©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of the C-terminal Domain of IB in Protein Degradation and Stabilization (*)

(Received for publication, October 26, 1995; and in revised form, January 16, 1996)

Pierre Beauparlant (§) Rongtuan Lin (¶) John Hiscott (**)

From the Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital and the Departments of Microbiology and Medicine, McGill University, Montreal, Quebec, H3T 1E2, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the present study, the role of the IkappaBalpha C terminus in NF-kappaB/IkappaBalpha regulation was examined in NIH 3T3 cells engineered to inducibly express wild type or mutated human IkappaBalpha proteins under the control of the tetracycline responsive promoter. Deletion studies demonstrated that the last C-terminal 30 amino acids (amino acids (aa) 288 to aa 317, deleted in IkappaBalphaDelta3), including most of the PEST domain, were dispensible for IkappaBalpha function. However, deletions from aa 261 to 317 or aa 269 to 317 (IkappaBalphaDelta1 and IkappaBalphaDelta2 respectively), lacked the ability to dissociate NF-kappaB/DNA complexes in vitro and were unable to inhibit NF-kappaB dependent transcription. Moreover, IkappaBalphaDelta1 and IkappaBalphaDelta2 mutants were resistant to inducer-mediated degradation. Analysis of IkappaBalpha deletions in the presence of protein synthesis inhibitors revealed that, independently of stimulation, IkappaBalphaDelta1 and IkappaBalphaDelta2 had a half-life four times shorter than wild type IkappaBalpha and the interaction of IkappaBalphaDelta1 and IkappaBalphaDelta2 with p65 was dramatically decreased in vivo as measured by co-immunoprecipitation. Interestingly, protease inhibitors which block inducer-mediated degradation of IkappaBalpha also stabilized the turnover of IkappaBalphaDelta1 and IkappaBalphaDelta2. Based on these studies, we propose that in the absence of stimulation, the C-terminal domain between aa 269 and 287 may play a role to protect IkappaBalpha from a constitutive protease activity.


INTRODUCTION

The NF-kappaB/Rel transcription factors participate in the activation of immune regulatory genes including cytokines, cell surface receptors, and acute phase proteins, as well as the HIV-1 (^1)long terminal repeat (for review, see (1) and (2) ). NF-kappaB/Rel proteins are present in most cell types in an inactive cytoplasmic form, complexed to inhibitory IkappaB proteins that bind to and mask a nuclear translocation signal within the Rel homology domain(3, 4) . A number of IkappaB proteins have been identified, all of which contain multiple ankyrin repeats, including IkappaBalpha(5) , IkappaBbeta(6) , IkappaB(7, 8) , Bcl-3(9, 10) , and the precursors p105 (11) and p100(12) .

The role of IkappaBalpha in the regulation of NF-kappaB DNA binding activity has been extensively studied (for review, see (13) ). All NF-kappaB/Rel heterodimers, as well as p65 and c-Rel homodimers can interact with IkappaBalpha, although IkappaBalpha preferentially associates with p65(11, 14, 15, 16, 17, 18) . IkappaBalpha has a half-life of 1-2 h when complexed with NF-kappaB but is less stable when free in the cytoplasm(19, 20, 21) . The short half-life of IkappaBalpha may be due to the presence of a C-terminal domain rich in proline, glutamic acid, serine, and threonine amino acids called the PEST domain(5, 22) . Activating agents, such as double strand RNA, phorbol esters, tumor necrosis factor-alpha (TNF-alpha), interleukin-1, and lipopolysaccharide (LPS), accelerate the degradation of cytosolic IkappaBalpha, thereby promoting release and nuclear translocation of NF-kappaB/Rel dimers(1, 2, 3, 4, 23) . Nuclear NF-kappaB/Rel dimers transactivate target gene expression, including transcriptional up-regulation of the MAD3 (IkappaBalpha) gene, thereby establishing an autoregulatory loop in which newly synthesized IkappaBalpha restores the cytoplasmic pool of latent NF-kappaB(1, 21, 24, 25) .

Following inducer mediated stimulation, IkappaBalpha becomes hyperphosphorylated, detectable in immunoblots as a slowly migrating form, sensitive to phosphatase treatment(23, 24) . Hyperphosphorylation does not impair the ability of IkappaBalpha to associate with NF-kappaB but represents a signal for subsequent degradation by the proteasome pathway(26, 27, 28, 29) . Central to the proteasome machinery is the ATP-dependent, 26 S multisubunit protease, which can operate in a ubiquitin-dependent or independent fashion (30) . Ubiquitination of IkappaBalpha following TNF-alpha stimulation has been demonstrated (13) and only proteasome inhibitors have been shown to prevent IkappaBalpha degradation induced by TNF-alpha(27, 28, 29, 31) . Proteasome inhibitors such as PSI and MG115 prevent IkappaBalpha degradation but not IkappaBalpha hyperphosphorylation, illustrating that these two events are independent(29, 31) . Recent studies demonstrate that phosphorylation of the N-terminal serine residues Ser-32 and/or Ser36 is the signal that leads to rapid inducer-mediated degradation of IkappaBalpha(32, 33) . Substitution of these residues prevents IkappaBalpha phosphorylation, ubiquitination, and degradation(13) . Similarly, C-terminal truncation of IkappaBalpha has been shown to prevent inducer-mediated degradation(32, 33, 34, 35) . However, the function of IkappaBalpha C terminus remains undefined.

In this study we have examined the role of the IkappaBalpha C terminus in inducer-mediated degradation. NIH 3T3-derived cell lines were generated that express human wild type or mutant IkappaBalpha proteins under the control of a tetracycline responsive promoter(36) . We demonstrate that C-terminal deletions from aa 269 to 287 abolish inducer-mediated degradation by rendering IkappaBalpha constitutively unstable and diminish the association of IkappaB with p65. Stabilization of C-terminal IkappaBalpha mutants with proteasome inhibitors, suggests that in unstimulated cells, the C-terminal domain functions to protect IkappaBalpha from proteasome action.


MATERIALS AND METHODS

Plasmids

A cDNA encoding the hybrid transactivator tTA (36) , a fusion protein between the tetracycline repressor and the HSV VP16 transactivator, was inserted between the HindIII and BamHI sites in the expression plasmid pREP4 (Invitrogen). In the resulting plasmid (pPREP-4-tTA), tTA transcription is controlled by the Rous sarcoma virus promoter, and tTA activity is inhibited by tetracycline(36) , at concentrations ranging from 0.1 to 1.0 µg/ml. Wild type IkappaBalpha cDNA was cloned downstream of the tetracycline responsive promoter CMV(t)(36) and inserted into the pREP9 expression plasmid between the XhoI and BamHI sites, therefore replacing the existing Rous sarcoma virus promoter. Mutated IkappaBalpha expression vectors were created by replacing wild type IkappaBalpha in pREP-9-IkappaBalpha(wt) plasmid with deleted or mutated versions. Briefly, the C-terminal deletion mutants IkappaBalpha(Delta1), IkappaBalpha(Delta2), IkappaBalpha(Delta3), and IkappaBalpha(Delta4) were generated by inserting an artificial stop codon in the human IkappaBalpha gene at amino acid positions 261, 269, 288, and 296, respectively. Cassettes encoding IkappaBalpha mutants were also inserted in the mammalian expression vector pSVK3 (Pharmacia)(37) . IkappaBalpha(DM) is a full-length human IkappaBalpha in which serine 283 and threonine 291 were substituted for alanine residues; in IkappaBalpha(3C), serine 283, threonine 291, and threonine 299 were also substituted for an alanine residue.

Generation of tTA and IkappaBalpha-expressing Cell Lines

Plasmid pREP4-tTA was introduced in NIH 3T3 cells by lipofection (Lipofectamine, Life Technologies, Inc.) according to the manufacturer's instructions. Cells were selected beginning at 48 h in Dulbecco's modified Eagle's medium containing 10% calf serum and 300 µg/ml hygromycin B (Boeringer Mannheim). Resistant cells carrying the pREP4-tTA plasmid (tTA-3T3 cells) were then transfected with the various pREP9-CMV(t)-IkappaBalpha plasmids. Cells were selected and maintained in Dulbecco's modified Eagle's medium containing 10% calf serum, 300 µg/ml hygromycin B, and 400 µg/ml G418 (Life Technologies, Inc.). Colonies of resistant cells (tTA-IkappaBalpha) were expanded individually or pooled together to create polyclonal population. At all times, cell lines were maintained in the presence of tetracyline (1 µg/ml) to repress exogenous IkappaBalpha expression.

Expression and Phosphorylation of Recombinant IkappaBalpha

GST-IkappaBalpha fusion proteins from Escherichia coli were isolated as described previously(37, 38) . Phosphorylation of IkappaBalpha recombinant proteins (2 ng) by CKII was performed for 30 min at 30 °C with 5 units of recombinant CKII enzyme (New England Biolabs) in a buffer containing 25 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM MnCl(2), 1 mM MgCl(2), and 10 mM ATP.

Electromobility Shift Assay

Nuclear extracts (39) were prepared from tTA-3T3 cells treated 30 min with TNF-alpha (0.5 ng/ml) and subjected to electromobility shift assay using an interferon-beta PRDII probe as described previously(40) . To demonstrate the specificity of the protein-DNA complex formation, 200-fold molar excess of either a mutated PRDII oligonucleotide (Mut) or wild type PRDII oligonucleotide (Wt) was added to the nuclear extract before addition of the PRDII probe. To evaluate the DNA binding inhibitory activity of IkappaBalpha or the various mutants, 2 ng of recombinant IkappaBalpha or mutant IkappaBalpha was added to the extract 10 min before the probe was added. Similarly, CKII phosphorylated recombinant IkappaBalpha proteins (2 ng) were tested for their ability to inhibit NF-kappaB DNA binding activity.

Inhibition of NF-kappaB-dependent Transcription

Using the calcium phosphate method(41) , N-Tera-2 cells were co-transfected with pHIV-CAT reporter plasmid (3 µg) along with CMV-p65 plasmid (3 µg) and various pSVK3 plasmids (Pharmacia) encoding wild type or mutated IkappaBalpha (9 µg). In the pHIV-CAT plasmid, the chloramphenicol acetyltransferase gene is under the control of a minimal SV40 promoter fused to one copy of the HIV-1 enhancer. CMV-p65 is a CMV promoter-driven expression plasmid encoding NF-kappaBp65. Thirty hours after transfection, cells were stimulated with 25 ng/ml phorbol 12-myristate 13-acetate (Sigma). Forty-eight hours after transfection, cells were harvested and lysed. Protein extracts (100 µg) were then assayed during 4 h at 37 °C for CAT activity.

Immunoblot Analysis of IkappaBalpha Turnover

tTA-IkappaBalpha expressing cells cultured in tetracycline-free Dulbecco's modified Eagle's medium supplemented with 10% calf serum, were stimulated with 5 ng/ml TNF-alpha (Life Technologies, Inc.) or 10 µg/ml LPS (Sigma), either with or without addition of 50 µg/ml cycloheximide. In some experiments, cells were pretreated for 1 h with either 100 µM calpain inhibitor I (ICN), 40 µM MG132 proteasome inhibitor (kindly supplied by MyoGenics Inc.)(31) , or an equivalent volume (8 µl/ml) of their respective solvent (ethanol and dimethyl formamide, respectively) as a control. Cells were washed with phosphate-buffered saline and lysed in 10 mM TrisbulletCl, pH 8.0, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin (WBL buffer). Equivalent amounts of protein (15 µg) were electrophoresed on a 10% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose and IkappaBalpha was detected using affinity purified IkappaBalpha antibody AR20(42) .

Immunoprecipitation of p65 and IkappaBalpha

Cells were washed with phosphate-buffered saline and labeled for 60 min in methionine-free RPMI 1640 containing 400 µCi/ml TranS-label (Amersham). Cells were collected in 40 mM TrisbulletCl, pH 7.5, 1 mM EDTA, pH 8.0, 150 mM NaCl, 2 mM phenylmethylsulfonyl fluoride (TEN buffer) and lysed in 20 mM TrisbulletCl, pH 7.5, 200 mM NaCl, 0.5% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride (TNT buffer). Cell lysates (300 µg) were incubated with 10 µl of IkappaBalpha antibody (AR20) or p65 antibody (AR28) (43) and 30 µl of protein A-Sepharose beads (Pharmacia) for 3 h at 4 °C. Beads were washed five times with TNT buffer and the immunoprecipitates were eluted by boiling the beads 3 min in 1% SDS, 0.5% beta-mercaptoethanol. The eluate was diluted with 1 volume of TNT buffer and incubated overnight at 4 °C with 10 µl of IkappaBalpha or p65 antibody. Beads were again washed five times with TNT buffer and the immunoprecipitate was eluted by boiling the beads 3 min in SDS sample buffer. Eluted proteins were electrophoresed on 10% SDS-polyacrylamide gel electrophoresis and detected by autoradiography. The specificity of IkappaBalpha antibody recognition was confirmed by competition with IkappaBalpha peptide (aa 2-16).


RESULTS

Inhibition of NF-kappaBbulletDNA Complex Formation

To investigate the role of the IkappaBalpha C terminus in NF-kappaB/IkappaBalpha regulation, we generated a series of C-terminal deletions of IkappaBalpha (Fig. 1). The IkappaBalpha proteins were generated by inserting an artificial stop codon in the human IkappaBalpha gene at aa 261(Delta1), aa 269(Delta2), aa 288(Delta3), and aa 296(Delta4), respectively. IkappaBalpha(DM) represents full-length human IkappaBalpha in which the Ser-283 and Thr-291 were substituted for alanine residues and IkappaBalpha(3C) contains the S283A, T291A, and T299A substitutions (Fig. 1). Wild type and C-terminal deletions of IkappaBalpha were examined for their ability to inhibit NF-kappaBbulletDNA complex formation in an electromobility shift assay. Extracts from tTA-3T3 cells stimulated with TNF-alpha for 30 min were analyzed for NF-kappaB binding activity using a P-labeled probe corresponding to the PRDII region of the interferon beta promoter (Fig. 2). Addition of recombinant wt IkappaBalpha, IkappaBalpha(Delta4), IkappaBalpha(Delta3), IkappaBalpha(DM), and IkappaBalpha(3C) proteins reduced the intensity of the NF-kappaB/PRDII band more than 10-fold (Fig. 2, lanes 2-4, 7, and 8), whereas addition of IkappaBalpha(Delta1) or IkappaBalpha(Delta2) had no affect on NF-kappaBbulletDNA complex formation (Fig. 2, lanes 5 and 6). This result demonstrated that the region located between aa 269 and 287 was important for inhibiting NF-kappaBbulletDNA complex formation in vitro.


Figure 1: Schematic representation of human IkappaBalpha and C-terminal deletion mutants. Human IkappaBalpha contains five internal ankyrin repeats (SWI6/ANK) involved in the binding to NF-kappaB molecules. At the N-terminal of IkappaBalpha are two phosphorylation sites (Ser-32 and Ser-36), shown previously to play a role in inducer-mediated degradation(32, 33) . A region rich in proline, serine, threonine, and glutamic acid, the PEST domain, spans aa 264-317; the C-terminal region of IkappaBalpha between aa 251 and 317 is expanded below the schematic to show the one-letter amino acid sequence. The C-terminal ends of the deletions IkappaBalpha(Delta1) (aa 1-260), IkappaBalpha(Delta2) (aa 1-268), IkappaBalpha(Delta3) (aa 1-287), and IkappaBalpha(Delta4) (aa 1-295) are depicted. In mutant IkappaBalpha(DM), Ser-283 and Thr-291 were substituted for alanines and in IkappaBalpha(3C), Thr-299 was also substituted for alanine. The C-terminal region involved in degradation (aa 279-287) is indicated in bold letters. The boundary aa 279 was determined in (35) ; the boundary aa 287 was determined in this study.




Figure 2: Dissociation of NF-kappaBbulletDNA complexes by recombinant IkappaBalpha. Nuclear extracts from tTA-3T3 cells (5 µg) stimulated for 30 min with TNF-alpha were incubated with P-labeled probe (0.2 ng) corresponding to the interferon-beta PRDII region(55) . The NF-kappaBbulletDNA complex was visualized on native 5% polyacrylamide gel (lane 1). The specificity of the complex formation was tested by adding a 200-fold molar excess of unlabeled wild type or mutated PRDII double stranded DNA to the reaction, prior to labeled probe addition (data not shown, see ``Materials and Methods''). Recombinant wt IkappaBalpha (lanes 2 and 9), IkappaBalpha(Delta4) (lanes 3 and 10), IkappaBalpha(Delta3) (lanes 4 and 11), IkappaBalpha(Delta2) (lanes 5 and 12), IkappaBalpha(Delta1) (lanes 6 and 13), IkappaBalpha(DM) (lanes 7 and 14), or IkappaBalpha(3C) (lanes 8 and 15) were added to the extracts prior to probe addition. The recombinant IkappaBalpha proteins were either untreated (lanes 2-8) or phosphorylated in vitro with recombinant casein kinase II prior to addition to the electromobility shift assay reactions (lanes 9-15).



In vivo, IkappaBalpha is constitutively phosphorylated at the C terminus by casein kinase II(37, 43) . Several previous reports demonstrated that the phosphorylation level of IkappaBalpha influenced the ability of IkappaBalpha to dissociate NF-kappaBbulletDNA complexes(15, 44, 45) . However, in vitro phosphorylation of wild type or mutant IkappaBalpha proteins with casein kinase II (Fig. 2, lanes 9-15) did not modulate the capacity of IkappaBalpha to inhibit NF-kappaBbulletPRDII DNA complex formation in vitro.

Inhibition of NF-kappaB-dependent Gene Expression

Next, the effect of IkappaBalpha C-terminal truncations on the inhibition of NF-kappaB dependent gene expression was examined. NF-kappaB activity was detected by measuring CAT activity derived from pHIV-CAT reporter plasmid in N-Tera-2 cells which are deficient in NF-kappaB activity(46, 47) . The level of NF-kappaB was increased in these cells by co-expressing RelA and treating with phorbol 12-myristate 13-acetate(33) . CAT activity was observed only when p65 was co-expressed and this activity was inhibited by excess wild type IkappaBalpha expression (Fig. 3). IkappaBalpha(Delta4) and IkappaBalpha(Delta3) expression also repressed NF-kappaB dependent CAT activity, whereas IkappaBalpha(Delta1) and IkappaBalpha(Delta2) expression did not reduce NF-kappaB dependent activity (Fig. 3). These results demonstrated that IkappaBalpha(Delta1) and IkappaBalpha(Delta2) were unable to suppress p65 dependent transcription, which correlates with their reduced ability to dissociate NF-kappaB complexes in vitro.


Figure 3: IkappaBalpha mediated repression of NF-kappaB dependent transcription. N-Tera-2 cells were co-transfected with pHIV CAT reporter plasmid (3 µg) along with the NF-kappaB p65 expression plasmid CMV-p65 (3 µg) and various SVK3-based plasmids expressing wild type or mutant IkappaBalpha (9 µg) as indicated. At 30 h after transfection, cells were stimulated with phorbol 12-myristate 13-acetate and CAT activity was analyzed at 48 h.



Tetracycline Control of IkappaBalpha Expression

To analyze IkappaBalpha deletions in stably transformed cells, IkappaBalpha expressing NIH-3T3 cells were generated using the tetracycline responsive system (see ``Materials and Methods''). Polyclonal tTA-IkappaBalpha cells, cultured in the absence of tetracycline for 72 h were examined for exogenous IkappaBalpha expression (Fig. 4A); IkappaBalpha(wt), IkappaBalpha(DM), IkappaBalpha(Delta1), IkappaBalpha(Delta2), IkappaBalpha(Delta3), and IkappaBalpha(Delta4) proteins were detected with apparent molecular masses of 38, 38, 31, 32, 35, and 36 kDa, respectively (Fig. 4A, lanes 2, 4, 6, 8, 10, and 12). Wild type human IkappaBalpha was distinguished from the 37-kDa murine homologue, due to a slight molecular weight difference (Fig. 4A, lanes 1 and 2). The human IkappaBalpha proteins were expressed in polyclonal populations at levels ranging from equivalent to the endogenous murine IkappaBalpha (IkappaBalphaDelta4) to levels 20-50-fold higher than the endogenous IkappaBalpha (IkappaBalphaDelta1). Exogenous IkappaBalpha levels were reduced when cells were cultured in the presence of tetracycline (1 µg/ml) for more than 24 h (Fig. 4A, lanes 1, 3, 5, 7, 9, and 11), although the degree of repression varied between cell lines. A representative analysis of selected individual clones of IkappaBalpha(wt) and IkappaBalphaDelta1 clones is shown in Fig. 4B.


Figure 4: Tetracycline-responsive expression of human IkappaBalpha in NIH 3T3 cells. A, human IkappaBalpha was detected by immunoblotting in extracts from polyclonal tTA-IkappaBalpha(wt) (lanes 1 and 2), tTA-IkappaBalpha(DM) (lanes 3 and 4), tTA-IkappaBalpha(Delta1) (lanes 5 and 6), tTA-IkappaBalpha(Delta2) (lanes 7 and 8), tTA-IkappaBalpha(Delta3) (lanes 9 and 10), tTA-IkappaBalpha(Delta4) (lanes 11 and 12) cells. tTA-IkappaBalpha cells were cultured in the presence (lanes 1, 3, 5, 7, 9, and 11) or absence (lanes 2, 4, 6, 8, 10, and 12) of tetracycline (1 µg/ml). Arrows indicate bands corresponding to endogenous murine IkappaBalpha and exogenous human IkappaBalpha. B, individual clones of tTA-IkappaBalpha(Delta1) (lanes 1-6) and tTA-IkappaBalpha(wt) (lanes 7-10) were grown in the presence (lanes 1, 3, 5, 7, and 9) or absence (lanes 2, 4, 6, 8, and 10) of tetracycline (1 µg/ml). Bands corresponding to wt IkappaBalpha and IkappaBalpha(Delta1) are indicated.



Association of IkappaBalpha with NF-kappaB p65 in Vivo

To determine if IkappaBalpha mutants could also associate with p65 in vivo, co-immunoprecipitation studies were performed with anti-p65 and anti-IkappaBalpha antibodies (Fig. 5). In tTA-3T3 cells, endogenous IkappaBalpha co-precipitated with anti-p65 antibody (Fig. 5, lane 2) and reciprocally, p65 co-precipitated with anti-IkappaBalpha antibody (Fig. 5, lane 4). IkappaBalpha peptide present in excess during IkappaBalpha immunoprecipitation prevented subsequent p65 immunoprecipitation, demonstrating the specificity of antibody recognition (Fig. 5, lane 5). In tTA-IkappaBalpha(wt) cells, human IkappaBalpha was detected following two sequential IkappaBalpha immunoprecipitations, and migrated just above the murine IkappaBalpha band (Fig. 5, lane 8), whereas in tTA-IkappaBalpha(Delta1) cells, human IkappaBalpha(Delta1) migrated below murine IkappaBalpha (Fig. 5, lane 13). In both cell lines, murine and human IkappaBalpha were co-immunoprecipitated with anti-p65 antibody (Fig. 5, lanes 7 and 12) and p65 was co-immunoprecipitated by anti-IkappaBalpha (Fig. 5, lanes 9 and 14). The reaction was specific since co-immunoprecipitation was abolished by the addition of excess IkappaBalpha peptide (Fig. 5, lanes 10 and 15). As with wt IkappaBalpha, the majority of IkappaBalpha(Delta3) and IkappaBalpha(Delta4) present in tTA-IkappaBalpha cells was associated with p65 (Table 1). However, the amount of IkappaBalpha(Delta1) complexed to p65 was significantly reduced compared with the total amount of IkappaBalpha(Delta1) present in the cell (Fig. 5, lanes 12 and 13). Similarly, only a small fraction of IkappaBalpha(Delta2) was associated with p65 (Table 1). These results indicated that wt IkappaBalpha, IkappaBalpha(Delta4), -(Delta3), and -(DM) (S283A,T291A) were stably associated with p65 in vivo, whereas the interaction of IkappaBalpha(Delta1) and IkappaBalpha(Delta2) with p65 was unstable in vivo.


Figure 5: In vivo association of IkappaBalpha with NF-kappaB p65. Polyclonal tTA-3T3 (lanes 1-5), tTA-IkappaBalpha(wt) (lanes 6-10), and tTA-IkappaBalpha(Delta1) (lanes 11-15) were metabolically labeled with [S]methionine for 1 h. Cell lysates were immunoprecipitated with p65 specific antibody (lanes 1, 2, 6, 7, 11, and 12) or IkappaBalpha specific antibody (lanes 3, 4, 5, 8-10, and 13-15). IkappaBalpha antibody recognition was competed by the addition of excess IkappaBalpha peptide (2 µg) to the reaction (lanes 5, 10, and 15). Immunoprecipitates were collected on protein A-Sepharose beads, washed stringently, and boiled in 1% SDS, 0.5% beta-mercaptoethanol. Supernatants were collected and immunoprecipitated again with p65 antibody (lanes 1, 4-6, 9-11, 14, and 15) or with IkappaBalpha antibody (lanes 2, 3, 7, 8, 12, and 13). Bands corresponding to p65, murine IkappaBalpha, human IkappaBalpha(wt), and IkappaBalpha(Delta1) are indicated.





Inducer-mediated Degradation of IkappaBalpha

To examine the effect of C-terminal deletion on inducer-mediated degradation of IkappaBalpha, tTA-IkappaBalpha cells were treated with TNF-alpha (5 ng/ml) or LPS (10 µg/ml) and cell extracts were analyzed for IkappaBalpha expression by immunoblotting ( Fig. 6and Table 1). Stimulation with TNF-alpha or LPS resulted in a rapid decrease and disappearance of murine and human IkappaBalpha from 15 to 60 min after stimulation, followed by de novo synthesis of IkappaBalpha (Fig. 6A and Table 1), as described previously(1, 21, 24, 25) . Inducer-mediated degradation of endogenous IkappaBalpha was observed in all tTA-IkappaBalpha expressing cells (Fig. 6, B-F). In response to either TNF-alpha or LPS stimulation, IkappaBalpha(Delta4), IkappaBalpha(Delta3), and IkappaBalpha(DM) degraded rapidly within 15-30 min (Fig. 6, B, C, F, and Table 1), whereas IkappaBalpha(Delta1) and IkappaBalpha(Delta2) did not undergo inducer-mediated degradation (Fig. 6, D and E, and Table 1). To eliminate effects associated with overexpression of exogenous IkappaBalpha, tTA-IkappaBalpha(Delta1) cells were cultured in the presence of tetracycline (0.1 µg/ml) which reduced IkappaBalpha(Delta1) expression to levels equivalent to endogenous murine IkappaBalpha. This experiment indicates that the region deleted in IkappaBalpha(Delta2) but present in IkappaBalpha(Delta3), aa 269-287, apparently plays a role in TNF-alpha and LPS mediated degradation.


Figure 6: Inducer-mediated degradation of IkappaBalpha. Polyclonal tTA-IkappaBalpha(wt) (A), tTA-IkappaBalpha(Delta4) (B), tTA-IkappaBalpha(Delta3) (C), tTA-IkappaBalpha(Delta2) (D), tTA-IkappaBalpha(Delta1) (E), and tTA-IkappaBalpha(DM) (F) cells were stimulated with TNF-alpha for 0 (lane 1), 15 (lane 2), 30 (lane 3), 60 (lane 4), 90 (lane 5), or 120 min (lane 6). Prior to stimulation, tTA-IkappaBalpha(Delta1) cells were cultured in the presence of tetracycline (0.1 µg/ml) to reduce the level of exogenous human IkappaBalpha. Endogenous murine and exogenous human IkappaBalpha were detected in whole cell extracts (15 µg) by immunoblotting using affinity purified AR20 antibody.



Intrinsic IkappaBalpha Stability

Since inducer-mediated degradation of IkappaBalpha does not require de novo protein synthesis(21, 48) , the turnover rate of IkappaBalpha in the absence (intrinsic stability) or presence of stimulus (inducer mediated degradation rate) was measured in cells treated with the protein synthesis inhibitor cycloheximide (50 µg/ml). tTA-3T3 and tTA-IkappaBalpha cells were stimulated with TNF-alpha for 2 h and levels of IkappaBalpha were measured and quantified (Fig. 7). The intrinsic stability of human wt IkappaBalpha was similar to that of murine IkappaBalpha (Fig. 7, A and B, lanes 1-6); without inducer, both proteins had a half-life of approximately 2 h (summarized in Table 1). IkappaBalpha(Delta3), IkappaBalpha(Delta4), and IkappaBalpha(DM) also had intrinsic stabilities similar to wt IkappaBalpha (Fig. 7, C and D, lanes 1-6; Table 1). However, the C-terminal deletion in IkappaBalpha(Delta1) (Fig. 7E, lanes 1-6) and IkappaBalpha(Delta2) (Table 1) destabilized IkappaBalpha by reducing IkappaBalpha half-life to approximately 30 min. Following TNF-alpha stimulation in the presence of cycloheximide, the half-life of wt IkappaBalpha, IkappaBalpha(DM), IkappaBalpha(Delta4), IkappaBalpha(Delta3), and murine IkappaBalpha was decreased to about 5 min (Fig. 7, A-D, lanes 7-12). In contrast, the degradation rate of IkappaBalpha(Delta2) and IkappaBalpha(Delta1) in the presence of TNF-alpha was similar to their respective intrinsic stabilities (Fig. 7E, lanes 7-12; Table 1). These experiments demonstrate that deletion of the amino acid domain 269-287 desensitized IkappaBalpha to TNF-alpha-mediated degradation and simultaneously accelerated IkappaBalpha turnover in unstimulated cells. Similar results were obtained when LPS was used as inducer (Table 1).


Figure 7: Analysis of IkappaBalpha turnover rate. Polyclonal tTA-3T3 (A), tTA-IkappaBalpha(wt) (B), tTA-IkappaBalpha(Delta4) (C), tTA-IkappaBalpha(Delta3) (D), and tTA-IkappaBalpha(Delta1) (E) cells were treated with cycloheximide (50 µg/ml) alone (lanes 1-6) or stimulated with TNF-alpha (5 ng/ml) in the presence of cycloheximide (lanes 7-12) for 0 (lanes 1 and 7), 15 (lanes 2 and 8), 30 (lanes 3 and 9), 60 (lanes 4 and 10), 90 (lanes 5 and 11), and 120 min (lanes 6 and 12). Endogenous murine and exogenous human IkappaBalpha were detected in whole cell extracts (15 µg) by immunoblotting, using affinity purified AR20 antibody.



Protection of IkappaBalpha Mutants by Protease Inhibitors

To determine if the reduced half-life of IkappaBalpha(Delta1) and IkappaBalpha(Delta2) mutants was related to inducer-mediated degradation and the proteasome pathway, tTA-IkappaBalpha(wt), tTA-IkappaBalpha(Delta1), and tTA-IkappaBalpha(Delta2) cells were pretreated for 1 h with calpain inhibitor I or the MG132 proteasome inhibitor, both of which are known to block inducer-mediated degradation of IkappaBalpha (Fig. 8). Cells were then treated with cycloheximide for 1 h and the level of IkappaBalpha remaining was determined by immunoblot and densitometric analysis. In the presence of cycloheximide, the amount of wt IkappaBalpha remaining at 60 min was approximately 65-70% of the level at time 0; pretreatment of wt IkappaBalpha expressing cells with protease inhibitors did not significantly increase the amount of remaining wt IkappaBalpha (60-80%). In contrast, after cycloheximide treatment for 1 h, the level of IkappaBalpha in IkappaBalpha(Delta1) and IkappaBalpha(Delta2) expressing cells was reduced 5-20% of the initial level, reflecting the rapid degradation of the Delta1 and Delta2 deletion mutants. However, in the presence of calpain inhibitor I or MG132, both IkappaBalpha(Delta1) and IkappaBalpha(Delta2) were dramatically stabilized and did not degrade significantly during the period of cycloheximide treatment. These results imply that IkappaBalpha(Delta1) and IkappaBalpha(Delta2) have a reduced half-life because they are constitutively degraded by proteases active during inducer-mediated degradation.


Figure 8: Stabilization of mutant IkappaBalpha by peptide aldehydes. tTA-IkappaBalpha(wt), tTA-IkappaBalpha(Delta1), and IkappaBalpha(Delta2) expressing cells were treated for 1 h with calpain inhibitor I (100 µM) or MG132 proteasome inhibitor (40 µM). Ethanol and dimethyl formamide, which are solvents for calpain inhibitor I and MG132, respectively, were added to the cells as controls. Cells were then treated with cycloheximide for 1 h. The percentage of exogenous IkappaBalpha remaining at the end of the 1-h cycloheximide treatment was determined by immunoblot analysis and compared to the amount of IkappaBalpha at time 0. The amount of wt IkappaBalpha (open bar), IkappaBalpha(Delta2) (solid bar), and IkappaBalpha(Delta1) (hatched bar) after a 1-h cycloheximide treatment is illustrated graphically.




DISCUSSION

In this study, we examined the biochemical and functional properties of C-terminal deletions in IkappaBalpha with respect to intrinsic protein stability, inducer-mediated degradation, dissociation of NF-kappaBbulletDNA complexes and association with p65 (RelA) in vitro and in vivo. Our results demonstrate that: 1) the C-terminal end of IkappaBalpha from aa 288 to 317 which includes most of the PEST domain is apparently dispensable for function since IkappaBalpha(Delta3) and IkappaBalpha(Delta4) behave like wt IkappaBalpha; 2) deletion of the region between aa 269 and 287 (IkappaBalphaDelta2) abolishes responsiveness to TNF-alpha and LPS-mediated degradation; 3) IkappaBalphaDelta1 and IkappaBalphaDelta2 mutants have a reduced intrinsic stability (t15-30 min) and are constitutively degraded by proteases that are inhibited by calpain inhibitor I and MG132; and 4) the domain between aa 269 and 287 is required for dissociation of NF-kappaBbulletDNA complexes in vitro, for strong interaction with p65 in vivo, and for efficient repression of NF-kappaB dependent transcription.

Recent studies by Brown et al.(33) demonstrated that residues Ser-32 or Ser-36 were required for TNF-alpha-mediated phosphorylation and degradation of IkappaBalpha, while Brockman et al.(32) further showed that S32A or S32A/S36A substitutions fully protected IkappaBalpha from HTLV-1 Tax mediated degradation. Furthermore, Chen et al.(13) demonstrated that S32A/S36A substitutions and to a lesser extent S32A substitution, prevented TNF-alpha induced ubiquitination of IkappaBalpha. These studies thus support a model in which IkappaBalpha is phosphorylated on residues Ser-32 and/or Ser-36 in response to multiple inducers and these phosphorylation events target IkappaBalpha for ubiquitination and subsequent degradation by the proteasome (13) .

Sequences within the C terminus of IkappaBalpha also play a role in inducer-mediated degradation(33, 34, 35) . Whiteside et al.(35) demonstrated that IkappaBalpha deletion from aa 279-317 abolished TNF-alpha and LPS-mediated degradation(35) . Our deletion studies also demonstrate that IkappaBalpha deleted from aa 269-317 (IkappaBalphaDelta2) had a similar phenotype, whereas IkappaBalpha deleted from aa 288-317 had a phenotype indistinguishable from wt IkappaBalpha, as measured in several functional assays. Therefore, based on these two studies, a C-terminal domain involved in IkappaBalpha degradation is located between aa 279 and 287: the sequence MLPESEDEE (outlined in bold in Fig. 1). This sequence contains one of the constitutive casein kinase II phosphorylation sites SEDE identified previously(37, 43, 51) , but mutation of the Ser-283 and Thr-291 sites did not affect IkappaBalpha activity(37) . Residues Glu-284, Asp-285, or Glu-286 were previously identified as being critical for the dissociation of NF-kappaBbulletDNA complexes(51) . Given the number of acidic amino acids in this short segment, it appears that the functional activity of this part of the C-terminal domain relates to its highly acidic nature. In fact, triple mutation of Ser-283, Thr-291, and Thr-299 increases the intrinsic stability of IkappaBalpha(37) .

Deletion of virtually the entire PEST domain in IkappaBalpha(Delta3) did not alter IkappaBalpha intrinsic stability or responsiveness to inducer-mediated degradation, since this mutant behaved like wt IkappaBalpha in several biochemical and functional assays (summarized in Table 1). However, deletion of the region adjacent to the PEST domain between aa 269 and 287 decreased IkappaBalpha stability from t

Based on our observations and the recent study of Sachdev et al.(53) demonstrating that C-terminal mutations in chicken pp40 decreased the interaction with p65(53) , we conclude that the region of IkappaBalpha from aa 269 to 287 may strengthen interaction with p65 in vivo. Biochemical characterization of the domain structure of IkappaBalpha demonstrated that IkappaBalpha contains a highly structured central domain that is resistant to proteolysis and flexible N- and C-terminal extensions that are sensitive to proteolytic digestion(54) . The C-terminal region was protected from proteolysis up to aa 275 when IkappaBalpha was bound to p65, suggesting that this region directly interfaced with p65 and was thus masked in the IkappaBalpha-p65 complex.

Inducer-mediated degradation is inhibited by peptidyl aldehydes such as MG132 and calpain inhibitor I(13, 28) . In this study, we show that these inhibitors also dramatically increased the intrinsic stability of mutant IkappaBalpha(Delta1) and (Delta2) but not wt IkappaBalpha. Chen et al.(13) showed that deletion of the C terminus did not prevent IkappaBalpha ubiquitination since the Delta243-317 mutant could still be ubiquitinated in vitro. We therefore propose that in unstimulated cells, the C terminus protects IkappaBalpha from constitutive proteasome-mediated degradation via p65 interaction. Upon stimulation, phosphorylation at the N terminus abolishes protection by the C terminus and targets IkappaBalpha for ubiquitination and degradation.

In view of the predominantly cytoplasmic localization of IkappaBalpha, the biological significance of NF-kappaBbulletDNA complex dissociation by IkappaBalpha is not yet understood, although IkappaBalpha has previously been identified in the nucleus(49) . Furthermore, in vitro transcription studies using purified NF-kappaB proteins demonstrated that addition of recombinant IkappaBalpha to the transcription reactions inhibited NF-kappaB dependent transcription(17, 38) . These experiments suggest that a novel nuclear role for newly synthesized IkappaBalpha may be to directly inhibit NF-kappaB dependent gene expression by dissociating NF-kappaBbulletDNA transcription complexes. This idea is supported by the recent observation that following induction de novo synthesized IkappaBalpha protein transiently appeared in the nucleus and negatively regulated NF-kappaB dependent transcription(50) .

The kinase activity responsible for inducer-mediated phosphorylation of Ser-32 and/or Ser-36 in the N-terminal signal response domain has not been identified. In light of the identification of casein kinase II as the activity responsible for constitutive phosphorylation at the C-terminal end of IkappaBalpha(37, 43) , it is a possibility that CKII also phosphorylates at positions Ser-32 and/or Ser-36 which are consensus CKII sites. However, at present no in vivo evidence for CKII phosphorylation within the signal response domain has been obtained. The resolution of the signaling events involved in IkappaBalpha regulation of NF-kappaB activity will require further characterization of the kinase activity involved in signal induced phosphorylation of IkappaBalpha.


FOOTNOTES

*
This work was supported in part by the Medical Research Council of Canada, the National Cancer Institute of Canada, and the Cancer Research Society, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Studentship from the Medical Research Council of Canada.

Recipient of a Fellowship from the Medical Research Council of Canada.

**
Recipient of a Scientist Award from the Medical Research Council of Canada. To whom correspondence should be addressed: Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec, H3T 1E2 Canada. Tel.: 514-340-8260 (ext. 5265); Fax: 514-340-7576.

(^1)
The abbreviations used are: HIV-1, human immunodeficiency virus-1; TNF-alpha, tumor necrosis factor-alpha; LPS, lipopolysaccharide; CAT, chloramphenicol acetyltransferase; aa, amino acid(s); wt, wild type.


ACKNOWLEDGEMENTS

We thank Drs. M. Gossen and H. Bujard for the tetracycline responsive plasmids and helpful information about selection of cells, as well as Dr. A. Cochrane for helpful discussions. We are grateful to MyoGenics Inc. for MG132. We also thank Normand Pepin for excellent technical assistance.


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