- Email: [email protected]
NIK and Cot cooperate to trigger NF-κB p65 phosphorylation
Biochemical and Biophysical Research Communications 371 (2008) 294–297
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
NIK and Cot cooperate to trigger NF-jB p65 phosphorylation Tobias Wittwer, M. Lienhard Schmitz * Institute of Biochemistry, Medical Faculty, Friedrichstrasse 24, Justus-Liebig-University, D-35392 Giessen, Germany
a r t i c l e
i n f o
Article history: Received 11 April 2008 Available online 23 April 2008
Keywords: NF-jB Transactivation Phosphorylation Kinase Transcription
a b s t r a c t The serine/threonine kinase Cot triggers NF-jB-dependent transactivation and activation of various MAPKinases. Here we identify Cot as a novel p65 interacting protein kinase. Cot expression induces p65 phosphorylation at serines 536 and 468 in dependence from its kinase function. Accordingly, shRNA-mediated knockdown of Cot expression interferes with TNF-induced NF-jB-dependent gene expression. Also the Cterminally truncated, oncogenic form of Cot is able to trigger p65 phosphorylation. In vitro kinase assays and dominant negative mutants revealed that NIK functions downstream of Cot to mediate p65 phosphorylation. Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved.
The NF-jB family of transcription factors consists of five different DNA-binding subunits that dimerize in different combinations to mediate gene expression. The two subunits p50 and p52 are synthesized as large precursor molecules called 105 and p100, respectively. The other three subunits (p65, RelB, and c-Rel) contain transactivation domains in their C-termini [1,2]. NF-jB is activated by diverse stressful conditions including proinflammatory stimuli such as tumor necrosis factor (TNF) and lipopolysaccharide (LPS). Canonical NF-jB activation by TNF involves the induction of signaling cascades that ultimately lead to the activation of IjB kinases (IKKs) which in turn phosphorylate the inhibitory IjBa protein, thus targeting it for ubiquitin/proteasome-mediated degradation [3]. The released NF-jB dimer is then free to translocate to the nucleus where it drives target gene expression. Efficient NF-jB-mediated transcription requires posttranslational modifications of the DNA-binding subunits, which are best characterized for the p65 protein. This subunit can be modified by ubiquitination, prolyl isomerization, acetylation or phosphorylation [4,5]. More than 10 different phosphorylation sites have been discovered for the p65 protein. They serve different functions ranging from the regulation of nuclear import, protein stability and coactivator binding [5–8]. The set of kinases with NF-jB activating properties includes the MAP3K family member Cot (Cancer Osaka thyroid). This kinase, in rats also known as Tpl-2, was initially identified in a C-terminally truncated form as a result of a screen for oncogenes [9]. Overexpression of a C-terminally truncated Cot isoform in T cells was sufficient to induce lymphomas, thus confirming the oncogenic
* Corresponding author. Fax: +49 641 9947589. E-mail address: [email protected] (M.L. Schmitz).
potential of this truncated kinase. Various stimuli including LPS and TNF trigger Cot activation which in turn allows the induction of the MAP kinases p38, JNK, and ERK and also NF-jB [10–12]. Knockout experiments revealed that Cot signals activate ERK, JNK, and NF-jB in a cell-type and stimulus-specific manner. Cot / mouse embryonic fibroblasts (MEFs) showed intact TNF-triggered IKK activation, while NF-jB-dependent transcription was impaired [13]. These results raise the possibility that the Cot-mediated effects are rather due to the regulation of NF-jB-dependent transcriptional activity.
Materials and methods Cell culture, expression vectors, and transient transfections. HEK 293 cells were cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, and 1% (v/v) penicillin/streptomycin. Cells were transfected using Rotifect (Roth) according to the manufacturer’s instructions. HA-Cot, HA-Cot inac., HA-CotD, Myc-Cot, Myc Cot KM [10,12], GFP-p65, and (jB)3-luc [14] were described. pSUPER-Cot and the point mutated control were cloned by standard methods. The following DNA sequence was used to allow the production of shRNA targeting Cot: GTGAAGAGCCAGCAGTTTA. The control sequence was mutated in three positions which are shown in bold: GTCAAGAGGCAGCAGTATA. Cell lysis and Western blotting. Cells were washed with ice-cold phosphate-buffered saline and collected by centrifugation. The pellet was resuspendet in NP-40 lysis buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% (v/v) NP-40, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 40 mM NaF, 2 mM sodium vanadate, leupeptin (10 lg/ml), and aprotinin (10 lg/ml)) and incubated on ice for 20 min. Cell debris was removed by centrifugation with 13,000 rpm at 4 °C. Proteins were separated by reducing SDS–PAGE and blotted onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). After blocking, membranes were incubated with appropriate primary and horseradish peroxidase-coupled secondary antibodies, followed by protein detection using the Perkin-Elmer Western lightning chemiluminescence system. Antibodies recognizing NF-jB p65 phospho-Ser 468
0006-291X/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.069
295
T. Wittwer, M.L. Schmitz / Biochemical and Biophysical Research Communications 371 (2008) 294–297 (3039) and phospho-Ser 536 (3031) were obtained from Cell Signaling Technology, NF-jB p65 (C-20), GST (Z-5), and Cot (M-20) antibodies were obtained from Santa Cruz. Antibodies recognizing Myc (9E10), GFP (clones 7.1 and 13.1), and HA (3F10) epitopes were from Roche. Purification of GST-p65 and in vitro kinase assays. The GST-p65 (354–551) fusion protein was expressed in Escherichia coli BL21 cells and purified by affinity chromatography on glutathione-coupled sepharose according to standard protocols. HA-Cot and Flag-IKKe were expressed in HEK 293 cells and purified by immunoprecipitation using aHA and aFlag antibodies, respectively. Precipitates were washed three times in NP-40 lysis buffer and two times in kinase buffer (20 mM Hepes/KOH, pH 7.4, 25 mM b-glycerophosphate, 2 mM dithiothreitol, and 20 mM MgCl2). The immune complex kinase assay was performed in a final volume of 20 ll kinase buffer containing 40 lM ATP and 1 lg of the purified GST-p65 substrate protein for 30 min at 30 °C. The reaction was terminated by the addition of SDS sample buffer and p65 phosphorylation was detected by immunoblotting. Coimmunoprecipitation. HEK 293 cells were transiently transfected with plasmids encoding GFP-p65 and Myc-Cot. The next day, cells were lyzed in NP-40 buffer and aliquots were analyzed for protein expression. The remaining lysates were incubated with aGFP or control antibodies and 30 ll protein A/G sepharose for 4 h on a spinning wheel at 4 °C. The immunoprecipitates were washed five times in NP-40 lysis buffer. Bound proteins were eluted by boiling in SDS sample buffer and detected by immunoblotting. Luciferase assays. Cells were seeded in 6-well plates, transfected and treated as specified. Cells were lyzed in reporter lysis buffer (25 mM Tris–Phosphate, 2 mM DTT, 2 mM CDTA, 10% (v/v) glycerol, and 1% (v/v) Triton X-100). Luciferase activity was determined in a luminometer (Duo Lumat LB 9507, Berthold) by adding 50 ll of assay buffer (40 mM Tricine, 2.14 mM (MgCO3)4Mg(OH)25H2O, 5.34 mM MgSO4, 0.2 mM EDTA, 66.6 mM DTT, 540 lM CoA, 940 lM luciferin, and 1.06 mM ATP) and measuring light emission for 10 s.
A
WB:
P GFP- p65
α P S468
P GFP-p65
p65 n.s. Cot n.s.
αCot
B -
GFP-p65 HA-Cot HA-CotΔ WB:
+ + + - + - - +
α P S536
P GFP- p65
α P S468
P GFP-p65
Cot binds to NF-jB p65 and triggers its phosphorylation
αHA
To test the impact of Cot on NF-jB-mediated gene expression, cells were cotransfected with a NF-jB-dependent luciferase gene
GFP-p65
αp65
αp65
Cot contributes to TNF-triggered expression of NF-jB target genes
+ + + - + - - +
α P S536
Results and discussion
Cot-deficient MEFs show impaired NF-jB-dependent gene expression but intact IKK activation and IjB phosphorylation [13]. To test a potential impact of Cot on p65 phosphorylation, cells were transfected to express active or inactive Cot along with low amounts of the GFP-tagged p65. Subsequent Western blotting with phospho-specific antibodies revealed that Cot expression triggered the phosphorylation of p65 at serine 536 and 468 dependent on its kinase function (Fig. 1A). This adds further evidence that these p65 phosphorylation sites can be modified by several kinases. The C-terminal serine 536 can be phosphorylated by multiple kinases, including IKKb, IKKe, TBK1, and RSK1 which are activated via distinct signaling pathways [15–18]. But not only the kinases, also the functions of serine 536 phosphorylation are quite diverse and range from enhanced transactivation to enhanced kinetics of p65 nuclear import and regulation of protein stability [6,7]. The oncogenic potential of Cot requires deletion of its C terminus, which is frequently associated with an increased kinase activity [19]. We thus compared the p65 phosphorylating activity between wildtype Cot and its C-terminally truncated version (CotD). These experiments revealed similar p65 phosphorylating activities of Cot and CotD (Fig. 1B), showing that the C-terminal part of the kinase is not affecting its ability to mediate p65 modification. We then explored the possible interaction between Cot and its substrate protein by coimmunoprecipitation experiments. Cells were transfected to express Myc-tagged Cot and/or GFP-p65, followed by precipitation of GFP-p65 and Western blotting. A fraction of Cot was detected in GFP-p65 immunoprecipitates (Fig. 2), showing the ability of the kinase to bind its substrate protein. However, the vast majority of Cot is stoichiometrically associated with p105 [20], so that only a fraction of the kinase will be able to bind the p65 protein.
-
GFP-p65 HA-Cot HA-Cot inac
GFP-p65 p65 Cot, CotΔ n.s.
Fig. 1. Cot induces p65 phosphorylation. (A). HEK 293 cells were transiently transfected with plasmids encoding GFP-p65 and HA-tagged forms of wildtype (HA-Cot) or inactive (HA-Cot inac.) Cot as shown. The next day, cells were lyzed and equal amounts of protein contained in cell lysates were analyzed by Western blotting (WB). Phosphorylation of p65 at serines 536 and 468 was revealed by phosphospecific antibodies, correct expression of transfected proteins was ensured with ap65 and aCot antibodies. A nonspecific (n.s.) band is indicated. (B). The experiment was done as in (A) with the exception that a C-terminally truncated version of Cot (CotD) was used.
and expression vectors encoding wildtype or inactive Cot proteins. The next day, cells were stimulated with TNF as shown and analyzed for luciferase activity (Fig. 3A). Expression of Cot
IP: GFP-p65 Myc-Cot
WB:
αIgG
+ -
+ -
+
+ +
αMyc
Cot IgG heavy chain
αGFP
GFP-p65
Input: GFP-p65 Myc-Cot
WB:
- + + +
αGFP
+ -
- + + +
αMyc
Cot
αGFP
GFP-p65
Fig. 2. Interaction of Cot and NF-jB p65. HEK 293 cells were transfected with plasmids encoding GFP-p65 and Myc-Cot as shown. The next day, cells were lyzed and extracts were either tested for correct expression of the tagged proteins (lower) or immunoprecipitated with aGFP or control antibodies. The eluted proteins were revealed by immunoblotting, the position of the respective proteins and the IgG heavy chain is indicated.
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T. Wittwer, M.L. Schmitz / Biochemical and Biophysical Research Communications 371 (2008) 294–297
(κB)3-luciferase
A
30
20
- fold
activation
40
10
0
HA-Cot
-
+
-
-
+
-
HA-Cot inac
-
-
+
-
-
+
TNF
-
-
-
+
+
+
B
C
(κB)3-luciferase
-fold activation
14 12
HA-Cot
-
10
shCot shCot-mut
-
8 6
αHA
4
αTubulin
+ + + + + -
- -
-
Cot Tubulin
2 0
shCot
-
+
-
+
shCot-mut
+
-
+
-
TNF
-
-
+
+
Fig. 3. Cot is required for efficient NF-jB transactivation. (A) HEK 293 cells were transfected with a NF-jB-dependent reporter gene along with empty vector or expression vectors for Cot and Cot inac. The next day, cells were stimulated for 12 h with TNF as shown and then analyzed for luciferase activity. Fold induction of gene expression is shown, luciferase activity of unstimulated cells was arbitrarily set as 1. Bars indicate standard deviations from three separate experiments. (B) Cells were transfected with the NF-jB reporter gene and plasmids directing the synthesis of a Cot-specific shRNA (shCot) or a point mutated control (shCot-mut). After 2 days, cells were stimulated for 8 h with TNF and analyzed for luciferase activity. (C) Cells were transfected to express low amounts of HA-tagged Cot along with increasing amounts of shCot or shCot-mut as indicated. After 2 days, cells were lyzed and extracts were analyzed for Cot expression and the occurrence of tubulin as a loading control.
slightly triggered NF-jB-mediated gene expression dependent on its kinase activity. Cot further enhanced TNF-triggered luciferase activity, thus revealing its stimulatory function on the NF-jB pathway. The relative contribution of human Cot for TNF-triggered NF-jB activation was investigated by a loss-of-function approach. Cells were cotransfected with a NF-jB-dependent reporter gene along with a vector directing the synthesis of a shRNA specific for Cot or a control vector encoding a point mutated shRNA. TNF-stimulated NF-jB activity was significantly impaired in the absence of Cot, thus revealing an important contribution of this kinase for NF-jB-dependent gene expression. Of note, TNF-induced gene expression was not fully abolished in the absence of Cot, although control experiments ensured the efficient elimination of this kinase by the shRNA (Fig. 3C). These results reveal a modulatory, but not essential function of Cot for TNF-triggered NF-jB activation. Cot-mediated p65 phosphorylation depends on NIK Can Cot phosphorylate p65 directly or does it cause the phosphorylation upon activation of a downstream kinase? To address this question, cells were transfected to express epitope-tagged Cot or IKKe as a positive control. The kinases were
purified via immunoprecipitation and then tested for their ability to phosphorylate the purified GST-p65 (354–551) protein using in vitro kinase assays. Analysis of p65 serine 536 and 468 phosphorylation by immunoblotting revealed potent phosphorylation of both sites in p65 by IKKe, but largely absent phosphorylation by Cot (Fig. 4A). These results raise the possibility that Cot-mediated p65 phosphorylation is exerted by another kinase downstream of Cot. As Cot directly binds to NIK [10] we tested the relevance of this kinase for Cot-mediated p65 modification. Cot-triggered p65 phosphorylation of serines 536 and 468 was completely blocked in the presence of kinase inactive NIK (Fig. 4B), which acts as a dominant negative protein [21]. These data are consistent with the in vitro kinase experiments and suggest that Cot requires NIK activity to exert its p65 phosphorylating function. Accordingly, expression of NIK alone also triggered p65 phosphorylation at both serines and coexpression of the two kinases together boosted phosphorylation even more (Fig. 4B). In summary, these data reveal an important contribution of Cot for NF-jB-dependent transcription that involves its ability to trigger p65 phosphorylation. Consistently, a recent study revealed that NIK and Cot cooperate to trigger transactivation by a Gal4-p65 fusion protein [22].
T. Wittwer, M.L. Schmitz / Biochemical and Biophysical Research Communications 371 (2008) 294–297
A
HA-Cot Flag-IKKε
KA & WB
-
+ -
+
α P S536
P GFP- p65
α P S468
P GFP-p65
WB
αHA
Cot
αFlag
IKKε GST-p65
αGST
B HA-Cot Flag-NIK Flag-NIK KK/AA WB:
+ + + - - + - + - +
+ -
α P S536
P GFP- p65
α P S468
P GFP-p65
αp65
p65
αFlag
NIK
αHA
Cot n.s.
Fig. 4. Cot and NIK cooperate for p65 phosphorylation. (A) HEK 293 cells were transfected with expression vectors for HA-Cot and Flag-IKKe, respectively. The kinases were immunoprecipitated from cell lysates, followed by immune complex kinase assays (KA) using recombinant and purified GST-p65-(354–551) as a substrate. Phosphorylation of p65 was determined by immunoblotting ensuring the indicated phospho-specific antibodies. The lower part shows control blots ensuring correct expression of input material. (B) Cells were transfected to express various combinations of Cot and NIK expression vectors as indicated. Extracts were analyzed for p65 phosphorylation and protein expression by immunoblotting.
Acknowledgments We thank Dres. S. Alemany (Madrid) and S.C. Ley (London) for generously providing plasmids. Our laboratory is supported by grants from the Deutsche Forschungsgemeinschaft projects SCHM 1417/4-1, SCHM 1417/5-1, SFB 547, and the ECCPS—Excellence Cluster Cardio-Pulmonary System. References [1] M.S. Hayden, S. Ghosh, Shared principles in NF-kappaB signaling, Cell 132 (2008) 344–362. [2] M.A. Calzado, S. Bacher, M.L. Schmitz, NF-kappaB inhibitors for the treatment of inflammatory diseases and cancer, Curr. Med. Chem. 14 (2007) 367–376.
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[3] N.D. Perkins, Integrating cell-signalling pathways with NF-kappaB and IKK function, Nat. Rev. Mol. Cell Biol. 8 (2007) 49–62. [4] K.J. Campbell, N.D. Perkins, Post-translational modification of RelA(p65) NFkappaB, Biochem. Soc. Trans. 32 (2004) 1087–1089. [5] M.L. Schmitz, I. Mattioli, H. Buss, M. Kracht, NF-kappaB: a multifaceted transcription factor regulated at several levels, Chembiochem 5 (2004) 1348– 1358. [6] T. Lawrence, M. Bebien, G.Y. Liu, V. Nizet, M. Karin, IKKalpha limits macrophage NF-kappaB activation and contributes to the resolution of inflammation, Nature 434 (2005) 1138–1143. [7] I. Mattioli, A. Sebald, C. Bucher, R.P. Charles, H. Nakano, T. Doi, M. Kracht, M.L. Schmitz, Transient and selective NF-kappa B p65 serine 536 phosphorylation induced by T cell costimulation is mediated by I kappa B kinase beta and controls the kinetics of p65 nuclear import, J. Immunol. 172 (2004) 6336– 6344. [8] H. Zhong, M.J. May, E. Jimi, S. Ghosh, The phosphorylation status of nuclear NFkappa B determines its association with CBP/p300 or HDAC-1, Mol. Cell 9 (2002) 625–636. [9] J. Miyoshi, T. Higashi, H. Mukai, T. Ohuchi, T. Kakunaga, Structure and transforming potential of the human cot oncogene encoding a putative protein kinase, Mol. Cell. Biol. 11 (1991) 4088–4096. [10] X. Lin, E.T. Cunningham Jr., Y. Mu, R. Geleziunas, W.C. Greene, The protooncogene Cot kinase participates in CD3/CD28 induction of NF-kappaB acting through the NF-kappaB-inducing kinase and IkappaB kinases, Immunity 10 (1999) 271–280. [11] D. Hagemann, J. Troppmair, U.R. Rapp, Cot protooncoprotein activates the dual specificity kinases MEK-1 and SEK-1 and induces differentiation of PC12 cells, Oncogene 18 (1999) 1391–1400. [12] E. Sanchez-Gongora, C. Lisbona, R. de Gregorio, A. Ballester, V. Calvo, L. PerezJurado, S. Alemany, COT kinase proto-oncogene expression in T cells: implication of the JNK/SAPK signal transduction pathway in COT promoter activation, J. Biol. Chem. 275 (2000) 31379–31386. [13] S. Das, J. Cho, I. Lambertz, M.A. Kelliher, A.G. Eliopoulos, K. Du, P.N. Tsichlis, Tpl2/cot signals activate ERK, JNK, and NF-kappaB in a cell-type and stimulusspecific manner, J. Biol. Chem. 280 (2005) 23748–23757. [14] I. Mattioli, H. Geng, A. Sebald, M. Hodel, C. Bucher, M. Kracht, M.L. Schmitz, Inducible phosphorylation of NF-kappa B p65 at serine 468 by T cell costimulation is mediated by IKK epsilon, J. Biol. Chem. 281 (2006) 6175– 6183. [15] H. Buss, A. Dorrie, M.L. Schmitz, E. Hoffmann, K. Resch, M. Kracht, Constitutive and interleukin-1-inducible phosphorylation of p65 NF-{kappa}B at serine 536 is mediated by multiple protein kinases including I{kappa}B kinase (IKK){alpha}, IKK{beta}, IKK{epsilon}, TRAF family member-associated (TANK)binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATAbinding protein-associated factor II31-mediated interleukin-8 transcription, J. Biol. Chem. 279 (2004) 55633–55643. [16] J. Bohuslav, L.F. Chen, H. Kwon, Y. Mu, W.C. Greene, p53 induces NF-kappaB activation by an IkappaB kinase-independent mechanism involving phosphorylation of p65 by ribosomal S6 kinase 1, J. Biol. Chem. 279 (2004) 26115–26125. [17] H. Sakurai, H. Chiba, H. Miyoshi, T. Sugita, W. Toriumi, IkappaB kinases phosphorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain, J. Biol. Chem. 274 (1999) 30353–30356. [18] F. Fujita, Y. Taniguchi, T. Kato, Y. Narita, A. Furuya, T. Ogawa, H. Sakurai, T. Joh, M. Itoh, M. Delhase, M. Karin, M. Nakanishi, Identification of NAP1, a regulatory subunit of IkappaB kinase-related kinases that potentiates NFkappaB signaling, Mol. Cell. Biol. 23 (2003) 7780–7793. [19] M.L. Gandara, P. Lopez, R. Hernando, J.G. Castano, S. Alemany, The COOHterminal domain of wild-type Cot regulates its stability and kinase specific activity, Mol. Cell. Biol. 23 (2003) 7377–7390. [20] M.P. Belich, A. Salmeron, L.H. Johnston, S.C. Ley, TPL-2 kinase regulates the proteolysis of the NF-kappaB-inhibitory protein NF-kappaB1 p105, Nature 397 (1999) 363–368. [21] L. Ling, Z. Cao, D.V. Goeddel, NF-kappaB-inducing kinase activates IKK-alpha by phosphorylation of Ser-176, Proc. Natl. Acad. Sci. USA 95 (1998) 3792– 3797. [22] C. Sanchez-Valdepenas, C. Punzon, B. San-Antonio, A.G. Martin, M. Fresno, Differential regulation of p65 and c-Rel NF-kappaB transactivating activity by Cot, protein kinase C zeta and NIK protein kinases in CD3/CD28 activated T cells, Cell Signal. 19 (2007) 528–537.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
NIK and Cot cooperate to trigger NF-jB p65 phosphorylation Tobias Wittwer, M. Lienhard Schmitz * Institute of Biochemistry, Medical Faculty, Friedrichstrasse 24, Justus-Liebig-University, D-35392 Giessen, Germany
a r t i c l e
i n f o
Article history: Received 11 April 2008 Available online 23 April 2008
Keywords: NF-jB Transactivation Phosphorylation Kinase Transcription
a b s t r a c t The serine/threonine kinase Cot triggers NF-jB-dependent transactivation and activation of various MAPKinases. Here we identify Cot as a novel p65 interacting protein kinase. Cot expression induces p65 phosphorylation at serines 536 and 468 in dependence from its kinase function. Accordingly, shRNA-mediated knockdown of Cot expression interferes with TNF-induced NF-jB-dependent gene expression. Also the Cterminally truncated, oncogenic form of Cot is able to trigger p65 phosphorylation. In vitro kinase assays and dominant negative mutants revealed that NIK functions downstream of Cot to mediate p65 phosphorylation. Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved.
The NF-jB family of transcription factors consists of five different DNA-binding subunits that dimerize in different combinations to mediate gene expression. The two subunits p50 and p52 are synthesized as large precursor molecules called 105 and p100, respectively. The other three subunits (p65, RelB, and c-Rel) contain transactivation domains in their C-termini [1,2]. NF-jB is activated by diverse stressful conditions including proinflammatory stimuli such as tumor necrosis factor (TNF) and lipopolysaccharide (LPS). Canonical NF-jB activation by TNF involves the induction of signaling cascades that ultimately lead to the activation of IjB kinases (IKKs) which in turn phosphorylate the inhibitory IjBa protein, thus targeting it for ubiquitin/proteasome-mediated degradation [3]. The released NF-jB dimer is then free to translocate to the nucleus where it drives target gene expression. Efficient NF-jB-mediated transcription requires posttranslational modifications of the DNA-binding subunits, which are best characterized for the p65 protein. This subunit can be modified by ubiquitination, prolyl isomerization, acetylation or phosphorylation [4,5]. More than 10 different phosphorylation sites have been discovered for the p65 protein. They serve different functions ranging from the regulation of nuclear import, protein stability and coactivator binding [5–8]. The set of kinases with NF-jB activating properties includes the MAP3K family member Cot (Cancer Osaka thyroid). This kinase, in rats also known as Tpl-2, was initially identified in a C-terminally truncated form as a result of a screen for oncogenes [9]. Overexpression of a C-terminally truncated Cot isoform in T cells was sufficient to induce lymphomas, thus confirming the oncogenic
* Corresponding author. Fax: +49 641 9947589. E-mail address: [email protected] (M.L. Schmitz).
potential of this truncated kinase. Various stimuli including LPS and TNF trigger Cot activation which in turn allows the induction of the MAP kinases p38, JNK, and ERK and also NF-jB [10–12]. Knockout experiments revealed that Cot signals activate ERK, JNK, and NF-jB in a cell-type and stimulus-specific manner. Cot / mouse embryonic fibroblasts (MEFs) showed intact TNF-triggered IKK activation, while NF-jB-dependent transcription was impaired [13]. These results raise the possibility that the Cot-mediated effects are rather due to the regulation of NF-jB-dependent transcriptional activity.
Materials and methods Cell culture, expression vectors, and transient transfections. HEK 293 cells were cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, and 1% (v/v) penicillin/streptomycin. Cells were transfected using Rotifect (Roth) according to the manufacturer’s instructions. HA-Cot, HA-Cot inac., HA-CotD, Myc-Cot, Myc Cot KM [10,12], GFP-p65, and (jB)3-luc [14] were described. pSUPER-Cot and the point mutated control were cloned by standard methods. The following DNA sequence was used to allow the production of shRNA targeting Cot: GTGAAGAGCCAGCAGTTTA. The control sequence was mutated in three positions which are shown in bold: GTCAAGAGGCAGCAGTATA. Cell lysis and Western blotting. Cells were washed with ice-cold phosphate-buffered saline and collected by centrifugation. The pellet was resuspendet in NP-40 lysis buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% (v/v) NP-40, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 40 mM NaF, 2 mM sodium vanadate, leupeptin (10 lg/ml), and aprotinin (10 lg/ml)) and incubated on ice for 20 min. Cell debris was removed by centrifugation with 13,000 rpm at 4 °C. Proteins were separated by reducing SDS–PAGE and blotted onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). After blocking, membranes were incubated with appropriate primary and horseradish peroxidase-coupled secondary antibodies, followed by protein detection using the Perkin-Elmer Western lightning chemiluminescence system. Antibodies recognizing NF-jB p65 phospho-Ser 468
0006-291X/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.069
295
T. Wittwer, M.L. Schmitz / Biochemical and Biophysical Research Communications 371 (2008) 294–297 (3039) and phospho-Ser 536 (3031) were obtained from Cell Signaling Technology, NF-jB p65 (C-20), GST (Z-5), and Cot (M-20) antibodies were obtained from Santa Cruz. Antibodies recognizing Myc (9E10), GFP (clones 7.1 and 13.1), and HA (3F10) epitopes were from Roche. Purification of GST-p65 and in vitro kinase assays. The GST-p65 (354–551) fusion protein was expressed in Escherichia coli BL21 cells and purified by affinity chromatography on glutathione-coupled sepharose according to standard protocols. HA-Cot and Flag-IKKe were expressed in HEK 293 cells and purified by immunoprecipitation using aHA and aFlag antibodies, respectively. Precipitates were washed three times in NP-40 lysis buffer and two times in kinase buffer (20 mM Hepes/KOH, pH 7.4, 25 mM b-glycerophosphate, 2 mM dithiothreitol, and 20 mM MgCl2). The immune complex kinase assay was performed in a final volume of 20 ll kinase buffer containing 40 lM ATP and 1 lg of the purified GST-p65 substrate protein for 30 min at 30 °C. The reaction was terminated by the addition of SDS sample buffer and p65 phosphorylation was detected by immunoblotting. Coimmunoprecipitation. HEK 293 cells were transiently transfected with plasmids encoding GFP-p65 and Myc-Cot. The next day, cells were lyzed in NP-40 buffer and aliquots were analyzed for protein expression. The remaining lysates were incubated with aGFP or control antibodies and 30 ll protein A/G sepharose for 4 h on a spinning wheel at 4 °C. The immunoprecipitates were washed five times in NP-40 lysis buffer. Bound proteins were eluted by boiling in SDS sample buffer and detected by immunoblotting. Luciferase assays. Cells were seeded in 6-well plates, transfected and treated as specified. Cells were lyzed in reporter lysis buffer (25 mM Tris–Phosphate, 2 mM DTT, 2 mM CDTA, 10% (v/v) glycerol, and 1% (v/v) Triton X-100). Luciferase activity was determined in a luminometer (Duo Lumat LB 9507, Berthold) by adding 50 ll of assay buffer (40 mM Tricine, 2.14 mM (MgCO3)4Mg(OH)25H2O, 5.34 mM MgSO4, 0.2 mM EDTA, 66.6 mM DTT, 540 lM CoA, 940 lM luciferin, and 1.06 mM ATP) and measuring light emission for 10 s.
A
WB:
P GFP- p65
α P S468
P GFP-p65
p65 n.s. Cot n.s.
αCot
B -
GFP-p65 HA-Cot HA-CotΔ WB:
+ + + - + - - +
α P S536
P GFP- p65
α P S468
P GFP-p65
Cot binds to NF-jB p65 and triggers its phosphorylation
αHA
To test the impact of Cot on NF-jB-mediated gene expression, cells were cotransfected with a NF-jB-dependent luciferase gene
GFP-p65
αp65
αp65
Cot contributes to TNF-triggered expression of NF-jB target genes
+ + + - + - - +
α P S536
Results and discussion
Cot-deficient MEFs show impaired NF-jB-dependent gene expression but intact IKK activation and IjB phosphorylation [13]. To test a potential impact of Cot on p65 phosphorylation, cells were transfected to express active or inactive Cot along with low amounts of the GFP-tagged p65. Subsequent Western blotting with phospho-specific antibodies revealed that Cot expression triggered the phosphorylation of p65 at serine 536 and 468 dependent on its kinase function (Fig. 1A). This adds further evidence that these p65 phosphorylation sites can be modified by several kinases. The C-terminal serine 536 can be phosphorylated by multiple kinases, including IKKb, IKKe, TBK1, and RSK1 which are activated via distinct signaling pathways [15–18]. But not only the kinases, also the functions of serine 536 phosphorylation are quite diverse and range from enhanced transactivation to enhanced kinetics of p65 nuclear import and regulation of protein stability [6,7]. The oncogenic potential of Cot requires deletion of its C terminus, which is frequently associated with an increased kinase activity [19]. We thus compared the p65 phosphorylating activity between wildtype Cot and its C-terminally truncated version (CotD). These experiments revealed similar p65 phosphorylating activities of Cot and CotD (Fig. 1B), showing that the C-terminal part of the kinase is not affecting its ability to mediate p65 modification. We then explored the possible interaction between Cot and its substrate protein by coimmunoprecipitation experiments. Cells were transfected to express Myc-tagged Cot and/or GFP-p65, followed by precipitation of GFP-p65 and Western blotting. A fraction of Cot was detected in GFP-p65 immunoprecipitates (Fig. 2), showing the ability of the kinase to bind its substrate protein. However, the vast majority of Cot is stoichiometrically associated with p105 [20], so that only a fraction of the kinase will be able to bind the p65 protein.
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GFP-p65 HA-Cot HA-Cot inac
GFP-p65 p65 Cot, CotΔ n.s.
Fig. 1. Cot induces p65 phosphorylation. (A). HEK 293 cells were transiently transfected with plasmids encoding GFP-p65 and HA-tagged forms of wildtype (HA-Cot) or inactive (HA-Cot inac.) Cot as shown. The next day, cells were lyzed and equal amounts of protein contained in cell lysates were analyzed by Western blotting (WB). Phosphorylation of p65 at serines 536 and 468 was revealed by phosphospecific antibodies, correct expression of transfected proteins was ensured with ap65 and aCot antibodies. A nonspecific (n.s.) band is indicated. (B). The experiment was done as in (A) with the exception that a C-terminally truncated version of Cot (CotD) was used.
and expression vectors encoding wildtype or inactive Cot proteins. The next day, cells were stimulated with TNF as shown and analyzed for luciferase activity (Fig. 3A). Expression of Cot
IP: GFP-p65 Myc-Cot
WB:
αIgG
+ -
+ -
+
+ +
αMyc
Cot IgG heavy chain
αGFP
GFP-p65
Input: GFP-p65 Myc-Cot
WB:
- + + +
αGFP
+ -
- + + +
αMyc
Cot
αGFP
GFP-p65
Fig. 2. Interaction of Cot and NF-jB p65. HEK 293 cells were transfected with plasmids encoding GFP-p65 and Myc-Cot as shown. The next day, cells were lyzed and extracts were either tested for correct expression of the tagged proteins (lower) or immunoprecipitated with aGFP or control antibodies. The eluted proteins were revealed by immunoblotting, the position of the respective proteins and the IgG heavy chain is indicated.
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T. Wittwer, M.L. Schmitz / Biochemical and Biophysical Research Communications 371 (2008) 294–297
(κB)3-luciferase
A
30
20
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activation
40
10
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HA-Cot
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HA-Cot inac
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TNF
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(κB)3-luciferase
-fold activation
14 12
HA-Cot
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shCot shCot-mut
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αHA
4
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shCot
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shCot-mut
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TNF
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Fig. 3. Cot is required for efficient NF-jB transactivation. (A) HEK 293 cells were transfected with a NF-jB-dependent reporter gene along with empty vector or expression vectors for Cot and Cot inac. The next day, cells were stimulated for 12 h with TNF as shown and then analyzed for luciferase activity. Fold induction of gene expression is shown, luciferase activity of unstimulated cells was arbitrarily set as 1. Bars indicate standard deviations from three separate experiments. (B) Cells were transfected with the NF-jB reporter gene and plasmids directing the synthesis of a Cot-specific shRNA (shCot) or a point mutated control (shCot-mut). After 2 days, cells were stimulated for 8 h with TNF and analyzed for luciferase activity. (C) Cells were transfected to express low amounts of HA-tagged Cot along with increasing amounts of shCot or shCot-mut as indicated. After 2 days, cells were lyzed and extracts were analyzed for Cot expression and the occurrence of tubulin as a loading control.
slightly triggered NF-jB-mediated gene expression dependent on its kinase activity. Cot further enhanced TNF-triggered luciferase activity, thus revealing its stimulatory function on the NF-jB pathway. The relative contribution of human Cot for TNF-triggered NF-jB activation was investigated by a loss-of-function approach. Cells were cotransfected with a NF-jB-dependent reporter gene along with a vector directing the synthesis of a shRNA specific for Cot or a control vector encoding a point mutated shRNA. TNF-stimulated NF-jB activity was significantly impaired in the absence of Cot, thus revealing an important contribution of this kinase for NF-jB-dependent gene expression. Of note, TNF-induced gene expression was not fully abolished in the absence of Cot, although control experiments ensured the efficient elimination of this kinase by the shRNA (Fig. 3C). These results reveal a modulatory, but not essential function of Cot for TNF-triggered NF-jB activation. Cot-mediated p65 phosphorylation depends on NIK Can Cot phosphorylate p65 directly or does it cause the phosphorylation upon activation of a downstream kinase? To address this question, cells were transfected to express epitope-tagged Cot or IKKe as a positive control. The kinases were
purified via immunoprecipitation and then tested for their ability to phosphorylate the purified GST-p65 (354–551) protein using in vitro kinase assays. Analysis of p65 serine 536 and 468 phosphorylation by immunoblotting revealed potent phosphorylation of both sites in p65 by IKKe, but largely absent phosphorylation by Cot (Fig. 4A). These results raise the possibility that Cot-mediated p65 phosphorylation is exerted by another kinase downstream of Cot. As Cot directly binds to NIK [10] we tested the relevance of this kinase for Cot-mediated p65 modification. Cot-triggered p65 phosphorylation of serines 536 and 468 was completely blocked in the presence of kinase inactive NIK (Fig. 4B), which acts as a dominant negative protein [21]. These data are consistent with the in vitro kinase experiments and suggest that Cot requires NIK activity to exert its p65 phosphorylating function. Accordingly, expression of NIK alone also triggered p65 phosphorylation at both serines and coexpression of the two kinases together boosted phosphorylation even more (Fig. 4B). In summary, these data reveal an important contribution of Cot for NF-jB-dependent transcription that involves its ability to trigger p65 phosphorylation. Consistently, a recent study revealed that NIK and Cot cooperate to trigger transactivation by a Gal4-p65 fusion protein [22].
T. Wittwer, M.L. Schmitz / Biochemical and Biophysical Research Communications 371 (2008) 294–297
A
HA-Cot Flag-IKKε
KA & WB
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+ + + - - + - + - +
+ -
α P S536
P GFP- p65
α P S468
P GFP-p65
αp65
p65
αFlag
NIK
αHA
Cot n.s.
Fig. 4. Cot and NIK cooperate for p65 phosphorylation. (A) HEK 293 cells were transfected with expression vectors for HA-Cot and Flag-IKKe, respectively. The kinases were immunoprecipitated from cell lysates, followed by immune complex kinase assays (KA) using recombinant and purified GST-p65-(354–551) as a substrate. Phosphorylation of p65 was determined by immunoblotting ensuring the indicated phospho-specific antibodies. The lower part shows control blots ensuring correct expression of input material. (B) Cells were transfected to express various combinations of Cot and NIK expression vectors as indicated. Extracts were analyzed for p65 phosphorylation and protein expression by immunoblotting.
Acknowledgments We thank Dres. S. Alemany (Madrid) and S.C. Ley (London) for generously providing plasmids. Our laboratory is supported by grants from the Deutsche Forschungsgemeinschaft projects SCHM 1417/4-1, SCHM 1417/5-1, SFB 547, and the ECCPS—Excellence Cluster Cardio-Pulmonary System. References [1] M.S. Hayden, S. Ghosh, Shared principles in NF-kappaB signaling, Cell 132 (2008) 344–362. [2] M.A. Calzado, S. Bacher, M.L. Schmitz, NF-kappaB inhibitors for the treatment of inflammatory diseases and cancer, Curr. Med. Chem. 14 (2007) 367–376.
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