- Email: [email protected]
Inhibition of NF-κB activation by a dominant-negative mutant of IκBα
Molecular Immunology. Vol. 33, No. 1. pp. 57-61, 1996 Copyright cl 1996Elsevier Science Ltd. All rights reserved Printed in Great Britain 0161-5890/96$15.00+0.00
Pergamon 0161-5890(95)00128-X
INHIBITION OF NF-KB ACTIVATION BY A DOMINANTNEGATIVE MUTANT OF kBc( CHAO-GUANG CHEN,* JIM MALLIAROS, MARINA KATERELOS, ANTHONY J. F. D’APICE and MARTIN J. PEARSE Immunology
Research Centre. St Vincent’s Hospital, Melbourne, Victoria, Australia
(First received 10 Ju1.v 1995; uccepted in reufsed,form 25 August 1995)
Abstract-The
activity of the transcription factor NF-KB is tightly regulated by the inhibitory molecule IKBK Upon stimulation, IlcBa is rapidly degraded and NF-KB translocates to the nucleus to induce gene expression. The IKBU degradation is preceded by phosphorylation, suggesting that this event plays a role in the activation of NF-KB. In this study, we have mutated three potential phosphorylation sites in porcine IKBU and found that expression of the Ser32mutant of IKBE (IS7’A), but not Tyr42 or Ser262mutants or wild-type IKBK blocked the activation of NF-KB by TNF-a. These results suggest that the Ser3’ residue, a potential casein kinase II phosphorylation site, is critical for NF-KB activation. Key words: NF-KB, IKBCZ,NF-KB activation. phosphorylation
al., 1993; Cordle et al.. 1993; Mellits et al., 1993). Treatment of cells with protease inhibitors which prevent IKBa degradation inhibits NF-KB activation. indicating a critical role for proteolytic degradation of IlcBa in NF-KB activation; however, the role of IlcBa phosphorylation is less clearly defined (Finco et al., 1994; Miyamoto et al., 1994; Palombella et al., 1994; Traenckner et al.. 1994; Alkalay et al., 1995; Didonato et al., 1995; Lin et al., 1995). In this study, we have identified a potential casein kinase II phosphorylation site in porcine IKBa, the mutation of which resulted in the formation of an ItiBc( protein that functioned as a dominant negative mutant.
INTRODUCTION The transcription factor NF-rcB was first identified as a nuclear protein bound to the immunoglobulin lc lightchain enhancer in B cells (Sen and Baltimore, 1986). It has subsequently become clear that NF-lcB is a pleiotropic factor involved in the regulation of a large number of genes during development and in the response to infection or stress in a variety of cell types (for reviews see Lenardo and Baltimore, 1989; Grilli et al., 1993). The NF-KB/Rel family of proteins includes c-Rel, p65 (RelA), RelB, p5O/p105, p49/plOO, ~551~98 and the Drosophila morphogen dorsal, and exist as homo- or heterodimers, with p50/65 heterodimers the most commonly found (Liou and Baltimore, 1993; Gerondakis and Grumont, 1994). These proteins share a conserved N-terminal region of approximately 300 amino acids, referred to as the Rel homology domain (RHD), which contains sequences important for DNA binding, protein dimerization, nuclear localization and association with inhibitor proteins of the IrcB family. The IKB family of proteins includes ItiBsr, IKB/?, IKB~ (C-terminal region of the ~98, ~100 and ~105 precursors), Bcl-3 and the Drosophila gene cactus (Liou and Baltimore, 1993; Gerondakis and Grumont, 1994). In quiescent cells, NF-KB is retained in the cytoplasm in an inactive form by IKB (with the exception of Bcl-3, which is a nuclear protein), which masks the NF-k-B nuclear localization signal (NLS) (Beg et al.. 1992). Upon activation with a variety of agents NF-KB is translocated to the nucleus and induces gene expression. The NF-liB activation is preceded by the phosphorylation and rapid degradation of bcBc( (Beg et *Author to whom correspondence
site.
MATERIALS AND METHODS Cell lines
Porcine aortic endothelial cells (EC) were obtained as described previously (Booyse et al., 1977). The COS7 (African green monkey fibroblast) and NIH3T3 (mouse fibroblast) cell lines were obtained from American Type Culture Collection. Preparation of NF-KB responsive luciferuse reporter gene
A double-stranded oligonucleotide containing three NF-KB consensus binding sites was subcloned upstream of the tk minimal promoter in pBLCAT2 (Luckow and Schiitz, 1987) to create an NF-KB responsive promoter, which was then subcloned upstream of the luciferase coding sequence in pGL2-basic (Promega, Madison, U.S.A.). This expression cassette was finally subcloned into the BglII site of the pcDNA3 vector (Invitrogen. California, U.S.A.) to generate pcDNA3.Luc.
should be addressed. 57
58
C.-G. CHEN et al.
Cloning and site-directed
mutagenesis
The cDNA encoding mouse ~65 was amplified by RTPCR from RNA isolated from murine pre-B cell line 18.81 using primers based on the published sequence (Nolan et al., 1991) and subcloned into pcDNA3 to generate pcDNA3/p65. Porcine IKBCZcDNA was also cloned by RT-PCR from RNA isolated from aortic endothelial cells using primers based on the published sequence (de Martin et al., 1993). The cDNA was extended at the 5’ end to include a sequence encoding the FlagTM (Kodak, New York, U.S.A.) tag. The cDNA was then subcloned into pAlter II plasmid (Promega) for site-directed mutagenesis. The Ser32 residue was changed to Ala, Tyr42 to Phe, and Ser26” to Gly to create IKB~ mutants IS32A,IY42Fand IS262Grespectively. The wild-type and mutant cDNAs were subcloned into the polylinker of pCDNA3.Luc downstream of the CMV promoter to create ~cDNA~.Luc/I~~~*, ~cDNA~.Luc/I~~~~ and ~cDNA~.Luc/I~~~~~. Transfection
and assay for luciferase activity
The NIH3T3 or COS cells were transfected with 5 pg/ml of plasmid DNA using the DEAE-dextran method (Kingston, 1988) and allowed to recover for 24 hr before stimulation with 10 ng/ml of TNF-a for 16 hr. Cell lysates were prepared, normalized for protein content and assayed for luciferase activity using Promega’s luciferase assay system. The luminescence level was determined on a TopCount microplate scintillation counter (Packard, Groningen, The Netherlands). Immunoprecipitation
IKBa:
-
LS32A
IW
I Y42F
1S262G
B
and Western blotting
Cells were washed and lysed in buffer containing 150 mM NaCl, 10 mM sodium phosphate pH7.4, 1 mM CaC12,0.5% Triton Xl00 and protease inhibitors: 1 mM PMSF (Calbiochem, California, U.S.A.), 10 pug/ml leupeptin (Sigma) and 10 pg/ml aprotinin (Sigma). FlagTMtagged IrcBcr was isolated from cytosolic extracts by incubation with 50 ~1 of agarose beads coupled to murine monoclonal anti-FlagTM antibody (Kodak, New York, U.S.A.). The ~65 protein was isolated with rabbit polyclonal anti-p65 antibody (Santa Cruz Biotechnology, Inc., California, U.S.A.) conjugated to protein G sepharose (Pharmacia, Uppsala, Sweden). Samples were fractionated by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred to nitrocellulose membranes (Amersham, Buckinghamshire, U.K.). Blots were probed with either anti-p65 antibody or monoclonal anti-FlagTM antibody. Immunoreactive polypeptides were detected using either rabbit anti-mouse Ig (Dako, Glosmup, Denmark) (1:2000) or protein A (Amersham) (1:5000) conjugated to horseradish peroxidase (HRP) using an enhanced chemiluminescence system (Amersham). Results Inhibition
fibroblast cell line NIH3T3. Forty-eight hours after transfection, TNF-cr stimulated and control non-stimulated cells were lysed and luciferase activity determined. Stimulation of cells with TNF-a resulted in a 4.1 -fold increase in luciferase activity, indicating that the promoter is responsive to NF-KB activation [Fig. l(A), column 1). To examine the effect of expressing wild-type Ik-Ba (IWT) on NF-KB activity, NIH3T3 cells were transfected with pcDNA3.Luc/lWT, a dual construct in which the luciferase reporter construct and ItiBa (under the control of the
of NF-KB activation
by IKBc~ Ser3’ mutant
The NF-k-B-dependent luciferase reporter construct in pcDNA3.Luc was tested by transfection of the murine
1 ~65: IKBa:
+ -
2
3
4
+
+ I S32A
-
IWT
Fig. 1. Effect of expressing IS3’Aon NF-h-B mediated gene expression. (A) Cells were transfected with 5 pg/ml of pcDNA3.Luc (-) or the IxBa constructs in pcDNA3.Luc plasmid as indicated, incubated for 24 hr and cultured overnight in the presence or absence of TNF-a (10 ng/ml). Results are expressed as fold induction of luciferase activity in TNF-a stimulated vs non-stimulated cells. (B) Cells were transfected with pcDNA3/p65 and pcDNA3.Luc (1) or pcDNA3/p65 in combination with pcDNA3.Luc/IWT (2) or pcDNA3.Luc/IS3’* (3). Cells transfected with pcDNA3.Luc alone (4) were included as a control. Luminescence expressed as counts per second (CPS). Experiments were performed in duplicate and expressed as means
(k SD). The results presented are representative three separate experiments.
of
IKBU mutagenesis analysis
59
CMV promoter) are physically linked. This avoids the not cell lysates is probably protein G. In a separate experipossibility of uneven uptake of separate constructs and ment, the anti-FlagTM antibody immunoprecipitated a 65 kDa protein (the expected size of p65), which was reactive ensures that all the luciferase activity detected is attributable to cells that have also been transfected with IKBU to anti-p65 antibody, from cells transfected with IWTand IS32AcDNAs but not from control cells [Fig. 2(B)]. The (wild-type or mutant). Following stimulation with TNFvariability in the intensity of the 65 kDa band in the ~1,luciferase activity increased by approximately 3.5fold different cell lysates is due to uneven protein loading. The [Fig. l(A), column 21. A comparison of the amino acid sequences of IKBCX 50 kDa band detected in all lanes containing immunofrom various species (de Martin et al., 1993) revealed the precipitated material but not cell lysates is most probably existence of three highly conserved potential phos- immunoglobulin heavy chain. phorylation sites, including a casein kinase II site (Ser32), a tyrosine phosphorylation site (Tyr4’) and a protein kinDISCUSSION ase C phosphorylation site (Se?62). To determine if any The transcription factor NF-KB plays a pivotal role in of these sites are involved in a phosphorylation-dependent NF-KB signalling pathway, mutant forms of IKBU the regulation of genes involved in a variety of immune and inflammatory responses (Lenardo and Baltimore, in which each of these sites was mutated were generated 1989; Grilli et al., 1993). The NF-KB activity is modulated by site-directed mutagenesis. The mutant cDNAs were subcloned into the polylinker of pcDNA3.Luc and used by IKBIXwhich binds to and retains NF-k-B in the cytoto transfect NIH3T3 cells. Cells expressing the Tyr42 plasm of quiescent cells. The 1~Bcris phosphorylated and (Iy42F) and Ser262(IS262G)mutants of IKBE responded to subsequently degraded following stimulation of cells with a variety of agents including TNF-sr (Beg et al., 1993; TNF-a stimulation in the same manner as cells transCordle et al., 1993; Mellits et al., 1993). Recent in vivo fected with IWTcDNA, resulting in a 2.5- and 2.7-fold induction of luciferase activity respectively [Fig. 1(A), studies have shown that inhibition of IJcB~ degradation columns 4 and 51. In contrast, little increase (0. l-fold) in by specific protease inhibitors prevents NF-k-B activation luciferase activity was observed following TNF-a stimu- without affecting 1~Ba phosphorylation (Finco et al., lation of cells transfected with the Ser32 mutant (IS”*) 1994; Miyamoto et al., 1994; Palombella et al., 1994; Traenckner et al., 1994; Alkalay et al., 1995; Didonato et cDNA [Fig. l(A), column 31. al., 1995; Lin et al., 1995) demonstrating that degradation of IKBU is essential for NF-KB nuclear transFunctional andph_vsical interaction of IS3’* andp65 in vivo location and that phosphorylation of 1~Bc( alone does The ability of 1~Ba to regulate the transcriptional not cause the dissociation of the NF-KB/IKBCXcomplex. activity of NF-KB is dependent on the physical associ- The question therefore remains as to the role of 1~Ba ation of the two molecules. The NIH3T3 cells co-transphosphorylation in the activation of NF-k-B. To directly fected with pcDNA3/p65 and pcDNA3. Luc gave examine this issue, porcine IKBZ mutants lacking one approximately 1OO-fold higher luciferase activity over the of the three potential phosphorylation sites, which are control cells transfected with the reporter plasmid conserved among IlcBa from different species, were used (pcDNA3.Luc) alone [Fig. l(B)]. Activation of luciferase to determine their effect on the activation of NF-KB. The gene expression by ~65 was completely inhibited by co- TNF-rx stimulation of control (pcDNA3.Luc transfected) transfection with either wild-type IKBUor the IS3**mutant cells resulted in a 4.1 -fold induction of endogenous NFcDNA [Fig. l(B)], suggesting a functional interaction KB activity, as determined using a KB-driven luciferase between the porcine wild-type IKBUand IS32A mutant with reporter construct. Transfection with either wild-type or the murine ~65 subunit of NF-KB. Tyr42 (Iy42”) and SerZ6*(IS26’G)mutants of IrcBa cDNA The TNF-a stimulation of cells co-transfected with caused only a slight reduction in the level of induction, ~65 and wild-type IKBE increased the level of luciferase to 3.5-, 2.5- and 2.7-fold, respectively. In contrast, NFactivity by approximately 3-fold, whereas there was no KB activation was completely inhibited by transfection increase in cells co-transfected with IS32A(data not with the Ser3’ mutant (IS3’*) cDNA, suggesting that this shown). potential phosphorylation site is critical for the activation To demonstrate the physical interaction of ~65 with of NF-KB. The lOO-fold increase in luciferase activity wild-type or mutant IKBu, cytoplasmic extracts from cells which occurred when cells were transfected with ~65 transfected with IWTor IS32A cDNAs were fractionated by cDNA was completely abolished by co-transfection with SDS-PAGE either directly or following immunoeither IWTor IS3”*cDNA. Furthermore, I’“” protein was precipitation with anti-p65 antibody. Proteins were blotco-precipitated with ~65 by antibodies specific either for ted onto a nitrocellulose membrane and probed with anti- ~65 or the FlagTM sequence used to tag the N-terminus FlagTM antibody. A 40 kDa protein band corresponding of1 S32A. Together these data indicate that the IS3’Amutant to the predicted molecular weight of porcine 1~BOZwas form of porcine 1~Ba retains the capacity to bind to and detected in both cell lysates and anti-p65 im- inactivate NF-k-B, but fails to respond to stimulation by munoprecipitated material from cells transfected with IWT TNF-cr. or IS32AcDNAs, but absent from control cells transfected The results presented in this study are consistent with with pcDNA3.Luc [Fig. 2(A)]. The 30 kDa band present those recently published by Brown et al. (1995). which in all lanes containing immunoprecipitated material but demonstrated that mutations at either Ser”’ or Ser3h of
60
C.-G. CHEN et nl.
A.
B.
Anti-Flag antibody
Anti-p65 (IP)
Cell lysates
Anti-p65 antibody
Anti-Flag (IP)
MW (kDa)
Cell lysates
i(*
p65-
IKBCI *i
-
97
..w -
65
-
45
-
30
Fig. 2. Physical association of IS32Amutant with endogenous NF-KB ~65. The COS cells were transfected with pcDNA3.Luc (control), pcDNA3.Luc/IWT (IWT) or pcDNA3.Luc/IS3** (IS3**).The immunoprecipitated (IP) proteins and cell lysates were fractionated by 7.5% SDS-PAGE and blotted. The IlcBa and ~65 proteins bands and molecular weight markers are indicated. (A) Proteins were immunoprecipitated with rabbit anti-p65 antibody conjugated to protein G sepharose and the blot probed with monoclonal anti-FlagTM antibody followed by peroxidase labelled rabbit anti-mouse Ig. (B) Proteins were immunoprecipitated with murine monoclonal anti-FlagTM antibody coupled to agarose beads and the blot probed with anti-p65 antibody followed by protein A conjugated to horseradish peroxidase.
human kBa prevent its phosphorylation and degradation following stimulation with PMA-ionomycin. These mutants, when co-expressed with ~65, prevented p65 from inducing the expression of an NF-rcB responsive reporter gene in NTera-2 cells, which lack endogenous NF-lcB and IKBE proteins. In the present study, however, we have used cells which express endogenous IKBE to demonstrate that NF-KB activation can be inhibited by expression of phosphorylation defective IKB~ in normal cells. Given the appropriate regulatory mechanisms, this dominant-negative mutant (IS32A)of IKB~ could be used to regulate NF-KB activity in uivo. We are currently test-
ing this using transgenic mice. Acknowledgements-We thank Drs D. Power and P. Cowan for critical review of this manuscript. REFERENCES Alkalay I., Yaron A., Hatzubai A., Jung S., Avraham A., Grelitz O., Pashut-Lavon I. and Ben-Neriah Y. (1995) In vivo stimulation of IrcB phosphorylation is not sufficient to activate NF-KB. Mol. Cell. Biol. 15, 1294-1301. Beg A. A., Finco T. S., Nantermet P. V. and Baldwin A. S. (1993) Tumor necrosis factor and interleukin- 1 lead to phosphorylation and loss of IlcBa: a mechanism for NF-rcB activation. Mol. Cell. Biol. 13, 3301-3310.
Beg A. A., Ruben S. M., Scheinman R. I., Haskill S., Rosen C. A. and Baldwin A. S. (1992) IJCBinteracts with the nuclear localization sequences of the subunits of NF-rcB: a mechanism for cytoplasmic retention. Gene Dev. 6, 1899-l 9 13. Booyse F. M., Quarfoot A. J., Bell S., Fass D. N., Lewis J. C., Mann K. G. and Bowie E. J. (1977) Cultured aortic endothelial cells from pigs with von Willebrand disease. Proc. natn. Acad. Sci. USA 74, 5702-5706. Brown K., Gerstberger S., Carlson L., Franzoso G. and Siebenlist U. (1995) Control of Ih-Ba proteolysis by site-specific signal-induced phosphorylation. Science 267, 1485-1488. Cordle S. R., Donald R., Read M. A. and Hawiger J. (1993) Lipopolysaccharide induces phosphorylation of MAD-3 and activation of c-Rel and related NF-rcB proteins in human monocytic THP- 1 cells. J. Biol. Chem. 268, 11803-l 1810. de Martin R., Vanhove B., Cheng Q., Hofer E., Csizmadia V., Winkler H. and Bach F. H. (1993) Cytokine-inducible expression in endothelial cells of an IkBa-like gene is regulated by NF-KB. EMBO J. 12,2773-2779. Didonato J. A., Mercurio F. and Karin M. (1995) Phosphorylation of IKBCXprecedes but is not sufficient for its dissociation from NF-KB. Mol. Cell. Biol. 15, 1302-l 3 11. Finco T. S., Beg A. A. and Baldwin A. S. (1994) Inducible phosphorylation of 1~Bolis not sufficient for its dissociation from NF-rcB and is inhibited by protease inhibitors. Proc. natn. Acad. Sci. USA 91, 11884-l 1888. Gerondakis S. and Grumont R. (1994) NF-KB: a bridge between cytoplasm and nucleus. Todav’s Life Sci. 6, 2428.
IKBCXmutagenesis
Grilli M., Chiu J.-S. and Lenardo M. (1993) NF-KB and Rel: participants in a multiform transcriptional regulatory system. ht. Rev. Cytol. 143, l-62. Kingston R. E. (1988) Current Protocols in Molecular Biology. Greene, New York. Lenardo M. J. and Baltimore D. (1989) NF-KB: a pleiotropic mediator of inducible and tissue-specific gene control. Cell 58,2277229. Lin Y.-C., Brown K. and Siebenlist U. (1995) Activation of NF-KB requires proteolysis of the inhibitor IrcBc(: signalinduced phosphorylation of IrcBc( alone does not release active NF-k-B. Proc. natn. Acad. Sci. USA 92, 552-556. Liou H.C. and Baltimore D. (1993) Regulation of the NFicB/Rel transcription factor and IKB inhibitor system. Current Opin. Cell Biol. 5,477487.
Luckow B. and Schtitz G. (1987) CAT constructs with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 15, 5400.
Mellits K. H., Hay R. T. and Goodbourn S. (1993) Proteolytic degradation of MAD-3 (Ih-Bc()and enhanced processing of
analysis
61
the NF-KB precursor ~105 are obligatory steps in the activation of NF-KB. Nucleic Acids Res. 21, 5059-5066. Miyamoto S., Maki M., Schmitt M. J., Hatanaka M. and Verma I. M. (1994) Tumor necrosis factor a-induced phosphorylation of IKB~ is a signal for its degradation but not dissociation from NF-KB. Proc. mtn. Acad. Sci. USA 91, 1274&12744.
Nolan G. P., Ghosh S., Liou H.-C., Tempst P. and Baltimore D. (1991) DNA binding and ItiB inhibition of the cloned ~65 subunit of NF-KB, a rel-related polypeptide. Cell 64,961-969. Palombella V. J., Rando 0. J., Goldberg A. L. and Maniatis T. (1994) The ubiquitin-proteasome pathway is required for processing the NF-KB~ precursor protein and the activation of NF-ICB. CeN 78, 7733785. Sen R. and Baltimore D. (1986) Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46, 705-716. Traenckner E. B.-M., Wilk S. and Baeuerle P. A. (1994) A proteasome inhibitor prevents activation of NF-KB and stabilizes a newly phosphorylated form of IKBCXthat is still bound to NF-KB. EMBO J. 13, 5433-5441.
Pergamon 0161-5890(95)00128-X
INHIBITION OF NF-KB ACTIVATION BY A DOMINANTNEGATIVE MUTANT OF kBc( CHAO-GUANG CHEN,* JIM MALLIAROS, MARINA KATERELOS, ANTHONY J. F. D’APICE and MARTIN J. PEARSE Immunology
Research Centre. St Vincent’s Hospital, Melbourne, Victoria, Australia
(First received 10 Ju1.v 1995; uccepted in reufsed,form 25 August 1995)
Abstract-The
activity of the transcription factor NF-KB is tightly regulated by the inhibitory molecule IKBK Upon stimulation, IlcBa is rapidly degraded and NF-KB translocates to the nucleus to induce gene expression. The IKBU degradation is preceded by phosphorylation, suggesting that this event plays a role in the activation of NF-KB. In this study, we have mutated three potential phosphorylation sites in porcine IKBU and found that expression of the Ser32mutant of IKBE (IS7’A), but not Tyr42 or Ser262mutants or wild-type IKBK blocked the activation of NF-KB by TNF-a. These results suggest that the Ser3’ residue, a potential casein kinase II phosphorylation site, is critical for NF-KB activation. Key words: NF-KB, IKBCZ,NF-KB activation. phosphorylation
al., 1993; Cordle et al.. 1993; Mellits et al., 1993). Treatment of cells with protease inhibitors which prevent IKBa degradation inhibits NF-KB activation. indicating a critical role for proteolytic degradation of IlcBa in NF-KB activation; however, the role of IlcBa phosphorylation is less clearly defined (Finco et al., 1994; Miyamoto et al., 1994; Palombella et al., 1994; Traenckner et al.. 1994; Alkalay et al., 1995; Didonato et al., 1995; Lin et al., 1995). In this study, we have identified a potential casein kinase II phosphorylation site in porcine IKBa, the mutation of which resulted in the formation of an ItiBc( protein that functioned as a dominant negative mutant.
INTRODUCTION The transcription factor NF-rcB was first identified as a nuclear protein bound to the immunoglobulin lc lightchain enhancer in B cells (Sen and Baltimore, 1986). It has subsequently become clear that NF-lcB is a pleiotropic factor involved in the regulation of a large number of genes during development and in the response to infection or stress in a variety of cell types (for reviews see Lenardo and Baltimore, 1989; Grilli et al., 1993). The NF-KB/Rel family of proteins includes c-Rel, p65 (RelA), RelB, p5O/p105, p49/plOO, ~551~98 and the Drosophila morphogen dorsal, and exist as homo- or heterodimers, with p50/65 heterodimers the most commonly found (Liou and Baltimore, 1993; Gerondakis and Grumont, 1994). These proteins share a conserved N-terminal region of approximately 300 amino acids, referred to as the Rel homology domain (RHD), which contains sequences important for DNA binding, protein dimerization, nuclear localization and association with inhibitor proteins of the IrcB family. The IKB family of proteins includes ItiBsr, IKB/?, IKB~ (C-terminal region of the ~98, ~100 and ~105 precursors), Bcl-3 and the Drosophila gene cactus (Liou and Baltimore, 1993; Gerondakis and Grumont, 1994). In quiescent cells, NF-KB is retained in the cytoplasm in an inactive form by IKB (with the exception of Bcl-3, which is a nuclear protein), which masks the NF-k-B nuclear localization signal (NLS) (Beg et al.. 1992). Upon activation with a variety of agents NF-KB is translocated to the nucleus and induces gene expression. The NF-liB activation is preceded by the phosphorylation and rapid degradation of bcBc( (Beg et *Author to whom correspondence
site.
MATERIALS AND METHODS Cell lines
Porcine aortic endothelial cells (EC) were obtained as described previously (Booyse et al., 1977). The COS7 (African green monkey fibroblast) and NIH3T3 (mouse fibroblast) cell lines were obtained from American Type Culture Collection. Preparation of NF-KB responsive luciferuse reporter gene
A double-stranded oligonucleotide containing three NF-KB consensus binding sites was subcloned upstream of the tk minimal promoter in pBLCAT2 (Luckow and Schiitz, 1987) to create an NF-KB responsive promoter, which was then subcloned upstream of the luciferase coding sequence in pGL2-basic (Promega, Madison, U.S.A.). This expression cassette was finally subcloned into the BglII site of the pcDNA3 vector (Invitrogen. California, U.S.A.) to generate pcDNA3.Luc.
should be addressed. 57
58
C.-G. CHEN et al.
Cloning and site-directed
mutagenesis
The cDNA encoding mouse ~65 was amplified by RTPCR from RNA isolated from murine pre-B cell line 18.81 using primers based on the published sequence (Nolan et al., 1991) and subcloned into pcDNA3 to generate pcDNA3/p65. Porcine IKBCZcDNA was also cloned by RT-PCR from RNA isolated from aortic endothelial cells using primers based on the published sequence (de Martin et al., 1993). The cDNA was extended at the 5’ end to include a sequence encoding the FlagTM (Kodak, New York, U.S.A.) tag. The cDNA was then subcloned into pAlter II plasmid (Promega) for site-directed mutagenesis. The Ser32 residue was changed to Ala, Tyr42 to Phe, and Ser26” to Gly to create IKB~ mutants IS32A,IY42Fand IS262Grespectively. The wild-type and mutant cDNAs were subcloned into the polylinker of pCDNA3.Luc downstream of the CMV promoter to create ~cDNA~.Luc/I~~~*, ~cDNA~.Luc/I~~~~ and ~cDNA~.Luc/I~~~~~. Transfection
and assay for luciferase activity
The NIH3T3 or COS cells were transfected with 5 pg/ml of plasmid DNA using the DEAE-dextran method (Kingston, 1988) and allowed to recover for 24 hr before stimulation with 10 ng/ml of TNF-a for 16 hr. Cell lysates were prepared, normalized for protein content and assayed for luciferase activity using Promega’s luciferase assay system. The luminescence level was determined on a TopCount microplate scintillation counter (Packard, Groningen, The Netherlands). Immunoprecipitation
IKBa:
-
LS32A
IW
I Y42F
1S262G
B
and Western blotting
Cells were washed and lysed in buffer containing 150 mM NaCl, 10 mM sodium phosphate pH7.4, 1 mM CaC12,0.5% Triton Xl00 and protease inhibitors: 1 mM PMSF (Calbiochem, California, U.S.A.), 10 pug/ml leupeptin (Sigma) and 10 pg/ml aprotinin (Sigma). FlagTMtagged IrcBcr was isolated from cytosolic extracts by incubation with 50 ~1 of agarose beads coupled to murine monoclonal anti-FlagTM antibody (Kodak, New York, U.S.A.). The ~65 protein was isolated with rabbit polyclonal anti-p65 antibody (Santa Cruz Biotechnology, Inc., California, U.S.A.) conjugated to protein G sepharose (Pharmacia, Uppsala, Sweden). Samples were fractionated by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred to nitrocellulose membranes (Amersham, Buckinghamshire, U.K.). Blots were probed with either anti-p65 antibody or monoclonal anti-FlagTM antibody. Immunoreactive polypeptides were detected using either rabbit anti-mouse Ig (Dako, Glosmup, Denmark) (1:2000) or protein A (Amersham) (1:5000) conjugated to horseradish peroxidase (HRP) using an enhanced chemiluminescence system (Amersham). Results Inhibition
fibroblast cell line NIH3T3. Forty-eight hours after transfection, TNF-cr stimulated and control non-stimulated cells were lysed and luciferase activity determined. Stimulation of cells with TNF-a resulted in a 4.1 -fold increase in luciferase activity, indicating that the promoter is responsive to NF-KB activation [Fig. l(A), column 1). To examine the effect of expressing wild-type Ik-Ba (IWT) on NF-KB activity, NIH3T3 cells were transfected with pcDNA3.Luc/lWT, a dual construct in which the luciferase reporter construct and ItiBa (under the control of the
of NF-KB activation
by IKBc~ Ser3’ mutant
The NF-k-B-dependent luciferase reporter construct in pcDNA3.Luc was tested by transfection of the murine
1 ~65: IKBa:
+ -
2
3
4
+
+ I S32A
-
IWT
Fig. 1. Effect of expressing IS3’Aon NF-h-B mediated gene expression. (A) Cells were transfected with 5 pg/ml of pcDNA3.Luc (-) or the IxBa constructs in pcDNA3.Luc plasmid as indicated, incubated for 24 hr and cultured overnight in the presence or absence of TNF-a (10 ng/ml). Results are expressed as fold induction of luciferase activity in TNF-a stimulated vs non-stimulated cells. (B) Cells were transfected with pcDNA3/p65 and pcDNA3.Luc (1) or pcDNA3/p65 in combination with pcDNA3.Luc/IWT (2) or pcDNA3.Luc/IS3’* (3). Cells transfected with pcDNA3.Luc alone (4) were included as a control. Luminescence expressed as counts per second (CPS). Experiments were performed in duplicate and expressed as means
(k SD). The results presented are representative three separate experiments.
of
IKBU mutagenesis analysis
59
CMV promoter) are physically linked. This avoids the not cell lysates is probably protein G. In a separate experipossibility of uneven uptake of separate constructs and ment, the anti-FlagTM antibody immunoprecipitated a 65 kDa protein (the expected size of p65), which was reactive ensures that all the luciferase activity detected is attributable to cells that have also been transfected with IKBU to anti-p65 antibody, from cells transfected with IWTand IS32AcDNAs but not from control cells [Fig. 2(B)]. The (wild-type or mutant). Following stimulation with TNFvariability in the intensity of the 65 kDa band in the ~1,luciferase activity increased by approximately 3.5fold different cell lysates is due to uneven protein loading. The [Fig. l(A), column 21. A comparison of the amino acid sequences of IKBCX 50 kDa band detected in all lanes containing immunofrom various species (de Martin et al., 1993) revealed the precipitated material but not cell lysates is most probably existence of three highly conserved potential phos- immunoglobulin heavy chain. phorylation sites, including a casein kinase II site (Ser32), a tyrosine phosphorylation site (Tyr4’) and a protein kinDISCUSSION ase C phosphorylation site (Se?62). To determine if any The transcription factor NF-KB plays a pivotal role in of these sites are involved in a phosphorylation-dependent NF-KB signalling pathway, mutant forms of IKBU the regulation of genes involved in a variety of immune and inflammatory responses (Lenardo and Baltimore, in which each of these sites was mutated were generated 1989; Grilli et al., 1993). The NF-KB activity is modulated by site-directed mutagenesis. The mutant cDNAs were subcloned into the polylinker of pcDNA3.Luc and used by IKBIXwhich binds to and retains NF-k-B in the cytoto transfect NIH3T3 cells. Cells expressing the Tyr42 plasm of quiescent cells. The 1~Bcris phosphorylated and (Iy42F) and Ser262(IS262G)mutants of IKBE responded to subsequently degraded following stimulation of cells with a variety of agents including TNF-sr (Beg et al., 1993; TNF-a stimulation in the same manner as cells transCordle et al., 1993; Mellits et al., 1993). Recent in vivo fected with IWTcDNA, resulting in a 2.5- and 2.7-fold induction of luciferase activity respectively [Fig. 1(A), studies have shown that inhibition of IJcB~ degradation columns 4 and 51. In contrast, little increase (0. l-fold) in by specific protease inhibitors prevents NF-k-B activation luciferase activity was observed following TNF-a stimu- without affecting 1~Ba phosphorylation (Finco et al., lation of cells transfected with the Ser32 mutant (IS”*) 1994; Miyamoto et al., 1994; Palombella et al., 1994; Traenckner et al., 1994; Alkalay et al., 1995; Didonato et cDNA [Fig. l(A), column 31. al., 1995; Lin et al., 1995) demonstrating that degradation of IKBU is essential for NF-KB nuclear transFunctional andph_vsical interaction of IS3’* andp65 in vivo location and that phosphorylation of 1~Bc( alone does The ability of 1~Ba to regulate the transcriptional not cause the dissociation of the NF-KB/IKBCXcomplex. activity of NF-KB is dependent on the physical associ- The question therefore remains as to the role of 1~Ba ation of the two molecules. The NIH3T3 cells co-transphosphorylation in the activation of NF-k-B. To directly fected with pcDNA3/p65 and pcDNA3. Luc gave examine this issue, porcine IKBZ mutants lacking one approximately 1OO-fold higher luciferase activity over the of the three potential phosphorylation sites, which are control cells transfected with the reporter plasmid conserved among IlcBa from different species, were used (pcDNA3.Luc) alone [Fig. l(B)]. Activation of luciferase to determine their effect on the activation of NF-KB. The gene expression by ~65 was completely inhibited by co- TNF-rx stimulation of control (pcDNA3.Luc transfected) transfection with either wild-type IKBUor the IS3**mutant cells resulted in a 4.1 -fold induction of endogenous NFcDNA [Fig. l(B)], suggesting a functional interaction KB activity, as determined using a KB-driven luciferase between the porcine wild-type IKBUand IS32A mutant with reporter construct. Transfection with either wild-type or the murine ~65 subunit of NF-KB. Tyr42 (Iy42”) and SerZ6*(IS26’G)mutants of IrcBa cDNA The TNF-a stimulation of cells co-transfected with caused only a slight reduction in the level of induction, ~65 and wild-type IKBE increased the level of luciferase to 3.5-, 2.5- and 2.7-fold, respectively. In contrast, NFactivity by approximately 3-fold, whereas there was no KB activation was completely inhibited by transfection increase in cells co-transfected with IS32A(data not with the Ser3’ mutant (IS3’*) cDNA, suggesting that this shown). potential phosphorylation site is critical for the activation To demonstrate the physical interaction of ~65 with of NF-KB. The lOO-fold increase in luciferase activity wild-type or mutant IKBu, cytoplasmic extracts from cells which occurred when cells were transfected with ~65 transfected with IWTor IS32A cDNAs were fractionated by cDNA was completely abolished by co-transfection with SDS-PAGE either directly or following immunoeither IWTor IS3”*cDNA. Furthermore, I’“” protein was precipitation with anti-p65 antibody. Proteins were blotco-precipitated with ~65 by antibodies specific either for ted onto a nitrocellulose membrane and probed with anti- ~65 or the FlagTM sequence used to tag the N-terminus FlagTM antibody. A 40 kDa protein band corresponding of1 S32A. Together these data indicate that the IS3’Amutant to the predicted molecular weight of porcine 1~BOZwas form of porcine 1~Ba retains the capacity to bind to and detected in both cell lysates and anti-p65 im- inactivate NF-k-B, but fails to respond to stimulation by munoprecipitated material from cells transfected with IWT TNF-cr. or IS32AcDNAs, but absent from control cells transfected The results presented in this study are consistent with with pcDNA3.Luc [Fig. 2(A)]. The 30 kDa band present those recently published by Brown et al. (1995). which in all lanes containing immunoprecipitated material but demonstrated that mutations at either Ser”’ or Ser3h of
60
C.-G. CHEN et nl.
A.
B.
Anti-Flag antibody
Anti-p65 (IP)
Cell lysates
Anti-p65 antibody
Anti-Flag (IP)
MW (kDa)
Cell lysates
i(*
p65-
IKBCI *i
-
97
..w -
65
-
45
-
30
Fig. 2. Physical association of IS32Amutant with endogenous NF-KB ~65. The COS cells were transfected with pcDNA3.Luc (control), pcDNA3.Luc/IWT (IWT) or pcDNA3.Luc/IS3** (IS3**).The immunoprecipitated (IP) proteins and cell lysates were fractionated by 7.5% SDS-PAGE and blotted. The IlcBa and ~65 proteins bands and molecular weight markers are indicated. (A) Proteins were immunoprecipitated with rabbit anti-p65 antibody conjugated to protein G sepharose and the blot probed with monoclonal anti-FlagTM antibody followed by peroxidase labelled rabbit anti-mouse Ig. (B) Proteins were immunoprecipitated with murine monoclonal anti-FlagTM antibody coupled to agarose beads and the blot probed with anti-p65 antibody followed by protein A conjugated to horseradish peroxidase.
human kBa prevent its phosphorylation and degradation following stimulation with PMA-ionomycin. These mutants, when co-expressed with ~65, prevented p65 from inducing the expression of an NF-rcB responsive reporter gene in NTera-2 cells, which lack endogenous NF-lcB and IKBE proteins. In the present study, however, we have used cells which express endogenous IKBE to demonstrate that NF-KB activation can be inhibited by expression of phosphorylation defective IKB~ in normal cells. Given the appropriate regulatory mechanisms, this dominant-negative mutant (IS32A)of IKB~ could be used to regulate NF-KB activity in uivo. We are currently test-
ing this using transgenic mice. Acknowledgements-We thank Drs D. Power and P. Cowan for critical review of this manuscript. REFERENCES Alkalay I., Yaron A., Hatzubai A., Jung S., Avraham A., Grelitz O., Pashut-Lavon I. and Ben-Neriah Y. (1995) In vivo stimulation of IrcB phosphorylation is not sufficient to activate NF-KB. Mol. Cell. Biol. 15, 1294-1301. Beg A. A., Finco T. S., Nantermet P. V. and Baldwin A. S. (1993) Tumor necrosis factor and interleukin- 1 lead to phosphorylation and loss of IlcBa: a mechanism for NF-rcB activation. Mol. Cell. Biol. 13, 3301-3310.
Beg A. A., Ruben S. M., Scheinman R. I., Haskill S., Rosen C. A. and Baldwin A. S. (1992) IJCBinteracts with the nuclear localization sequences of the subunits of NF-rcB: a mechanism for cytoplasmic retention. Gene Dev. 6, 1899-l 9 13. Booyse F. M., Quarfoot A. J., Bell S., Fass D. N., Lewis J. C., Mann K. G. and Bowie E. J. (1977) Cultured aortic endothelial cells from pigs with von Willebrand disease. Proc. natn. Acad. Sci. USA 74, 5702-5706. Brown K., Gerstberger S., Carlson L., Franzoso G. and Siebenlist U. (1995) Control of Ih-Ba proteolysis by site-specific signal-induced phosphorylation. Science 267, 1485-1488. Cordle S. R., Donald R., Read M. A. and Hawiger J. (1993) Lipopolysaccharide induces phosphorylation of MAD-3 and activation of c-Rel and related NF-rcB proteins in human monocytic THP- 1 cells. J. Biol. Chem. 268, 11803-l 1810. de Martin R., Vanhove B., Cheng Q., Hofer E., Csizmadia V., Winkler H. and Bach F. H. (1993) Cytokine-inducible expression in endothelial cells of an IkBa-like gene is regulated by NF-KB. EMBO J. 12,2773-2779. Didonato J. A., Mercurio F. and Karin M. (1995) Phosphorylation of IKBCXprecedes but is not sufficient for its dissociation from NF-KB. Mol. Cell. Biol. 15, 1302-l 3 11. Finco T. S., Beg A. A. and Baldwin A. S. (1994) Inducible phosphorylation of 1~Bolis not sufficient for its dissociation from NF-rcB and is inhibited by protease inhibitors. Proc. natn. Acad. Sci. USA 91, 11884-l 1888. Gerondakis S. and Grumont R. (1994) NF-KB: a bridge between cytoplasm and nucleus. Todav’s Life Sci. 6, 2428.
IKBCXmutagenesis
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Mellits K. H., Hay R. T. and Goodbourn S. (1993) Proteolytic degradation of MAD-3 (Ih-Bc()and enhanced processing of
analysis
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the NF-KB precursor ~105 are obligatory steps in the activation of NF-KB. Nucleic Acids Res. 21, 5059-5066. Miyamoto S., Maki M., Schmitt M. J., Hatanaka M. and Verma I. M. (1994) Tumor necrosis factor a-induced phosphorylation of IKB~ is a signal for its degradation but not dissociation from NF-KB. Proc. mtn. Acad. Sci. USA 91, 1274&12744.
Nolan G. P., Ghosh S., Liou H.-C., Tempst P. and Baltimore D. (1991) DNA binding and ItiB inhibition of the cloned ~65 subunit of NF-KB, a rel-related polypeptide. Cell 64,961-969. Palombella V. J., Rando 0. J., Goldberg A. L. and Maniatis T. (1994) The ubiquitin-proteasome pathway is required for processing the NF-KB~ precursor protein and the activation of NF-ICB. CeN 78, 7733785. Sen R. and Baltimore D. (1986) Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46, 705-716. Traenckner E. B.-M., Wilk S. and Baeuerle P. A. (1994) A proteasome inhibitor prevents activation of NF-KB and stabilizes a newly phosphorylated form of IKBCXthat is still bound to NF-KB. EMBO J. 13, 5433-5441.