Regulation of Ikappa Bbeta Degradation
SIMILARITIES TO AND DIFFERENCES FROM Ikappa Balpha *

(Received for publication, October 31, 1996)

Robert Weil Dagger , Christine Laurent-Winter § and Alain Israël

From the Unité de Biologie Moléculaire de l'Expression Génique, URA 1149 CNRS, and the § Laboratoire d'Electrophorèse Bidimensionnelle, Institut Pasteur, 75724 Paris Cedex 15, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The transcription factor NF-kappa B (nuclear factor-kappa B) is neutralized in nonstimulated cells through cytoplasmic retention by Ikappa B inhibitors. In mammalian cells, two major forms of Ikappa B proteins, Ikappa Balpha and Ikappa Bbeta , have been identified. Upon treatment with a large variety of inducers, Ikappa Balpha and Ikappa Bbeta are proteolytically degraded, resulting in NF-kappa B translocation into the nucleus. Recent observations suggest that phosphorylation of serines 32 and 36 and subsequent ubiquitination of lysines 21 and 22 of Ikappa Balpha control its signal-induced degradation. In this study we provide evidence that critical residues in the NH2-terminal region of Ikappa Bbeta (serines 19 and 23) as well as its COOH-terminal PEST region control Ikappa Bbeta proteolysis. However Lys-9, the unique lysine residue in the NH2-terminal region of Ikappa Bbeta , is not absolutely required for its degradation. We also demonstrate that following stimulation, an underphosphorylated nondegradable form of Ikappa Bbeta accumulates. Surprisingly, our data suggest that unlike Ikappa Balpha , Ikappa Bbeta is constitutively phosphorylated on one or two of the critical NH2-terminal serine residues. Thus, phosphorylation of these sites is necessary for degradation but does not necessarily constitute the signal-induced event that targets the molecule for proteolysis.


INTRODUCTION

The transcription factor NF-kappa B1 plays a central role in the regulation of genes implicated in the immune response and in inflammatory processes. NF-kappa B is composed of homo- and heterodimeric complexes of members of the Rel/NF-kappa B family of polypeptides. In vertebrates, this family comprises p50, p65 (RelA), c-Rel, p52, and RelB.

In resting cells, NF-kappa B is cytosolic, but the nuclear translocation of this factor can be induced by multiple stimuli that act at different levels in the cell. Some, like TNF-alpha , IL-1, LPS, or antibodies against the T cell receptor-CD3 complex, act on an extracellular receptor, whereas others, like PMA and double-stranded RNA, activate intracellular second messengers (for review, see Refs. 1 and 2).

The molecular mechanism responsible for the cytosolic retention of NF-kappa B involves its association with the inhibitory ankyrin repeat-containing members of the Ikappa B family of proteins. This family of inhibitors is mainly represented by Ikappa Balpha and Ikappa Bbeta (3, 4) but also includes Ikappa Bgamma , Bcl-3, p105, and p100 (for review, see Ref. 5). p105 and p100, which are also the precursors of the p50 and p52 subunits of NF-kappa B, function as Ikappa B proteins through association with p50, c-Rel or p65 (6-8). Among the different Ikappa Bs, Ikappa Balpha and Ikappa Bbeta play a major role in the regulation of NF-kappa B. These two proteins have been originally identified by partial purification (3, 4). A cDNA clone has been isolated which encodes a 36-kDa protein called MAD-3, which appears to be identical to Ikappa Balpha (9). The cloning of Ikappa Bbeta cDNA is more recent, and this molecule has thus been characterized less thoroughly (10).

Unlike Ikappa Bbeta , the Ikappa Balpha gene is positively regulated by NF-kappa B and glucocorticoids (10, 11-14). Ikappa Balpha and Ikappa Bbeta associate with p50-p65 heterodimers and prevent the nuclear translocation of these complexes by masking their nuclear localization sequence. These two molecules are structurally similar as they contain multiple ankyrin repeats and a COOH-terminal PEST domain, a sequence known to be highly correlated to rapid protein turnover. The PEST domain of Ikappa Balpha is involved in its degradation (15-19) and in its inhibition of the DNA binding activity of NF-kappa B (20).

Under the effect of a stimulus, Ikappa Balpha becomes phosphorylated and is subsequently degraded, allowing NF-kappa B to translocate into the nucleus. The use of protease inhibitors has shed some light on the proteases responsible for Ikappa Balpha degradation (21-27). In the presence of an NF-kappa B inducer, proteasome inhibitors stabilize a phosphorylated form of Ikappa Balpha characterized by a slow electrophoretic mobility. The observation that this retarded form is still associated with NF-kappa B invalidates the former hypothesis that Ikappa Balpha phosphorylation induces its dissociation from NF-kappa B.

Recently, the sites of phosphorylation of Ikappa Balpha have been identified as two closely spaced serines at positions 32 and 36 in the NH2-terminal part of the protein (15-19, 28). Mutation of these two serines to nonphosphorylatable residues prevents Ikappa Balpha degradation, suggesting that their phosphorylation is a prerequisite for degradation. In addition, an in vitro study has also shown that only the hyperphosphorylated form of Ikappa Balpha is degraded (29). It has been reported that degradation of Ikappa Balpha is triggered by ubiquitination (30). Ubiquitination occurs primarily on two adjacent lysines (Lys-21 and Lys-22) (31, 32).

In thymocytes, Ikappa Balpha is the main inhibitor of NF-kappa B, and the disruption of Ikappa Balpha gene by homologous recombination results in a constitutively elevated level of nuclear NF-kappa B (33). In contrast to hematopoietic cells, Ikappa Balpha -/- embryonic fibroblasts behave as wild type cells because of the major role of the Ikappa Bbeta molecule.

A recent study concludes that TNF-alpha or PMA induces degradation of Ikappa Balpha but not of Ikappa Bbeta , suggesting that these two proteins are regulated differentially (10). The mechanisms responsible for the differential behavior of the two Ikappa B molecules remain unresolved. We provide here some clues as to why these two molecules are regulated differentially. We first show that Ikappa Bbeta degradation is blocked by proteasome inhibitors, suggesting an involvement of the ubiquitin-proteasome pathway, similar to what has been described for Ikappa Balpha . These similarities are confirmed by the identification of two critical sites of phosphorylation (Ser-19 and Ser-23) whose mutations decrease the rate of signal-induced degradation of Ikappa Bbeta . However, Ikappa Bbeta contains only one lysine NH2-terminal to ankyrin repeats, and its mutation does not prevent the signal-induced degradation of the mutant protein. We also demonstrate that Ikappa Bbeta preexists as two electrophoretically different variants: the major, slow migrating form, is degraded following stimulation; the minor, faster migrating form accumulates. Our study also suggests that, unlike Ikappa Balpha , Ikappa Bbeta is phosphorylated on Ser-19 and/or Ser-23 in noninduced cells. Therefore, these results suggest that Ikappa Bbeta might differs from Ikappa Balpha in that the critical event that targets Ikappa Bbeta for degradation is not the induced phosphorylation of the two conserved serine residues located in the NH2-terminal region of the molecule.


MATERIALS AND METHODS

Cells

The 70Z/3 murine pre-B cell line, EL-4 murine T cell line (kindly provided by G. Milon, Pasteur Institute), E29.1, a CD4-negative/CD8-negative mouse T cell hybridoma (34), and 293T (embryonic kidney cells) were maintained in RPMI medium supplemented with 10% fetal calf serum and 50 µM beta -mercaptoethanol. 70Z/3 derivatives expressing human Ikappa Balpha (hIkappa Balpha ) have been described previously (10). 70Z/3 HA Ikappa Bbeta wt represents a stable transformant expressing hemagglutinin-tagged, wild type Ikappa Bbeta . 70Z/3 expressing Ikappa Bbeta HA-tagged variants: HA A19, HA A23, HA A19 A23, HA R9, and HA R9 A19 A23 expressed HA Ikappa Bbeta containing the following point mutations: (A19, Ser-19 right-arrow Ala; A23, Ser-23 right-arrow Ala; R9, Lys-9 right-arrow Arg). 70Z/3 HA wt Delta PEST and 70Z/3 HA A19 A23 Delta PEST expressed amino acids 1-306 of HA Ikappa Bbeta wt and HA Ikappa Bbeta A19 A23, respectively.

Antisera

The antisera used were the following. Anti-RelA antiserum 1226 (kindly provided by N. Rice), was raised against amino acids 537-550 of human RelA. Ikappa Balpha immunoblots were probed with anti-Ikappa Balpha 52008, generated against recombinant human Ikappa Balpha or with an antibody kindly provided by M. Karin. Anti-Ikappa Bbeta immunoblots were probed with rabbit polyclonal anti-Ikappa Bbeta sera 37015 and 41276 raised against a glutathione S-transferase fusion protein containing amino acids 258-360 of mouse Ikappa Bbeta or an antibody raised against recombinant Ikappa Bbeta (10).

Plasmids

Expression vectors for transfection into 70Z/3 cells were obtained by subcloning cDNAs encoding Ikappa Balpha or its derivatives into the plasmids pRc-CMV or pcDNA-3 (Invitrogen). Some of the mutants were first cloned into the plasmid pT7beta globin-HA,2 and then the fragment containing the beta -globin 5'-untranslated region, the HA epitope, and Ikappa Bbeta coding sequences was subcloned into pRc-CMV or pcDNA-3. Deletion of the PEST region of Ikappa Bbeta was obtained by digestion with HindIII. Mutations of Ikappa Bbeta were constructed by site-directed mutagenesis using polymerase chain reaction and verified by sequencing. All constructions were linearized with ScaI before electroporation into 70Z/3 cells.

Transfections

70Z/3 were pelleted and resuspended in complete medium at 5 × 106 cells/0.5 ml, and the cells were electroporated in 4-mm cuvettes with a Eurogentec Cellject electroporator at 260 V, 1,500 microfarads, and infinite resistance. Cells were diluted into 5 ml of complete medium and were left to recover overnight before the addition of 1 mg/ml G418. Once established, clones were maintained in complete medium supplemented with 0.5 mg/ml G418.

Extracts of Treated and Nontreated Cells

Cells (5 × 106) were incubated for the indicated time at 37 °C in 1 ml of complete medium containing 100 units/ml TNF-alpha (Pharmingen), 15 µg/ml LPS (Sigma), 50 ng/ml PMA (Sigma), 10 µg/ml cycloheximide (Sigma), 150 nM calyculin A (Sigma), 100 µM N-acetyl-Leu-Leu-norleucinal (calpain inhibitor I, Sigma) as indicated in the figure legends.

After these treatments, cells were washed twice in phosphate-buffered saline and lysed in 250 µl of 1 × TNE (50 mM Tris, pH 8.0, 1% Nonidet P-40, and 2 mM EDTA), supplemented with 10 µg/ml each of the protease inhibitors leupeptin, aprotinin, N-tosyl-L-phenylalanine chloromethyl ketone, N-p-tosyl-L-lysine chloromethyl ketone, and phenylmethylsulfonyl fluoride as well as the phosphatase inhibitors sodium fluoride (50 mM) and sodium orthovanadate (1 mM).

Immunoprecipitations

Cells were lysed as described above. Specific polypeptides were then recovered by immunoprecipitation of equivalent amounts of cellular protein, using either of the following antibodies: anti-Ikappa Balpha serum 52008, affinity-purified anti-Ikappa Bbeta serum 41276, anti-RelA serum 1226, or mouse anti-HA 12CA5 monoclonal antibody. Immune complexes were collected with Staphylococcus aureus protein A (Pansorbin, Calbiochem) or protein G (Sigma), washed three times in lysis buffer, and resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. For certain experiments, RelA and associated proteins were eluted from specific antibodies with an excess of the 1226 peptide, and the eluate was mixed with 2 × sample buffer. Subsequent immunoblots were performed using the protocol outlined below.

Immunoblots

Proteins were transferred to Immobilon membranes (Millipore), and immunoblots were incubated with anti-Ikappa Balpha , anti-Ikappa Bbeta , or anti-RelA serum diluted 1/1,000 (for ECL) or 1/200 (for 125I-protein A), as indicated in the figure legends, and revealed with the Amersham ECL system (for direct immunoblotting of total cell extracts) or by incubation with 125I-protein A (Amersham) (for immunoblotting of immunoprecipitates). Immunoreactive products were detected by autoradiography, and 125I-labeled proteins were quantitated with a PhosphorImager (Molecular Dynamics).

Two-dimensional Gel Electrophoresis

For two-dimensional analysis of cell extracts, 15 × 106 cells were lysed with 30 µl of 1 × TNE supplemented with inhibitors as above. Cytoplasmic extracts were reduced with 1% beta -mercaptoethanol and denatured with 0.3% SDS. For immunoprecipitations, 5 × 107 cells were lysed with 300 µl of 1 × TNE supplemented with inhibitors and immunoprecipitated as above, except that the beads were resuspended in 30 µl of SDS buffer (0.3% SDS, 1% beta -mercaptoethanol, and 50 mM Tris, pH 8.0). Samples were boiled for 3 min and flash frozen in liquid nitrogen. After lyophilization, samples were resuspended in 30 µl of sample buffer (9.95 M urea, 4% Nonidet P-40, 2% ampholytes, pH 5-7, and 100 mM dithiothreitol) and centrifuged for 2 min. Samples were then loaded onto the isoelectrofocusing gel (pH 4-8; Millipore) and run for 20,000 Vh. The second dimension was performed as described previously on a 10% acrylamide gel (35). Relative isoelectric points were determined by parallel migration of a carbamylated muscle creatine phosphokinase standard (BDH), and the relative molecular weights of the proteins were determined according to molecular weight markers applied to an adjacent slot on the same gel. After SDS-polyacrylamide gel electrophoresis, proteins were blotted as for the one-dimensional polyacrylamide gels.

Metabolic Labeling and Peptide Mapping Studies

For 32Pi labeling, 293T cells were transiently transfected with HA Ikappa Bbeta wt or HA A19 A23 Ikappa Bbeta plasmids and were incubated for 3 h in phosphate-free RPMI medium supplemented with 0.8 mCi of 32Pi (carrier free; ICN)/ml and 2% dialyzed fetal calf serum. They were then washed with phosphate-free medium and lysed in TNE buffer containing protease and phosphatase inhibitors, as described above. Ikappa Bbeta polypeptides were recovered by immunoprecipitation and resolved in 8% SDS-polyacrylamide gels. Trypsin cleavage was performed, and phospholabeled Ikappa Bbeta polypeptides were resolved in Tricine-SDS-polyacrylamide gels electrophoresis as described elsewhere (36) and detected by autoradiography.


RESULTS

TNF-induced Degradation of Ikappa Bbeta in E29.1 T Cell Hybridoma

In E29.1 T cell hybridoma, the two inhibitors Ikappa Balpha and Ikappa Bbeta behaved differently in response to TNF-alpha . TNF-alpha treatment resulted in rapid (15 min) and transient degradation of Ikappa Balpha (data not shown, and see Fig. 2), whereas Ikappa Bbeta was degraded slowly (Fig. 1) and incompletely resynthesized 3 h after induction (lanes 7 and 9). Increasing the time of stimulation to 6, 9, and 24 h led to the reappearance of Ikappa Bbeta (lanes 10-12). This longer time course allowed the identification of two different molecular species of Ikappa Bbeta : the upper form migrates at 50 kDa and is totally degraded upon stimulation, whereas persistent activation causes the slow accumulation of a lower molecular mass product of approximately 48 kDa.


Fig. 2. Effect of TNF-alpha , LPS, and PMA stimulation on Ikappa Bbeta degradation in various cell lines. Different cell lines were treated with NF-kappa B inducers for the indicated periods of time. Panel A, TNF-alpha causes the degradation of Ikappa Bbeta in E29.1 T cell hybridoma. Cell lysates (20 µg of proteins) from TNF-alpha treated cells were prepared and subjected to parallel Ikappa Balpha (right panel) or Ikappa Bbeta (left panel) immunoblotting. Panel B, Ikappa Bbeta is not degraded upon TNF-alpha stimulation of EL-4 T cells. Cell lysates (20 µg of proteins) from TNF-alpha -treated cells were prepared as described in panel A and subjected to parallel Ikappa Balpha (right panel) or Ikappa Bbeta (left panel) immunoblotting. Panel C, LPS and PMA cause the degradation of Ikappa Bbeta in 70Z/3 cells. After lysis, 20 µg of proteins for each lane was probed with anti-Ikappa Bbeta (left panel) or anti-Ikappa Balpha (right panel) antibodies. All of the anti-Ikappa Bbeta blots were probed with 37015 antiserum except for the PMA experiment (panel C), which was blotted with an anti-Ikappa Bbeta antibody provided by S. Ghosh. The positions of Ikappa Balpha , Ikappa Bbeta , and nonspecific products are shown by an arrow on the right of each panel.
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Fig. 1. Western blot analysis of TNF-alpha -stimulated E29.1 cells reveals the presence of two electrophoretically different forms of Ikappa Bbeta . 20 µg of total cell extracts from E29.1 cells exposed to TNF-alpha for the indicated times were separated by SDS-polyacrylamide gel electrophoresis, transferred to Immobilon, and probed with anti-Ikappa Bbeta (37015) antiserum.
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Ikappa Bbeta Is Not Degraded in All T Cell Lines in Response to TNF-alpha Stimulation

To evaluate the discrepancy concerning the effect of TNF-alpha on Ikappa Bbeta degradation (10, 33), we examined the consequence of TNF-alpha treatment on a second murine T cell line, EL-4 (Fig. 2B). Our results suggest that there may be some cell specificity in Ikappa Bbeta degradation in response to TNF-alpha since this treatment affected only Ikappa Balpha in EL-4 T cells, whereas it resulted in the loss of Ikappa Balpha and Ikappa Bbeta in E29.1 (compare Fig. 2, A and B). However, treatment of EL-4 T cells with IL-1 or PMA induced a complete degradation of Ikappa Bbeta (data not shown). We also confirmed the detection of a doublet of bands in the murine preB 70Z/3 cell line in response to LPS and PMA (Fig. 2C). This doublet, noted previously in the Ikappa Bbeta original cloning paper (10), represents specific forms of Ikappa Bbeta since it is detected with our Ikappa Bbeta antibody 37015 (Fig. 2, A and C), with an antibody kindly provided by S. Ghosh (Fig. 2C) and also by immunoblotting p65 immunoprecipitates with anti-Ikappa Bbeta antibodies (Fig. 3B).


Fig. 3. Characterization of the two electrophoretically different forms of Ikappa Bbeta . Panel A, Ikappa Bbeta is de novo synthesized as a low molecular mass product following stimulation. E29.1 T cell were incubated with TNF-alpha for the indicated times with (lanes 9-12) or without (lanes 5-8) cycloheximide. Cycloheximide was added 30 min prior to induction for lanes 9-12. To normalize cell treatments, cells were also treated with cycloheximide alone for the indicated time (lanes 1-4). Total lysates were prepared as described under "Materials and Methods" and assayed by immunoblotting with anti-Ikappa Bbeta (37015) (top panel) or anti-Ikappa Balpha (with an antibody provided by M. Karin) (bottom panel). Ikappa Balpha degradation cannot be totally observed in the absence of cycloheximide as it is already resynthesized at 30 min (see Fig. 2A). Panel B, the two forms of Ikappa Bbeta are associated with NF-kappa B. E29.1 cells were stimulated for the indicated time with TNF-alpha alone (lanes 3-6) or in the presence of ALLN (lane 7). After immunoprecipitation with normal rabbit serum (NRS) or 1226 anti-p65 antiserum, coprecipitated proteins were eluted from bound antibodies with an excess of the 1226 peptide. The eluates were subjected to 8% SDS-polyacrylamide gel electrophoresis and probed with anti-p65 (1226) (top panel) or anti-Ikappa Bbeta (37015) (bottom panel) antisera. These two immunoblots were derived from the same gel. The migration of p65 and Ikappa Bbeta is indicated on the left.
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Evidence of Two Functionally Different Forms of Ikappa Bbeta

To determine whether one of the two variants of Ikappa Bbeta described above could be derived from the other, we tested the effect of the protein synthesis inhibitor cycloheximide on the TNF-alpha -induced degradation of Ikappa Bbeta (Fig. 3A, top panel). To perform this experiment, we pretreated E29.1 T cells with cycloheximide for 30 min. Alone this treatment had no effect on the Ikappa Balpha and Ikappa Bbeta levels (lanes 1-4); but, followed by stimulation with TNF-alpha for the indicated period (lanes 9-12), we observed a complete loss of the low molecular mass product of Ikappa Bbeta , whereas the upper form disappeared with kinetics similar to non-cycloheximide-treated cells (compare lanes 5-8 with lanes 9-12). We thus consider the low molecular mass variant of Ikappa Bbeta to be a newly synthesized product. As expected, Ikappa Balpha had a rapid turnover and was entirely resynthesized after 60 min (Fig. 3A, bottom panel, lanes 5-8) but did not reappear when protein synthesis was blocked by cycloheximide (lanes 10-12).

We then asked whether the newly synthesized form of Ikappa Bbeta was specifically bound to NF-kappa B. To this end, p65-containing complexes were precipitated with anti-p65 antiserum 1226, eluted with the cognate peptide, and analyzed by immunoblotting with anti-p65 (Fig. 3B, top panel) or anti-Ikappa Bbeta antibody (bottom panel). The two Ikappa Bbeta variants were not present in the immune complexes obtained using preimmune serum (lane 1, bottom panel) but were both present in p65 immunoprecipitates (lanes 2-6), indicating that both variants were associated with p65 with a similar affinity. In contrast to the rapid disappearance of the slower migrating form of Ikappa Bbeta , the newly synthesized form accumulated following TNF-alpha stimulation. We also demonstrated that a proteasome inhibitor, ALLN, efficiently blocked degradation of Ikappa Bbeta and did not lead to its dissociation from NF-kappa B (lane 7).

Inhibition of Serine-Threonine Phosphatase Activity Targets Ikappa Balpha for Proteolysis and Releases Ikappa Bbeta from NF-kappa B

Earlier studies demonstrated that treatment of cells with the phosphatase inhibitors okadaic acid and calyculin A induced phosphorylation changes of Ikappa Balpha which contribute to its degradation (18, 24, 27, 28). To investigate the impact of phosphatase inhibitor treatment on Ikappa Bbeta stability and association with NF-kappa B, coimmunoprecipitations were performed. E29.1 cells were treated for different periods of time with calyculin A. NF-kappa B·Ikappa B complexes were recovered by immunoprecipitations using anti-Ikappa Balpha , anti-p65, or affinity-purified anti-Ikappa Bbeta antibodies. The abundance of Ikappa Balpha , Ikappa Bbeta , and p65 in these immunoprecipitates was established by parallel immunodetection of these proteins. As predicted (Fig. 4), calyculin A caused a rapid depletion of Ikappa Balpha concomitant with the disappearance of associated p65 (lanes 2-4). However, calyculin A treatment induced a progressive shift of Ikappa Bbeta mobility, with almost no associated degradation. The association of Ikappa Bbeta with NF-kappa B, monitored by its association with p65, was abolished by calyculin A treatment (lanes 6-8), suggesting that this modified form of Ikappa Bbeta is stable even when not associated with NF-kappa B. Calyculin A treatment also induced the appearance of slow migrating forms of p65 which likely correspond to hyperphosphorylated molecules (lanes 9-12).


Fig. 4. Calyculin A dissociates Ikappa Bbeta from p65 but does not induce its degradation. E29.1 T cells were stimulated for the indicated period with calyculin A. Immunoprecipitates with anti-Ikappa Balpha , anti-Ikappa Bbeta , or anti-p65 were analyzed by immunoblotting in the same conditions used in Fig. 1A. The migration of prestained molecular size markers is shown on the right, with the different antibodies used to blot the corresponding piece of the membrane. The positions of p65, heavy chain of IgG (Ig), Ikappa Balpha , and Ikappa Bbeta are indicated on the left.
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Role of Serine 19 and 23 and Lysine 9 in the Process Leading to Ikappa Bbeta Proteolysis

It has been shown recently that two serine residues (Ser-32 and Ser-36) spaced by a three-amino acid GLD motif and two adjacent lysines (Lys-21 and Lys-22) play a critical role in signal-induced proteolysis of Ikappa Balpha (8, 15, 16, 19, 30-32). Analysis of the Ikappa Bbeta protein sequence reveals two serines (Ser-19 and Ser-23) in a similar environment and only one lysine (Lys-9) in the entire NH2-terminal domain of Ikappa Bbeta . It was thus tempting to examine whether Ser-19 and Ser-23 as well as Lys-9 play a similar role in signal-induced degradation of Ikappa Bbeta .

Using site-directed mutagenesis, Ser-19 and Ser-23 were replaced by nonphosphorylatable alanine residues, and the resulting Ikappa Bbeta mutants were stably introduced into murine 70Z/3 pre-B cells. To distinguish between endogenous Ikappa Bbeta and exogenous, mutated versions of Ikappa Bbeta , the latter were tagged at the NH2 terminus with an HA epitope that is specifically recognized by the 12CA5 monoclonal antibody (anti-HA).

We first examined the impact of Ser-19 and Ser-23 mutations on Ikappa Bbeta proteolysis (Fig. 5A). After treatment with LPS, cells were lysed, and the "tagged" mutants were specifically immunoprecipitated with anti-HA antibody and then detected by anti-Ikappa Bbeta immunoblotting. We also verified that transfected proteins remained associated with NF-kappa B by immunoblotting the immune complexes with an antibody to the p65 subunit of NF-kappa B. As shown above for endogenous Ikappa Bbeta , we also observed two forms of the exogenously expressed Ikappa Bbeta derivatives. The slower migrating species of exogenously expressed wild type Ikappa Bbeta was the only species to be degraded efficiently following a 1-h treatment of cells with LPS (Fig. 5A, lanes 2 and 3). In contrast, the amount of the faster migrating form was strongly increased after 2 h of stimulation (lane 4). On the other hand, mutation of either Ser-19 or Ser-23 or both completely abolished the degradation of Ikappa Bbeta in response to LPS (lanes 5-16). As indicated by parallel p65 immunoblotting, these mutants were still capable of interacting with NF-kappa B. We verified that the observed lack of degradation of these mutants was not a result of differences in the level of proteins, nor in LPS induction variability, by directly probing the total cell extracts with anti-p65 or anti-Ikappa Bbeta antiserum (data not shown).


Fig. 5. Ser-19 and Ser-23 but not Lys-9 are necessary for Ikappa Bbeta proteolysis in response to LPS. 70Z/3 cells were stably transfected with HA-tagged wt or mutated murine Ikappa Bbeta expression vectors. Cells were induced with LPS for the indicated periods. Panel A, extracts from 70Z/3 cells which stably express tagged wt and serine 19 and 23 to alanine double (A19 A23) or single point mutants (A19 and A23) were immunoprecipitated with anti-HA monoclonal antibody and transferred to an Immobilon membrane that was then separated in two pieces to allow anti-p65 (1226) or anti-Ikappa Bbeta (37015) immunoblotting. Panel B, 70Z/3 clones expressing tagged wt, R9 A19 A23 Ikappa Bbeta or R9 Ikappa Bbeta mutants were pretreated for 30 min with cycloheximide before LPS stimulation. Extracts were then immunoprecipitated with anti-HA monoclonal antibody, transferred to an Immobilon membrane, and probed with anti-Ikappa Bbeta (37015) antiserum.
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To test whether the mutation of Ser-19 and Ser-23 affected Ikappa Bbeta degradation in response to other inducers, we analyzed the effect of IL-1 and PMA. We found that, like LPS stimulation, IL-1 and PMA caused a complete degradation of endogenous and HA Ikappa Bbeta wt but not of the Ser-19 and/or Ser-23 mutants (data not shown).

Using the same type of analysis, we were able to demonstrate that the presence of Lys-9 was not required for the signal-induced proteolysis of Ikappa Bbeta . To measure the effect of Lys-9 mutation on Ikappa Bbeta half-life upon LPS treatment, experiments using cycloheximide were carried out (Fig. 5B). Under these conditions, we found that HA Ikappa Bbeta wt was completely degraded in 60 min, whereas the triple mutation of Lys-9, Ser-19, and Ser-23 prevented its degradation. Surprisingly, following quantification of the blots by PhosphorImager, we did not observe detectable changes when Ikappa Bbeta was mutated on Lys-9 alone (the basal levels of expression of the two molecules are different, but compare lanes 1 and 2 with lanes 11 and 12). These results suggest that, in contrast to Lys-21 and Lys-22 of Ikappa Balpha , Lys-9 does not seem to be an exclusive site of ubiquitination of Ikappa Bbeta , but, as for Ikappa Balpha , two phosphorylatable serines are required to target Ikappa Bbeta for degradation (see "Discussion").

Role of Ikappa Bbeta PEST Region

It has been reported that signal-dependent degradation of Ikappa Balpha requires the carboxyl-terminal PEST sequence (16, 17, 19). To determine whether Ikappa Bbeta PEST sequences are also necessary for signal-dependent proteolysis, we constructed truncated proteins lacking amino acids 307-360 (Delta PEST) in the context of both Ikappa Bbeta wt and Ikappa Bbeta A19 A23. Cell lysates were prepared and subjected to immunoprecipitation using anti-HA antibody followed by anti-Ikappa Bbeta immunoblotting (Fig. 6, A and B, top panels). We observed that both tagged constructs, HA wt Delta PEST and HA A19 A23 Delta PEST, were stable upon LPS stimulation (lanes 2, 3, 7, and 8). However, during the 1-h treatment with LPS, the amount of HA wt Delta PEST did not increase, in contrast to HA A19 A23 Delta PEST, suggesting that the former construct was still partially susceptible to signal-induced degradation. This was confirmed by pretreatment of the cells with ALLN (Fig. 6A, top panel, compare lane 3 with 5, and lane 8 with 10). Immunoblotting of total cell extracts with anti-p65 antibody indicated that protein loading was identical in each lane (middle and bottom panels). To analyze this point further, cells were pretreated with cycloheximide for 1 h and then stimulated with LPS (Fig. 6B). Under these conditions Ikappa Balpha was not resynthesized (bottom panel, lanes 3, 4, 7, and 8), and endogenous Ikappa Bbeta was degraded completely (middle panel, lanes 3, 4, 7, and 8). This experiment revealed a small decrease in the amount of HA wt Delta PEST after 1 h (upper panel, lane 3) but no change for HA A19 A23 Delta PEST (lanes 6-8). However, compared with the effect of cycloheximide on HA wt (Fig. 5B), this degradation was minor. We thus consider that the deletion of Ikappa Bbeta PEST sequence largely protects Ikappa Bbeta from proteolysis and that this effect is reinforced by Ser-19 and Ser-23 mutations.


Fig. 6. The PEST sequence is required for signal-dependent degradation of Ikappa Bbeta . Panel A, 70Z/3 cells stably transformed with HA-tagged wt Delta PEST and A19 A23 Delta PEST were induced with LPS for the indicated periods following (lanes 4-5, 9-10) a 1-h pretreatment with ALLN. Top panel, HA wt Delta PEST and HA A19 A23 Delta PEST were immunoprecipitated with anti-HA monoclonal antibody, transferred to an Immobilon membrane, and then immunoblotted with an anti-Ikappa Bbeta anti-serum provided by S. Ghosh. Middle panel, direct anti-p65 (1226) immunoblot of total cell extracts. Bottom panel, direct anti-Ikappa Bbeta (37015) immunoblot of total cell extracts. The positions of tagged Ikappa Bbeta mutants (HA Ikappa Bbeta ), endogenous Ikappa Bbeta , and p65 are shown on the left. The altered mobility of the HA A19 A23 Delta PEST construct versus wt has been reproducibly observed. Panel B, 70Z/3 cells stably transformed with HA-tagged wt Delta PEST and A19 A23 Delta PEST were pretreated with cycloheximide and stimulated with LPS for the indicated periods. Cell extracts were analyzed by immunoprecipitation with anti-HA monoclonal antibody, transferred to an Immobilon membrane, and then immunoblotted with an anti-Ikappa Bbeta antiserum provided by S. Ghosh (top panel). Western blotting was performed on total cell extracts with anti-Ikappa Bbeta antibody (middle panel), the part corresponding to the transfected HA-tagged molecule has been cut out) or anti-Ikappa Balpha antibody (with an antibody provided by M. Karin) (bottom panel). The positions of a nonspecific band (NS), of tagged Ikappa Bbeta mutants (HA Ikappa Bbeta ), and of endogenous Ikappa Bbeta and Ikappa Balpha are shown on the left.
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Two-dimensional Gel Analysis of Ikappa Bbeta

To get a better separation of Ikappa Bbeta isoforms, total lysates from unstimulated E29.1 T cells were subjected to two-dimensional gel analysis and immunoblotted with anti-Ikappa Bbeta antiserum (Fig. 7A). As for Ikappa Balpha (16, 19) multiple isoforms were detected which likely represent differentially phosphorylated isoforms of Ikappa Bbeta . Upon stimulation with TNF-alpha , the spots disappeared progressively (panels B and C). Strikingly, the simultaneous presence of ALLN did not lead to the appearance of additional spots (panel D). This result was confirmed by two-dimensional analysis of mixed lysates from unstimulated and TNF-alpha -induced cells in the presence of ALLN (panel E). This is in contrast to the situation observed with Ikappa Balpha , which, upon signaling, becomes phosphorylated on two critical serines, resulting in the appearance of additional isoforms of higher apparent molecular mass and more acidic migration.3 (16, 19). This intriguing observation led us to evaluate the possibility that Ikappa Bbeta was constitutively phosphorylated on Ser-19 and/or Ser-23. To this end, we decided to use 70Z/3 cells expressing Delta PEST constructs in the hope that, as for Ikappa Balpha , it would simplify the overall pattern of isoforms (19). Interestingly, both HA wt Delta PEST and HA A19 A23 Delta PEST constructs still gave rise to several isoforms of different apparent molecular mass and isoelectric points (panels F and H), suggesting that unlike Ikappa Balpha , additional phosphorylations take place in the ankyrin repeat region (see "Discussion"). In agreement with what we observed for wt Ikappa Bbeta (panels D and E), pretreatment of HA wt Delta PEST with ALLN did not result in the appearance of additional spots in response to LPS (panels G and I). But the most striking observation was that the HA A19 A23 Delta PEST isoforms present a distribution identical to that of the HA wt Delta PEST isoforms, except that they exhibit a much more basic isoelectric point (panel J). The simplest explanation for this observation is that all of the isoforms are constitutively phosphorylated on Ser-19 and/or Ser-23 in HA wt Delta PEST constructs.


Fig. 7. Analysis of constitutive and inducible phosphorylation of Ikappa Bbeta by two-dimensional gel electrophoresis. E29.1 T cells were analyzed before stimulation (panel A) and after 30 min (panel B) and 90 min of induction by TNF-alpha in the absence (panel C) or presence (panel D) of ALLN. Cell extracts from treated and untreated cells were subjected to two-dimensional gel electrophoresis as described under "Materials and Methods." Endogenous Ikappa Bbeta proteins were analyzed by immunoblotting with anti-Ikappa Bbeta antiserum 37015. A mixing experiment (panel E) of lysates obtained from uninduced and TNF-alpha -stimulated cells in the presence of ALLN demonstrated the identical migration of Ikappa Bbeta isoforms observed in these two conditions. 70Z/3 cells expressing HA wt Delta PEST (panels F and G) or HA A19 A23 Delta PEST (panel H) were treated as indicated. Total cell extracts were precipitated with anti-HA antibody and subjected to two-dimensional gel electrophoresis as described under "Materials and Methods." Immunoblots were probed with an anti-Ikappa Bbeta antiserum provided by S. Ghosh. Extracts analyzed in panels F and G were mixed in panel I; extracts analyzed in panels F and H were mixed in panel J. For each panel, the horizontal axis represents the isoelectric focusing dimension; the vertical axis represents the molecular mass dimension. The acidic side of the gel is on the right.
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Serine 19 and/or Serine 23 Is Detectably Phosphorylated in Uninduced 293T Cells

To define the level of phosphorylation of wt and A19 A23 Ikappa Bbeta molecules, 293T cells were transiently transfected with HA wt or HA A19 A23 Ikappa Bbeta and were metabolically labeled with 32Pi. Then, phospholabeled Ikappa Bbeta molecules were recovered by immunoprecipitation as described under "Materials and Methods" (Fig. 8A). We found that wt Ikappa Bbeta polypeptides were prominently phosphorylated in 293T cells, to an extent apparently similar to that of A19 A23 Ikappa Bbeta . However, to define precisely the sites of phosphorylation on these molecules, phospholabeled Ikappa Bbeta molecules were subjected to peptide mapping studies using trypsin (Fig. 8B). Trypsin digestion removes from Ikappa Bbeta a unique 103-amino acid fragment of approximately 13 kDa which contains serines 19 and 23. Strikingly, we found that a 13-kDa peptide was phosphorylated in wt Ikappa Bbeta molecules but was devoid of phosphorylation in A19 A23 Ikappa Bbeta molecules. This is the largest peptide generated by trypsin digestion (the next largest is 37 amino acids).


Fig. 8. Phosphorylation state of wt Ikappa Bbeta and A19 A23 Ikappa Bbeta in 293T cells. Panel A, in vivo 32Pi labeling of nontransfected (NT) or transiently transfected 293T cells (HA wt and HA A19 A23). Cells were labeled with 32Pi, and Ikappa Bbeta molecules were recovered by immunoprecipitation with affinity-purified anti-Ikappa Bbeta serum 41276. Exposure, 1 h. Panel B, trypsin peptide mapping studies. In vivo phosphorylation of serines 19 and 23 was determined by trypsin cleavage of 32Pi-labeled wt Ikappa Bbeta and A19 A23 Ikappa Bbeta . The migration of molecular mass markers is indicated on the right. Exposure, 6 days.
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DISCUSSION

Recent data have provided some insight into the mechanisms leading to Ikappa Balpha degradation (15-19, 21, 30-32, 37). The signal-induced degradation of this molecule is dependent upon the presence of an intact COOH-terminal PEST region as well as the induced phosphorylation of serine residues 32 and 36. This phosphorylation probably targets the molecule for ubiquitination on multiple residues, lysines 21 and 22 playing a central role (31, 32). This in turn targets the molecule for degradation by the 26 S proteasome (30), which takes place in the absence of dissociation between the inhibitor and NF-kappa B.

The Ikappa Bbeta molecule was originally purified and shown to be inactivated in vitro by dephosphorylation, in contrast to the situation observed with Ikappa Balpha (38). The recent cloning of Ikappa Bbeta (10) demonstrated that the protein contained, like Ikappa Balpha , a COOH-terminal PEST region, several ankyrin repeats, and an NH2-terminal region with two serines (at positions 19 and 23) in an environment similar to Ikappa Balpha . A unique lysine residue was present in this region, at position 9. However, some differences could be observed between the two molecules following stimulation. First, although PMA, TNF, LPS, and IL-1 all induce degradation of Ikappa Balpha , only LPS and IL-1 induced degradation of Ikappa Bbeta in 70Z/3 cells (10). Second, the Ikappa Balpha molecule is resynthesized rapidly following degradation, partly because the promoter of the Ikappa Balpha gene is positively regulated by NF-kappa B. On the contrary, the Ikappa Bbeta molecule is not resynthesized before the stimulus has ceased. This suggests that the promoter of the Ikappa Bbeta gene does not respond to NF-kappa B. However, a more recent study seems to indicate that in different cell types the Ikappa Bbeta molecule is also degraded in response to TNF-alpha (33).

Therefore, to get some insight into the mechanisms responsible for these different behaviors, we carried out a systematic analysis of the events leading to Ikappa Bbeta degradation. In contrast to previous work (10), we found that Ikappa Bbeta could be degraded in response to TNF-alpha and PMA in various murine cell lines (Fig. 2). However, the EL-4 T cell line shows a different susceptibility to TNF-alpha since only Ikappa Balpha is degraded. This observation raised some important questions concerning the signaling pathway leading to the loss of Ikappa Balpha and Ikappa Bbeta . If the two inhibitors were targeted for proteolysis by the same pathway, they would be affected equally in response to a given stimulus. It is however possible that the weakness of the signal via the TNF-alpha receptor in this cell line could account for the phenomenon observed, since Ikappa Bbeta can indeed be degraded by other stimuli (IL-1 and PMA) (data not shown).

Following treatment of E29.1 murine T cells with TNF-alpha , two forms of Ikappa Bbeta become detectable: a major upper form (form I), which is degraded progressively; and a minor lower form (form II), which accumulates progressively and appears to be resistant to degradation in response to TNF-alpha stimulation. The same kinetics of degradation is observed during treatment of 70Z/3 cells by LPS or PMA. The two forms of Ikappa Bbeta are both associated with p65. Using cycloheximide, we demonstrated that form II is not derived from form I but requires ongoing protein synthesis to accumulate (Fig. 3A).

Treatment of form I with alkaline phosphatase results in a partial conversion into a protein that comigrates with form II, suggesting that form I is a hyperphosphorylated form of form II (data not shown). These data suggest that this hyperphosphorylation might target form I for degradation.

Based on the results obtained with Ikappa Balpha , we asked whether serines 19 and 23 were the sites of phosphorylation responsible for Ikappa Bbeta proteolysis. In accordance with this model, we observed that mutation of either of these two serines into an alanine abolished Ikappa Bbeta degradation (Fig. 5). These results are consistent with a recent report (39). However, the A19 A23 Ikappa Bbeta mutant still exists as form I and form II, suggesting that form II is not derived from form I by dephosphorylation on Ser-19 and/or Ser-23 (Fig. 5, A and B).

The Ikappa Balpha PEST sequence is constitutively phosphorylated by a highly ubiquitous conserved kinase, casein kinase II. Ikappa Balpha possesses several consensus sites for casein kinase II, and mutation of one of these sites increased the Ikappa Balpha half-life (40). It has thus been hypothesized that the role of basal phosphorylation is to allow degradation of excess free Ikappa Balpha by reducing its half-life. This PEST sequence also plays an important role in signal-dependent degradation of Ikappa Balpha (16, 17, 19). As reported for Ikappa Balpha , we also find that Ikappa Bbeta -inducible proteolysis requires the PEST sequence present in its COOH-terminal region. However, deletion of the PEST sequence results in the disappearance of form II (Fig. 6). This suggests that form II differs from form I in its level of phosphorylation in the PEST region.

In contrast to Ikappa Balpha , mutation of lysine at position 9 inhibited weakly, if at all, Ikappa Bbeta degradation, indicating that if this is a site of ubiquitination, its mutation does not prevent ubiquitination at other sites (Fig. 5). Furthermore, no upshift of the molecule could be observed following stimulation in the presence of ALLN, a proteasome inhibitor that prevents degradation of Ikappa Balpha and Ikappa Bbeta and allows accumulation of a hyperphosphorylated form of Ikappa Balpha . However this lack of upshift does not necessary imply a lack of hyperphosphorylation of the molecule, as hyperphosphorylated murine Ikappa Balpha migrates like its hypophosphorylated counterpart and is identified only by two-dimensional gel electrophoresis3 (19). To reveal hyperphosphorylated forms of Ikappa Bbeta , we performed a two-dimensional gel analysis following various treatments of 70Z/3 cells. We observed that Ikappa Bbeta preexists as multiple isoforms that most likely correspond, as for Ikappa Balpha , to differentially phosphorylated forms. Interestingly, the isoforms observed in the presence of LPS and ALLN coincide exactly with those present in untreated cells, suggesting that their net charge does not change following stimulation.

From these results we can conclude that serines 19 and 23 are critical determinants of Ikappa Bbeta degradation but that their phosphorylation status does not seem to change following activation. One intriguing possibility is that they could be constitutively phosphorylated in untreated cells. To test this hypothesis, we compared HA wt Delta PEST and HA A19 A23 Delta PEST proteins, as the number of isoforms should be reduced following deletion of the PEST region. As hypothesized, we observe that mutation of serines 19 and 23 to alanines shifted all isoforms toward more basic pIs. Since these serine to alanines substitutions per se do not result in a modification of calculated pI, the observed difference in pI between the two proteins (0.1 pH unit) suggests that Ser-19 and/or Ser-23 is constitutively phosphorylated. Consequently, we found that the peptide containing serines 19 and 23 was constitutively phosphorylated in wt Ikappa Bbeta molecules but not when serine 19 and 23 were mutated (Fig. 8). However this result has been obtained in transiently transfected cells as we never obtained enough radioactivity from 32P-labeled cells to carry out the analysis of Ikappa Bbeta -derived phosphopeptides. The intriguing observation that Ser-19 and/or Ser-23 is constitutively phosphorylated is supported by the the fact that following treatment with calyculin A, a potent inhibitor of serine/threonine phosphatases, Ikappa Balpha is degraded quickly, whereas the Ikappa Bbeta molecule is not; but, surprisingly, it dissociates from p65 (Fig. 4). Although this situation is not very physiological, since treatment with calyculin A induces multiple nonspecific phosphorylations (see the multiple bands observed for p65 in Fig. 4, lanes 10-12), it tells us that hyperphosphorylation is unable to induce Ikappa Bbeta degradation, thus supporting our hypothesis that Ser-19 and/or Ser-23 is constitutively phosphorylated and that this is not sufficient to trigger Ikappa Bbeta proteolysis. In this context, several speculations can be made concerning the signaling pathways leading to the inducible degradation of Ikappa Balpha and Ikappa Bbeta . First, if the critical serine residues of Ikappa Balpha and Ikappa Bbeta are phosphorylated by the same kinase, then there should exist a mechanism maintaining serines 32 and 36 of Ikappa Balpha unphosphorylated in uninduced cells. Since degradation of Ikappa Balpha is induced by phosphatase inhibitors, it might be that a constitutive phosphatase, which is inactivated upon induction, is involved but does not recognize Ikappa Bbeta . Alternatively, serines 32 and 36 of Ikappa Balpha might be masked in resting cells and thus inaccessible to the kinase activity. Finally, the kinase might be constitutively associated with Ikappa Bbeta but recruited to Ikappa Balpha upon NF-kappa B activation. Alternatively, different kinases may phosphorylate the two Ikappa B molecules.

Second, if Ikappa Bbeta is constitutively phosphorylated on Ser-19 and/or Ser-23, the nature of the signal responsible for targeting Ikappa Bbeta for degradation remains to be identified. Because we have been unable to detect any inducible modification of Ikappa Bbeta mobility after two-dimensional electrophoresis, we think it is unlikely that other Ikappa Bbeta -specific phosphorylation events are involved. Alternatively, it might be that degradation per se is induced. For example, enzymes responsible for the ubiquitination of Ikappa Bbeta could be activated following induction. A possible candidate would be the specific E3 involved. Indeed it has been demonstrated that in the case of E6-AP, activity can be regulated (41, 42). A change in activity of the 26 S proteasome following induction could also constitute the inducible signal, as has been shown in the case of stimulation by interferon-gamma (43).


FOOTNOTES

*   This research was sponsored in part by grants from l'Association pour la Recherche sur le Cancer, l'Agence Nationale de Recherche contre le SIDA, INSERM, and the ligue Nationale Française contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Recipient of long term fellowship from ANRS.
   To whom correspondence should be addressed: Unité de Biologie Moléculaire de l'Expression Génique, URA 1149 CNRS, Institut Pasteur, 25 Rue du Dr. Roux, 75724 Paris Cedex 15, France. Tel.: 33-1-4568-8553; Fax: 33-1-4061-3040; E-mail: aisrael{at}pasteur.fr.
1   The abbreviations used are: NF-kappa B, nuclear factor-kappa B; TNF, tumor necrosis factor; IL, interleukin; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; HA, hemagglutinin; wt, wild type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ALLN, N-acetyl-Leu-Leu-norleucinal.
2   C. Brou, unpublished data.
3   S. T. Whiteside, C. Laurent-winter, and A. Israël, unpublished observations.

ACKNOWLEDGEMENTS

We thank Simon Whiteside, Gilles Courtois, and Sylvie Mémet for helpful discussions; Christel Brou for technical advice and for the plasmid pT7beta globin-HA; Michael Karin, Nancy Rice, and Sankar Ghosh for the antisera; and Paolo Truffa-Bachi and Geneviève Milon for the E29.1 and EL-4 T cells.


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