicated times

with IL-1 (10ng/ml), TNF (20ng/ml) or PMA (20ng/ml) plus Ionomycin (0,5µg/ml). Cells

were lysed in SDS sample buffer and phosphorylation of p65 NF-κB at ser536 was analysed

by western blotting. Equal loading of all lanes was confirmed using antibodies against p65

(not shown).

Fig.2. MG132 and IL-1 induce ser536 phosphorylation of p65 NF-κB through the IKK

complex

HeLa cells were treated with MG132 at various concentrations for one hour (A) or with

MG132 (10µM) for the indicated times (B) or left untreated. p65 NF-κB phosphorylated at

ser536, phosphorylated IκBα and p65 were detected by western blotting of SDS lysates. In C)

cells were treated with MG132 (10µM) for 1h or with IL-1 (10ng/ml) for 10min or left

untreated. The IKK complex was purified from extracts of these cells by immunoprecipitation

with an anti IKKα antibody and used to phosphorylate a GST-p65 fusion protein containing

the transactivation domain (amino acids 354-551) in vitro.

Fig.3.Ser536 phosphorylation of p65 NF-κB occurs independent of IKKα and β

A) HeLa cells were left untreated, treated with transfection reagent alone (oligofectamine) or

transfected with siRNA (100nM) directed against luciferase, IKKα or IKKβ. After 72h cells

were stimulated for 10min with IL-1 (10ng/ml) or left untreated.

B) HeLa cells were left untreated, or, treated for one hour with 0.02% DMSO or SC-514.

Then, cells were stimulated for 10min with IL-1 (10ng/ml) or left untreated.

In A) and B) phosphorylation of ser536 of p65 and of IκBα and the expression of IKKα,

IKKβ and p65 was determined by western blotting of SDS lyates.

C) HEK293 IL-1R cells were transfected with pCS3MT vector alone (lane 1, lane 5), or

expression plasmids coding for GFP-p65 (0,5µg), wild type IKKα and IKKβ, kinase inactive

IKKα and IKKβ, or FLAG- IκBα (3µg each) as indicated. NF-κB(3)luc (0,25µg) and 0,5µg

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SV40 beta gal were cotransfected. Shown is one out of two experiments that gave identical

results. Cells were lysed in luciferase assay buffer and expression of the transfected proteins

analysed by antibodies against phospho ser536 of p65, p65, IKKα, IKKβ, phospho IκBα and

IκBα. The arrows indicate position of endogenous ΙκΒα that is induced by GFP-p65 and of

the co-transfected FLAG-tagged IκBα.

Fig.4.Chromatographic separation of ser536 kinase actvities identifies IKKα, IKKβ,

TBK1, and IKKε and one unknown kinase as ser536 kinases

A) HeLa cells were stimulated for 10min with IL-1 (10ng/ml) or left untreated. Cells were

lysed and the total activity of all cellular kinases that phosphorylate GST-p65354-551

(indicated by an arrow) was determined by radioactive in vitro kinase assay.

B) Total extracts obtained from cells treated as in A) were loaded onto a Resource Q

anionexchange chromatography column. Proteins were eluted with an increasing NaCl (0-

0.75 M) gradient. Aliquots of individual fractions were assayed for GST-p65 (354-551)

kinase activity in vitro. An autoradiography containing phosphorylated GST-p65 (354-551) is

shown. Two broad peaks of GST-p65 kinase activity (fractions 5-9 and 12-15) were detected.

C) Aliquots of individual fractions from the experiment shown in B) were analysed for the

coelution of IKKα, IKKβ, TBK1 and IKKε antigens with the ser536 kinase activities by

western blotting. Arrows indicate positions of specific antigens.

D)The specificity of the enzymes of the two fractions (6, 12) from the experiment shown in

B) that contained the peak of IL-1-stimulated p65 kinase activities were analysed by in vitro

kinase assays (ka) without exogenous substrate, with GST-p65 (354-551) wildtype (wt) or

with versions mutated to alanine (A) at the indicated serine (S) residues. Shown is the region

of the autoradiography containing phosphorylated GST-p65 (354-551) and of the GST-p65

(354-551) protein band stained with coomassie brilliant blue (CBB).

Fig.5.IKKε and TBK1 stimulate ser536 phosphorylation of p65 in vivo and are direct

536 kinases in vitro

A) HEK293 IL-1R cells were transfected with pCS3MT vector alone (lane 1, lane 7), or,

expression plasmids coding for GFP-p65 (0,5µg), wild type IKKε or TBK1 and kinase

inactive IKKε or TBK1 (3µg each) as indicated. NF-κB (3)luc (0,25µg) and 0,5µg SV40 beta

gal were cotransfected. After 24h cells were stimulated for 5h with IL-1 (10ng/ml) or left

untreated as indicated. Luciferase activities were determined in cell lysates, normalized and

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expressed relative to the unstimulated vector control. The graph shows the mean luciferase

activities of two independent experiments performed in duplicates +/- SEM. Expression of

transfected proteins was analysed by western blotting of SDS lysates from cell cultures that

were transfected in parallel. To analyse phosphorylation of ser536 of p65 half of the cells

were treated for 10min with IL-1 (10ng/ml) prior to lysis. Shown is one out of the two

experiments that gave identical results.

B) IKKε and TBK1 were expressed in HEK293 cells as described in A). Overexpressed

kinases from untreated cells and cells treated for 10 min with IL-1 (10ng/ml) were

immunprecipitated with anti FLAG antibodies and analysed for GST-p65 (354-551) kinase

activity in vitro using wildtype p65 or the ser536 alanine (S536A) mutant. Shown is the

region of the autoradiography containing phosphorylated GST-p65 (354-551) and of the GST-

p65 (354-551) protein band stained with coomassie brilliant blue (CBB).

Fig.6. p65 phosphorylated at ser536 occurs in the nucleus and is recruited to the

endogenous IL-8 promoter

A) HeLa cells were stimulated for 10 min with IL-1 (10ng/ml) or left untreated. Nuclear and

cytsolic extracts were prepared and activity of kinases that phosphorylate GST-p65(354-551)

or a version in which ser536 was mutated to alanine (S536A) was determined by in vitro

kinase assay. Phosphorylation of ser536 was detected by immunoblotting of reaction

mixtures, the blot membrane was reprobed with an anti p65 antibodies to confirm equal

loading. Lanes 9+10 contained only GST-p65 fusion proteins without cell extracts.

B) HeLa cells were stimulated for the indicated times with IL-1 (10ng/ml) or left untreated.

Nuclear extracts were analysed for p65 and ser536 phosphorylation of p65 by western

blotting

C) Nuclear extracts from unstimulated HeLa cells or cells stimulated for 10 min with IL-1

(10ng/ml) were incubated with a 32P-labelled oligonucleotide containig a consensus NF-κB

binding sites in the presence or absence of antibodies against p65 or phospho ser536 of p65.

Solid arrows indicate the two IL-1-induced protein-DNA complexes, open arrows indicate

forms of these complexes supershifted by the antibodies.

D) KB cells were stimulated for the indicated times with IL-1 (10ng/ml) or left untreated.

After in vivo cross-linking soluble chromatin was prepared and antibodies against p65 and

phospho ser536 of p65 were used to immunoprecipitate protein-DNA complexes. IL-8

promoter DNA bound to p65 was amplified by PCR from the immune complexes, separated

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by agarose gel electrophoresis and visualized by ethidium bromide staining. Im parallel PCR

was performed on chromatin prior to immunoprecipitation (input).

Fig.7.Generation and functional analysis of p65 ser536 mutants

A), B) Predicted primary and secondary structure of FXXΦΦ motifs in the p65

transactivation domain. A) Shown is a comparison of the FXXΦΦ motif that has been

identified in p53 (52) and of two similar regions in p65. H indicates an α-helical secondary

structure as found for p53 by x-ray crystallography (54) or for p65 by CD spectroscopy (40).

Known (for p53) or putative TAFII31 binding sites (for p65) are shown in blue.. The

phosphorylation sites in p53 (thr18) and in p65 (ser536) are underlined, acidic amino acids

participating in hydrogen-bonds to these residues are shown in red. Position of amino acids

are shown in brackets.

B) Predicted secondary structure of ser536 mutants. Using the molecular modelling program

DEEP VIEW version 3.7 (55;56) the amino acid region surrounding ser536 of p65 (DFSSIA)

was modelled onto the published α-helical structure of the peptide TFSDLWK of p53 (54).

Indicated by a dashed green line is a hydrogen-bond that forms between ser536 and asp533.

The effects of A, D, and N substitutions of ser536 on this hydrogen-bond are shown. Green

indicates the peptide backbone, the depicted amino acids are displayed as sticks (carbon, gray;

nitrogen, blue,; oxygen, red). 3D-rendering was made by using Povray 3.5 (www.povray.org).

C)Wild type (wt) p65 or the indicated mutants were transcribed and translated in vitro.

Aliquots of the reaction mixtures were analysed for the expression of p65 by western blotting

(upper panel) and then analysed for binding to a NF-κB consensus site containing

oligonucleotide by EMSA. The region of the autoradiography containing the NF-κB -DNA

complexes is shown (lower panel). As a control reticulocyte lysates without p65 expression

plasmids (-) were analysed in parallel.

D) 1µg of pMT7 expression plasmids for wild type p65 (wt) and the indicated ser536 mutants

were transiently expressed in p65-deficient fibroblasts (-/-). Half of the cells were stimulated

for 10min with IL-1 (10ng/ml) and phosphorylation and expression of p65 proteins analysed

by the anti phospho ser536 and by anti p65 antibodies by western blotting.Antibodies against

ERK were used to confirm equal loading.

E) 0.3 µg of expression plasmids for wild type p65 (wt) and the indicated ser536 mutants

were cotransfected with 0,25 µg pUHC13.3IL-8 prom.luc. and 1µg SV40-beta gal. After 24h

the cells were stimulated for four hours with IL-1 (10ng/ml) or left untreated. Thereafter

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25

luciferase activities were determined and normalized for beta gal activity. Shown are the

mean values +/- S.E.M. from five independent experiments performed in duplicates relative to

the unstimulated vector control.

Fig.8.Ser536 participates in binding of p65 to TAFII31 and AES in vitro

A) 35S-labelled wild type (wt) p65 or the indicated mutants were transcribed and translated in

vitro. Equal amounts of the reaction mixtures were incubated with GST or GST-TAFII31 that

had been immobilized on GSH-sepharose. Bound proteins were eluted with SDS sample

buffer separated by SDS PAGE and detected by autoradiography. 1/10 of the input samples

were analysed in parallel. B) 35S-labelled p65 (wt), the indicated p65 mutants, or AES were

synthesized in vitro as described above. Binding of labelled p65 proteins to immobilized

GST-TAFII31 was analysed without or in the presence of equal amounts of labelled AES as

indicated (right panel). 1/10 of the input samples were analysed in parallel (left panel).

Fig.9.Regulation of IL-8 transcription by TAFII31 or AES requires p65 and is affected

by mutations of ser536

A) P65 deficient fibroblasts were transfected with 0.3µg of pMT7 p65, 3µg pCMV-AES, 3µg

pMTN-HATAFII31, or empty vector as indicated. 0,25 µg pUHC13.3IL-8 prom.luc. and 1µg

SV40-beta gal were cotransfected. After 48 h the cells were stimulated for four hours with IL-

1 (10ng/ml) or left untreated. Thereafter luciferase activities were determined and normalized

for beta gal activity. Shown are the mean values +/- S.E.M. from three independent

experiments performed in duplicates relative to the unstimulated vector control.

B) p65-deficient fibroblasts were transfected with various combinations of 0.3µg of

pMT7p65S536D, pMT7p65S536N plus 1.5µg empty vector (black bars), 1.5µg pMTN-

HATAFII31 (light gray bars) and 1.5µg pCMV-AES (dark gray bars) as indicated. 0,5 µg

pUHC13.3IL-8 prom.luc. and 1µg SV40-beta gal were cotransfected. After 24h the cells were

lysed, luciferase activities were determined and normalized for beta gal activity. The relative

effects of cotransfection of TAFII31 or AES on activity of the indicated p65 mutants

compared to cotransfection of empty vector alone (set as 100%) is indicated.Results represent

the mean values from two independent experiments performed in duplicate.

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Buss et al., Fig.1

P p65

+ PD98059P p65

P p65 + SP600125

P p65 + SB203580

P p65 + Wortmannin

P p65 + MG132

- 10 30 - 10 30 - 10 30 (min) PMA/IONO TNF IL-1

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P p65

p65

P IκBα

- 0.1 1 10 MG132 (µM)

- MG132 IL-1

32P-GST-p65

Buss et al., Fig.2

A)

B)

C)

- 10 30 60 120 MG132 (min)

P p65

p65

P IκBα

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untransfected

siRNA IKKα + IKKβ

IL-1

+

oligofectamine

++

++

+

++

++

siRNA Luciferase

+ +

p65

P p65

P IκBα

IKKα

IKKβ

IL-1 +SC-514 (µM)

+

++ +

DMSO

p65

P p65

P IκBα

+ + + + +50 100 10050

Buss et al., Fig.3

A)

B)

C)

GFP-p65IKKα + IKKβ

+d.n.IKKα + d.n.IKKβ

IκBα

+ + ++

++

1 2 3 4 5

P GFP-p65

GFP-p65

P IκBα

FLAG-IκBα

IKKα

IKKβ

end. IκBα

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IL-1 -

5 6 7 8 9 10 11 12 13 14 15 16 17 18 Fraction

-

IL-1kinase assay

5 6 7 8 9 10 11 12 13 14 15 16 17 18 Fraction

IKKα

IKKβ

TBK1

IKKε

Buss et al., Fig.4

A)

B)

C)

D)

- wt

536A

529A

536A

529A

+

fraction 6

fraction 12

kaCBB

kaCBB

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1 2 3 4 5 6 7 8 9 10 11 12

NF-

κB(3

)luc.

act

ivity

(- fo

ld)

0

20

40

60

80

100

120

140

160

P GFP-p65

GFP-p65

IKKε

ERK

TBK1

97

9797

6697

45

kD

1 2 3 4 5 6 7 8 9 10 11 12 GFP-p65

IKKε

IL-1

+d.n.IKKε

+ + + + + + + +++

++

+

++

++

+ + + + + +d.n.TBK1

TBK1

IKKε IKKεTBK1 TBK1

kinase assayCBB

GST-p65+

+ IL-1 +

++ +

+ ++

+ + +

GST-p65-S536A

Buss et al., Fig.5

A)

B)

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NF-κB

++

IL-1

++

+ + +α P p65

α p65

- .2 .5 1 2 3 4 16 24 IL-1 (h) p65

P p65

IL-1 (min) - 10 30 180 Input

p65

P p65

Buss et al., Fig.6

A)

B)

C)

D)

p65

P p65

GST-p65 ++

IL-1 ++ + +

+ + ++ + +

GST-p65-S536A+

+

nucleus cytosol

1 2 3 4 5 6 7 8 9 10

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Buss et al., Fig.7

A)

B)

FXXΦΦp53 (9) SVEPPLSQETFSDLWKLLP (27)α-helix HHHHHHHHH

FXXΦΦ FXXΦΦp65 (531) DEDFSS IADMDFSALLSQISS (551)

HHHHHHHHHHHHHHHHHHHHHα-helix

S536N S536A

WT S536D

Phe534Asp533

Ser535

Ser536Ile537

Ala538

Asn536 Ala536

Asp536

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- fol

d

020406080

100120140

wt

S536

D

S536

D

S536

N

S536

A

S536

N

S536

A

S536

A wt

- / -

- / -

+ IL-1

- / - wt

- / - wt

+ IL-1

p65

P p65

ERK2

S536

A

S536

A

S536

D

S536

D

S536

N

S536

N

Buss et al., Fig.7

C)

D)

E)

- wt

S536

D

S536

N

S536

A

EMSA

westernblot

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wtS536D

S536N

Inpu

t

GST

GST

-TA

FII3

1

Buss et al., Fig.8

A)

B) Input72

55

40

p65

p65

S536

D

p65

S563

NA

ES

30

24

GSTGST-TAFII31

wt

S536D

S536N

AES - + - +

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Buss et al., Fig.9

- fol

d

050

100150200250300600800

1000

1 2 3 4 5 6 7 8 9 10 11 12

p65 IL-1

+

++

+

+ +

+

+++

++

+

+ + + + + +

AESTAFII31

vector++

A)

B)

IL-8

pro

mot

er a

ctiv

ity (%

)

0

50

100

150

200

250

S536D S536N

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Michael KrachtHolger Buss, Anneke Dörrie, M.Lienhard Schmitz, Elke Hoffmann, Klaus Resch and

unknown kinase and couples p65 to TAFII31-mediated IL-8 transcriptionSRC="/math/epsilon.gif" ALIGN="BASELINE" ALT="epsilon ">, TBK1 and an

, IKK<IMGβ, IKKbαmediated by multiple protein kinases including IKKConstitutive and IL-1-inducible phosphorylation of p65 NF-kB at serine 536 is

published online October 15, 2004J. Biol. Chem. 

  10.1074/jbc.M409825200Access the most updated version of this article at doi:

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