©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of IB Ubiquitination in Signal-induced Activation of NF-B in Vivo(*)

(Received for publication, October 5, 1995; and in revised form, January 11, 1996)

Marilynn Roff (1) Jill Thompson (1)(§) Manuel S. Rodriguez (2)(¶) Jean-Marc Jacque (2) Francoise Baleux (3) Fernando Arenzana-Seisdedos (2) Ronald T. Hay (1)(**)

From the  (1)School of Biological and Medical Sciences, University of St. Andrews, Fife KY16 9AL, Scotland and the (2)Unité d'Immunologie Virale and (3)Unité de Chimie Organique, Institut Pasteur, 28 Rue du Dr. Roux, Paris 75015, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In unstimulated cells, the transcription factor NF-kappaB is held in the cytoplasm in an inactive state by the inhibitor protein IkappaBalpha. Stimulation of cells results in rapid phosphorylation and degradation of IkappaBalpha, thus releasing NF-kappaB, which translocates to the nucleus and activates transcription of responsive genes. Here we demonstrate that in cells where proteasomal degradation is inhibited, signal induction by tumor necrosis factor alpha results in the rapid accumulation of higher molecular weight forms of IkappaBalpha that dissociate from NF-kappaB and are consistent with ubiquitin conjugation. Removal of the high molecular weight forms of IkappaBalpha by a recombinant ubiquitin carboxyl-terminal hydrolase and reactivity of the immunopurified material with a monoclonal antibody specific for ubiquitin indicated that IkappaBalpha was conjugated to multiple copies of ubiquitin. Western blot analysis of immunopurified IkappaBalpha from cells expressing epitope-tagged versions of IkappaBalpha and ubiquitin revealed the presence of multiple copies of covalently bound tagged ubiquitin. An S32A/S36A mutant of IkappaBalpha that is neither phosphorylated nor degraded in response to signal induction fails to undergo inducible ubiquitination in vivo. Thus signal-induced activation of NF-kappaB involves phosphorylation-dependent ubiquitination of IkappaBalpha, which targets the protein for rapid degradation by the proteasome and releases NF-kappaB for translocation to the nucleus.


INTRODUCTION

The NF-kappaB/rel family of transcription factors are involved in the activation of a wide variety of genes, including the HIV-1 provirus, that respond to immune and inflammatory signals (for review, see (1) and (2) ). In humans, the family of proteins consists of p50 (3, 4) , p52(5, 6, 7) , p65(8, 9) , c-Rel(10) , and RelB(11) . While almost all combinations of homo- and heterodimer can exist, the typical form of NF-kappaB that is activated in response to extracellular signals is a heterodimer of p50 and p65. Disruption of genes coding for the p50 or RelB components of NF-kappaB complexes results in transgenic animals with defects in immune and inflammatory responses(12, 13) . NF-kappaB proteins share a highly conserved region known as the rel homology domain, which is responsible for DNA binding, dimerization, and nuclear localization. DNA is recognized by NF-kappaB in an unusual way involving base and backbone contacts with the DNA over one complete helical turn(14, 15) . Structural analysis of p50 homodimers bound to DNA reveals that the protein recognizes DNA by an extended network of loops that arise from noncontiguous regions of the protein(16, 17) . Although p50 does not possess a transcriptional activation domain, its p65 partner does have an acidic activation domain that accounts for the transcriptional activity of the NF-kappaB heterodimer(18, 19) .

p50 represents the amino-terminal region of a p105 precursor from which it is processed, by a pathway thought to involve ubiquitination of the protein(20) . The carboxyl-terminal region of p105 contains multiple repeats of a 30-35-amino acid sequence present in the erythrocyte protein ankyrin(21) . In lymphoid cells the carboxyl-terminal region of p105 has been identified as an independent entity known as IkappaB (22, 23) that preferentially inhibits the DNA binding activity of p50 homodimers. In p105, the carboxyl-terminal region is thought to function as a cis-acting inhibitor of DNA binding activity (24) . NF-kappaB activity is regulated by its association with the inhibitor protein IkappaBalpha or MAD3(25, 26) , which like the carboxyl-terminal region of p105, the proto-oncogene bcl-3 (27) , and the recently described IkappaBbeta (28) contains multiple ankyrin repeats. How IkappaB proteins inhibit both nuclear translocation and DNA binding of NF-kappaB proteins has not been established, but the nuclear localization signals of p50 and p65 are occluded by bound IkappaB and IkappaBalpha, respectively(29, 24, 30) . Mutational analysis of IkappaBalpha, IkappaB, and pp40 has demonstrated that both the ankyrin repeats and carboxyl-terminal acidic domains are required for interaction with the corresponding NF-kappaB proteins(31, 32, 33) . IkappaBalpha displays a tripartite organization with a central domain containing five ankyrin repeats, an unstructured amino-terminal extension, and a small highly acidic carboxyl-terminal domain connected to the core of the protein by a protease-sensitive linker that is occluded by bound p65(34) .

In unstimulated cells, NF-kappaB is held in the cytoplasm by IkappaBalpha, but signal induction releases NF-kappaB, which translocates to the nucleus and activates responsive genes. Following signal induction, IkappaBalpha is rapidly phosphorylated and degraded(35, 36, 37, 38, 39, 40, 41) . Mutational analysis has indicated that residues Ser-32 and Ser-36 are the likely sites of inducible phosphorylation(36, 42, 43) , which targets the protein for degradation but does not disrupt complexes of NF-kappaB and IkappaBalpha(44, 45, 46, 47, 48, 49, 50) . Inhibition of protein degradation via the 26 S proteasome results in accumulation of the hyperphosphorylated form of IkappaBalpha and a failure to activate NF-kappaB, indicating that IkappaBalpha proteolysis is a necessary step in NF-kappaB activation(20, 50) . Degradation of IkappaBalpha is rapidly followed by induction of IkappaBalpha mRNA in a mechanism that is regulated by interaction of NF-kappaB with the promoter of the IkappaBalpha gene(51, 52, 41) . Resynthesized IkappaBalpha protein appears transiently in the nucleus where it negatively regulates NF-kappaB dependent transcription(53) .

To determine the pathway of signal-induced degradation of IkappaBalpha, we have made use of a peptide aldehyde inhibitor that blocks the proteolytic activity of the proteasome(54) . In the presence of this inhibitor, signal induction results in the accumulation of phosphorylated and ubiquitinated forms of IkappaBalpha and a failure to activate NF-kappaB. Although the phosphorylated forms of IkappaBalpha remain bound to NF-kappaB, the multiply ubiquitinated forms of IkappaBalpha were not associated with the transcription factor. It is likely that prior phosphorylation is required for signal-induced ubiquitination as an S32A/S36A IkappaBalpha mutant is neither phosphorylated, degraded, nor ubiquitinated. Our observations emphasize the importance of signal-induced protein degradation in activation of the NF-kappaB transcription factor and demonstrate a crucial role for the ubiquitin-proteasome pathway in this process.


EXPERIMENTAL PROCEDURES

Materials

Z-LLL-H was synthesized as described previously (55) , isolated by reverse-phase high performance liquid chromatography (>95% purity) and the structure confirmed by NMR spectroscopy. Human recombinant TNFalpha (^1)was provided by the MRC ADP reagent program. Okadoic acid was purchased from Sigma.

Plasmids

The plasmid expressing HA-tagged ubiquitin under control of the cytomegalovirus immediate early promoter (56) was obtained from D. Bohmann. The plasmid expressing IkappaBctag also under control of the cytomegalovirus immediate promoter was as described previously(57) . Plasmid DNA was prepared from saturated cultures of Escherichia coli using Qiagen columns as described by the manufacturer. A plasmid containing the gene for a Drosophila ubiquitin carboxyl-terminal hydrolase fused to the glutathione S-transferase gene (58) was obtained from M. Bownes.

Cell Culture and Transfections

HeLa S3 cells were grown in suspension in minimal essential medium without calcium, containing 5% calf serum and supplemented with penicillin and streptomycin (culture medium). COS-7 cells were grown in Dulbecco's modified Eagle medium containing 5% fetal calf serum and passaged every 3 days. A total of 60 µg of plasmid DNA was transfected for 16 h in subconfluent COS-7 cells seeded in 150 cm^2 flasks using Lipofectamine according to instructions provided by the manufacturer (Life Technologies, Inc.). The transfection mix contained 48 µg of HA-ubi made up to a total of 60 µg with either empty vector (pCDNA1, Invitrogen) or IkappaBctag. 16 h after transfection, cells were trypsinized and aliquots were seeded in 9-cm plates and cultured for an additional 24 h. Cells were treated with Z-LLL-H or TNFalpha and Z-LLL-H or were untreated, and extracts were prepared as described below. The expression of both wild-type and S32A/S36A IkappaBctag proteins was stabilized in HeLa cells using neomycin selection with DNA vectors driven by an immediate early cytomegalovirus promoter (pCDNAIII, InVitrogen) or an Rous sarcoma virus promoter (RcRSV, InVitrogen), respectively. Single cell clones were obtained by limiting dilution of the neomycin resistant cells and selected on the basis of tagged protein expression.

Preparation of Cytoplasmic and Nuclear Extracts

HeLa cells were concentrated to 5 times 10^6 cells ml prior to treatment with the indicated concentrations of Z-LLL-H or the Me(2)SO vehicle for 45 min. Human recombinant TNFalpha was added to a final concentration of 10 ng ml, and incubation at 37 °C continued for the indicated time. Cells were collected by centrifugation, washed once with ice-cold phosphate-buffered saline (PBS), and cytoplasmic extracts were prepared by lysis in 20 mM sodium phosphate buffer, pH 7.5, 50 mM sodium fluoride, 5 mM tetrasodium pyrophosphate, 10 mM beta-glycerophosphate, 2 mM EDTA, 0.5% Nonidet P-40, protease inhibitors (1 µM leupeptin, 1 µM pepstatin, 1 mM pefablock, 20 µML-1-tosylamido-2-phenylethyl chloromethyl ketone, 40 µg ml bestatin), and 10 mM iodoacetamide. After centrifugation to remove nuclei, the iodoacetamide was quenched by the addition of dithiothreitol to 10 mM. To prepare nuclei, cells were lysed as above, but iodoacetamide was omitted and nuclei were extracted by resuspension in the above buffer containing 425 mM NaCl. After 30 min at 4 °C, the lysate was clarified by centrifugation at 14,000 rpm for 15 min at 4 °C in an Eppendorf microcentrifuge.

Gel Electrophoresis DNA Binding Assays

NF-kappaB DNA binding assays were performed as described previously(53) , and contained 8 µg of nuclear protein and a P-labeled double-stranded DNA representing the kappaB motif present in the human immunodeficiency virus type-1 enhancer. Free DNA was resolved from DNA-protein complexes on a native 6% polyacrylamide gel, and the positions of the radioactive species were determined by autoradiography of the dried gel.

Western Blot Analysis

Cytoplasmic extracts (40 µg) of protein were resolved in a 10% polyacrylamide gel containing SDS, transferred to polyvinylidine difluoride membranes (Sigma) by electroblotting, and processed for Western blotting as described previousloy(53) . Where indicated, membranes were stripped by incubation in 50 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM beta-mercaptoethanol at 70 °C for 15 min. After extensive washing in PBS containing 0.1% Tween 20, membranes were processed as described previously(53) . Primary antibodies used to detect IkappaBalpha were monoclonal antibody MAD3 10B, which recognizes an epitope located between amino acids 21 and 48 of IkappaBalpha (34) and affinity-purified immunoglobulins obtained from a rabbit immunized with recombinant IkappaBalpha. p50 was detected with affinity-purified immunoglobulins obtained from an immune polyclonal rabbit serum generated by immunization with recombinant p50 (residues 35-381). A monoclonal antibody (4F3) recognizing ubiquitin was obtained from Dr. L. Guarino(59) . Polyclonal antibodies specific for ubiquitin were a kind gift from R. J. Mayer. The SV5 Pk tag (60) monoclonal antibody recognizes the sequence GKPIPNPLLGLDST and was obtained from Dr. R. E. Randall. Monoclonal antibody 12CA5, specific for the 9-amino acid HA peptide sequence YPYDVPDYA from influenza HA was obtained from BabCo. An affinity-purified rabbit polyclonal anti-actin antibody (A-2066) was purchased from Sigma.

Immunoprecipitation

Cells were collected by centrifugation and washed once with ice-cold PBS and cytoplasmic extracts prepared as described above. Immunoglobulins from a preimmune rabbit serum or from anti-IkappaBalpha or anti-NF-kappaBp50 rabbit antisera were covalently cross-linked to Protein A-Sepharose using dimethyl pimelimidate. 10 µl packed volume of Protein A beads bound to antibody were incubated with 1 mg of cytoplasmic protein for 2 h at room temperature, and the beads were washed 4 times with 10 ml of lysis buffer without iodoacetamide and once with PBS containing 0.1% Tween 20. Immunoprecipitation under denaturing conditions was performed in RIPA buffer as described previously(39) . Immunoprecipitated proteins were fractionated in a 10% polyacrylamide gel containing SDS, transferred to polyvinylidine difluoride, and detected by Western blotting using ECL as described above.

Treatment with Ubiquitin Carboxyl-terminal Hydrolase

Cytoplasmic extracts were prepared as described, but iodoacetamide was omitted from the lysis buffer. A Drosophila ubiquitin carboxyl-terminal hydrolase (58) was expressed in E. coli as a GST fusion protein (GST-UCH) and purified over glutathione-agarose essentially as described for NF-kappaB p50(61) . GST alone was purified in an identical fashion. Cytoplasmic protein (50 µg) was incubated at 37 °C for 30 min in a buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 mM dithiothreitol, and 10 µM GST or GST-UCH. Reaction products were fractionated in an 8% polyacrylamide gel containing SDS and electroblotted onto polyvinylidine difluoride, and IkappaBalpha was detected by Western blotting using MAD3 10B monoclonal antibody and ECL. The membrane was stripped and reprobed with a rabbit polyclonal anti-ubiquitin antibody (62) that was detected by ECL. The membrane was again stripped as described above and probed with an affinity-purified rabbit polyclonal anti-actin antibody (Sigma, A-2066).


RESULTS

Proteasome Inhibitor Z-LLL-H Blocks TNFalpha-induced Activation of NF-kappaB

Activation of NF-kappaB and signal-induced degradation of IkappaBalpha can be blocked by inhibiting the activity of the multicatalytic protease or proteasome(20, 50) . To define events that lead to targeting of IkappaBalpha for degradation, the peptide aldehyde Z-LLL-H was synthesized as a tool to accumulate intermediates in the degradative process. HeLa suspension cells pretreated with Z-LLL-H were exposed to TNFalpha, and the cells were fractionated into nucleus and cytoplasm. NF-kappaB DNA binding activity, determined in a native polyacrylamide gel, was low in nuclei of unstimulated cells but was present at an elevated level in cells treated with TNFalpha (Fig. 1a). Characterization by specific competition with unlabeled DNA, recognition by p50 and p65 antibodies, and inhibition by recombinant IkappaBalpha indicated that the TNFalpha-induced DNA-protein complex in HeLa cells was composed of p50, p65 heterodimers, and p65 homodimers(53) . This TNFalpha-induced increase was largely, but not completely, abrogated by pretreatment of the cells with either 10 or 25 µM Z-LLL-H (Fig. 1a). Western blot analysis of nuclear extracts with an antibody recognizing p50 revealed a low level of nuclear p50 in untreated cells and high levels of nuclear p50 in TNFalpha-induced cells. Preincubation of the cells with Z-LLL-H prior to TNFalpha induction blocked the nuclear accumulation of p50 (Fig. 1b). Z-LLL-H inhibition of nuclear translocation was not specific to TNF but was also observed with other inducers such as interleukin-1beta (data not shown).


Figure 1: Effect of proteasome inhibition on activation and nuclear translocation of NF-kappaB. a, HeLa cells were pretreated with the proteasome inhibitor Z-LLL-H and exposed to TNFalpha as indicated. NF-kappaB DNA binding activity in nuclear extracts was determined in a gel electrophoresis DNA binding assay with the positions of DNA-protein complexes (B) and free DNA (F) indicated. b, as in a, but nuclear and cytoplasmic extracts were fractionated in an 8% polyacrylamide gel containing SDS and transferred to polyvinylidine difluoride, and the p50 subunit of NF-kappaB and its p105 precursor protein were detected by ECL Western blotting using an affinity-purified polyclonal antibody raised against the p50 protein.



Slowly Migrating Forms of IkappaBalpha Accumulate in the Presence of TNFalpha and Z-LLL-H

Western blot analysis, with monoclonal or affinity-purified polyclonal antibodies recognizing IkappaBalpha(34) , reveals that TNFalpha induces rapid degradation of IkappaBalpha, but this process is inhibited in cells previously treated with Z-LLL-H (Fig. 2, short exposure). As expected(20, 50) , phosphorylated IkappaBalpha accumulates in the presence of the inhibitor (Fig. 2, short exposure). Further exposure of the Western blot reveals that in the presence of Z-LLL-H, TNFalpha induction results in accumulation of a ladder of more slowly migrating forms of IkappaBalpha, with a major form that is 8 kD larger than IkappaBalpha (Fig. 2, long exposure). Also detected are more rapidly migrating forms of IkappaBalpha that are likely to be intermediates in the degradation of IkappaBalpha. With Z-LLL-H alone, small amounts of more slowly migrating forms of IkappaBalpha are also detected (Fig. 2, long exposure), suggesting that even in the resting state, IkappaBalpha is being turned over by the same mechanism that operates after TNFalpha induction. The appearance and molecular weight of the more slowly migrating forms of IkappaBalpha that are detected in the presence of the proteasomal inhibitor are highly suggestive of multiple additions of ubiquitin. This phenomenon was also observed in monocytic cells (U937) and with other inducers of NF-kappaB (interleukin-1beta) when cells were preincubated with Z-LLL-H (data not shown).


Figure 2: Effect of proteasome inhibition on signal-induced degradation of IkappaBalpha. HeLa cells were pretreated with Z-LLL-H and exposed to TNFalpha as indicated. Cytoplasmic extracts (40 µg) were fractionated by SDS-PAGE, and IkappaBalpha protein was analyzed by ECL Western blotting using monoclonal antibody MAD3 10B (34) . The positions of prestained molecular weight markers (Sigma), IkappaBalpha and the phosphorylated form of the protein (P) are indicated. More slowly migrating and faster migrating forms of IkappaBalpha are indicated by the brackets. A short exposure is displayed to demonstrate Z-LLL-H inhibition of TNFalpha-induced IkappaBalpha degradation, while a long exposure is shown to display the IkappaBalpha species that accumulate in the presence of Z-LLL-H.



Ubiquitination of IkappaBalpha in Vivo

To demonstrate that the more slowly migrating forms of IkappaBalpha are a result of multiple additions of ubiquitin, extracts from cells treated with TNFalpha and Z-LLL-H were incubated with either a recombinant ubiquitin carboxyl-terminal hydrolase (58) (GST-UCH) or GST. Western blot analysis indicates that the more slowly migrating forms of IkappaBalpha present in the 20-min sample are lost after treatment with GST-UCH but remain after treatment with GST alone (Fig. 3a). In contrast phosphorylated IkappaBalpha and more rapidly migrating IkappaBalpha forms are unchanged after incubation with either GST-UCH or GST (Fig. 3a). Thus the more slowly migrating forms of IkappaBalpha represent the addition of one or more copies of ubiquitin, whereas faster migrating IkappaBalpha species are not linked to ubiquitin and may be intermediates in IkappaBalpha degradation. To determine the fate of the bulk of cellular ubiquitin conjugates, the blot in Fig. 3a was reprobed with an antibody recognizing protein-ubiquitin conjugates. Cells incubated with Z-LLL-H accumulate high molecular weight conjugates, some of which are removed by the GST-UCH, but not by GST alone (Fig. 3b). An unidentified 47-kDa species is detected after incubation with GST but not GST-UCH (Fig. 3b), presumably as a result of ubiquitin removal. Thus the Drosophila UCH has a limited specificity for protein-ubiquitin conjugates. Reprobing the blot with an anti-actin antibody revealed an equivalent signal in each lane (Fig. 3c), confirming the absence of nonspecific proteolysis.


Figure 3: Ubiquitination of IkappaBalpha in vivo. a-c, HeLa S3 cells either treated with 10 µM Z-LLL-H for 45 min or untreated were exposed to 10 ng ml TNFalpha for the indicated time. Cytoplasmic extracts were incubated with either 10 µM purified GST or 10 µM of a GST-ubiquitin carboxyl-terminal hydrolase fusion protein (GST-UCH) at 37 °C for 30 min. a, reaction products were analyzed by Western blotting with the IkappaBalpha-specific monoclonal antibody MAD3 10B. IkappaBalpha, its phosphorylated derivative, and anomalously migrating forms are indicated as described in the legend to Fig. 2. b, the blot displayed in a was stripped and reprobed with an anti-ubiquitin antibody(62) . c, the same blot was again stripped and reprobed with an anti-actin antibody (Sigma). d, cytoplasmic extracts were prepared and immunoprecipitated with either rabbit preimmune serum (PI), rabbit anti-IkappaBalpha serum, rabbit anti-p50 serum, or rabbit anti-p65 serum cross-linked to protein A-Sepharose. Immunoprecipitates were fractionated by SDS-PAGE and analyzed by ECL Western blotting with anti-ubiquitin monoclonal antibody 4F3. The positions of molecular mass markers are shown.



To independently prove that IkappaBalpha is linked to ubiquitin, cells were treated with TNFalpha and Z-LLL-H, and extracts were immunoprecipitated with antibodies to IkappaBalpha, NF-kappaB p50, NF-kappaB p65, or nonimmune serum prior to detection of bound ubiquitin by Western blotting with a monoclonal antibody directed against ubiquitin(59) . In TNFalpha-treated cells, most IkappaBalpha is degraded, but ubiquitinated adducts on the remaining protein are detected in immunoprecipitates with IkappaBalpha antibodies but not with antibodies to p50, p65, or nonimmune serum (Fig. 3d). In the presence of TNFalpha and Z-LLL-H, a considerable increase in ubiquitinated forms of IkappaBalpha are detected in immunoprecipitates with antibodies specific to IkappaBalpha. Again antibodies to p50 and p65 only precipitate amounts of ubiquitinated IkappaBalpha that are comparable with that obtained with the preimmune serum, even although they can precipitate bound IkappaBalpha that is not linked to ubiquitin (see Fig. 5). The failure of NF-kappaB antibodies to immunoprecipitate material recognized by the ubiquitin antibody (Fig. 3d), suggests that IkappaBalpha-ubiquitin conjugates dissociate from NF-kappaB.


Figure 5: Ubiquitinated IkappaBalpha dissociates from NF-kappaB whereas phosphorylated IkappaBalpha remains bound to NF-kappaB. a, HeLa S3 cells were treated with either 10 µM Z-LLL-H for 45 min (ZLLLH), 10 ng of ml TNFalpha for 15 min (TNFalpha), 10 µM Z-LLL-H for 45 min followed by a further 15 min in the presence of 10 ng ml TNFalpha (TNFalpha + ZLLLH) or 0.1% Me(2)SO vehicle alone for 45 min. Cytoplasmic extracts were prepared and immunoprecipitated with either rabbit preimmune serum (PI), rabbit anti-IkappaBalpha serum (a-IkB), or rabbit anti-p50 serum (a-p50). Immunoprecipitates were fractionated by SDS-PAGE and analyzed by Western blotting with anti-IkappaBalpha monoclonal antibody MAD3 10B. Molecular mass markers and the position of IkappaBalpha and its phosphorylated derivative are indicated. IkappaBalpha-ubiquitin conjugates are indicated by the upper bracket, while IkappaBalpha degradation products are indicated by the lower bracket. b, HeLa cells were pretreated with Z-LLL-H and exposed to TNFalpha. Cytoplasmic extracts were immunoprecipitated with anti-p50 antibodies and bound IkappaBalpha detected by Western blotting.



To independently confirm these results, an HA-tagged version of human ubiquitin (56) was introduced into cells by transfection (Fig. 5a). Cells were transfected with either empty vector DNA, the plasmid expressing HA-tagged ubiquitin, or cotransfected with plasmids expressing epitope-tagged IkappaBalpha (57) and HA-tagged ubiquitin. Transfected cells were trypsinized, seeded into three separate dishes and after 16 h treated with either TNFalpha, TNFalpha plus Z-LLL-H or untreated. Cytoplasmic extracts were denatured in SDS, immunoprecipitated with polyclonal antibodies to IkappaBalpha or preimmune serum, and analyzed by Western blotting using first the monoclonal antibody recognizing the HA tag and reprobed with an IkappaBalpha-specific monoclonal antibody. HA immunoreactive material is not detected in cells transfected with the empty vector (Fig. 4b, upper left panel) but in cells transfected with the plasmid expressing HA tagged ubiquitin, the expressed protein appears to be associated with endogenous IkappaBalpha even in the absence of TNFalpha and Z-LLL-H (Fig. 4b, upper left panel), again indicating that IkappaBalpha is being constantly turned over. Multiply ubiquitinated IkappaBalpha species are relatively rare, but they can be detected by the HA antibody as a large number of epitopes are present. In contrast, these species are difficult to detect with the IkappaBalpha antibody as only a single epitope is present. Although treatment of the cells with TNFalpha results in a proportion of the IkappaBalpha being degraded (Fig. 4b, upper right panel), this does not translate into a reduction in the amount of multiply HA-tagged, ubiquitinated IkappaBalpha detected (Fig. 4b, upper left panel). While the rate of IkappaBalpha degradation is increased in the presence of TNFalpha, the rate of ubiquitination of IkappaBalpha is also increased, with the consequence that the steady-state level of ubiquitinated IkappaBalpha is unaltered. Endogenous IkappaBalpha is thus covalently linked to HA-tagged ubiquitin as part of a rapidly turning over pool of modified IkappaBalpha that is stabilized by blocking degradation with Z-LLL-H.


Figure 4: HA-tagged ubiquitin is covalently bound to IkappaBalpha in vivo. a, structure of the HA-tagged polyubiquitin gene (HA-ubi, (56) ) with the amino-terminal tag shaded. The IkappaBctag gene (57) is displayed with the ankyrin repeats (filled boxes), a region involved in interaction with p65 (shaded box), the acidic region (open box) and the carboxyl-terminal tag (hatched box). Both genes used for transfections are under the control of the cytomegalovirus immediate early promoter. b, COS7 cells in 150-cm^2 flasks were transfected with the indicated plasmids, and after 16 h the cells were trypsinized and split into three, and growth continued in 9-cm plates. After a further 24 h of growth, cells were either untreated, treated with 10 ng/ml TNFalpha for 15 min, or pretreated for 45 min with 10 µM Z-LLL-H and then incubated with 10 ng/ml TNFalpha for a further 15 min. At the end of the incubation period, cytoplasmic extracts were prepared, denatured in the presence of SDS, and immunoprecipitated with matrix-bound immunoglobulins from either rabbit preimmune serum (IP PI, lower panels) or a polyclonal serum specific for IkappaBalpha (IP Ab IkappaBalpha, upper panels). Immunoprecipitates were fractionated by SDS-PAGE and analyzed by Western blotting with the HA tag-specific monoclonal antibody 12CA5 (Blot Ab HA, left panels). After ECL development, the blots were stripped and reprobed with the IkappaBalpha-specific monoclonal antibody MAD3 10B (Blot Ab IkappaBalpha, right panels).



A similar situation is apparent when HA-tagged ubiquitin is cotransfected with a plasmid expressing epitope-tagged IkappaBalpha (IkappaBctag). Only a small proportion of the highly expressed IkappaBctag is degraded (Fig. 4b, upper right panel), indicating that the IkappaBalpha modification and degradation machinery has a limited capacity. Thus, only a small amount of immunopurified IkappaBalpha is associated with HA-tagged ubiquitin in the absence or presence of TNFalpha (Fig. 4b, upper right panel), but when TNFalpha and Z-LLL-H are present, a substantial quantity of HA-tagged ubiquitin is linked to IkappaBalpha (Fig. 4b, upper right panel). Preimmune serum did not immunoprecipitate material reactive with the HA-specific antibody (Fig. 4b, lower left panel). Thus IkappaBalpha is covalently linked to HA-tagged ubiquitin when signal-induced degradation is blocked by proteasomal inhibitors.

IkappaBalpha, Ubiquitinated in Vivo, Is Not Associated with NF- kappaB

To confirm that ubiquitinated IkappaBalpha was not associated with NF-kappaB, cell extracts were immunoprecipitated with antibodies specific for either IkappaBalpha, p50, or preimmune serum and bound IkappaBalpha detected by Western blotting. In the presence of Z-LLL-H, TNFalpha induction results in accumulation of ubiquitinated forms of the protein that are immunoprecipitated with antibodies to IkappaBalpha (Fig. 5a). While antibodies to p50 immunoprecipitate IkappaBalpha, they fail to precipitate ubiquitinated IkappaBalpha (Fig. 5a), indicating that ubiquitination of IkappaBalpha releases NF-kappaB. In contrast, phosphorylated IkappaBalpha remains associated with NF-kappaB and is precipitated by antibodies to p50 (Fig. 5b). Although the TNFalpha-induced degradation of IkappaBalpha was incomplete, the presence of phosphorylated IkappaBalpha indicated that signal induction had taken place.

Residues Ser-32 and Ser-36 Are Required for Ubiquitination of IkappaBalpha in Vivo

Residues S32 and S36 are required for the signal-induced phosphorylation and degradation of IkappaBalpha(36, 42, 43) . To determine if phosphorylation of these residues was required for ubiquitination of IkappaBalpha, a cell line expressing an S32A/S36A IkappaBctag was selected. Both wild-type IkappaBctag and S32A/S36A IkappaBctag were expressed at levels that were comparable with that of the endogenous IkappaBalpha. Western blot analysis with the tag-specific monoclonal antibody revealed that the wild-type IkappaBctag was rapidly degraded in response to TNFalpha or TNFalpha plus okadaic acid, whereas okadaic acid alone did not induce degradation (Fig. 6b). In the presence of TNFalpha, okadaic acid and the proteasome inhibitor Z-LLL-H degradation was blocked (Fig. 6b), and ubiquitinated forms of the protein were detected (Fig. 6a). In contrast, the S32A/S36A mutant was neither degraded nor ubiquitinated (Fig. 6, a and b). Western blot analysis with an antibody, which detects both endogenous and tagged IkappaBalpha, revealed that in cells expressing S32A/S36A IkappaBctag, the endogenous IkappaBalpha was degraded. Thus it is likely that phosphorylation of Ser-32 and Ser-36 is required for ubiquitination and degradation of IkappaBalpha in vivo.


Figure 6: Residues Ser-32 and Ser-36 are required for ubiquitination of IkappaBalpha in vivo. HeLa cells stably expressing either wild-type IkappaBctag or S32A/S36A IkappaBctag were treated with either 10 ng/ml TNFalpha for 20 min (TNF), 0.5 µM okadaic acid for 20 min (OKA), 10 ng/ml TNFalpha plus 0.5 µM okadaic acid for 20 min (TNF + OKA), 10 µM Z-LLL-H for 45 min followed by a further 20 min in the presence of 10 ng/ml TNFalpha plus 0.5 µM okadaic acid (TNF + OKA + ZLLLH) or 0.1% Me(2)SO vehicle alone for 65 min(-). Cytoplasmic extracts were fractionated by SDS-PAGE and analyzed by Western blotting with either SV5 Pk tag monoclonal antibody (a, b) or anti-IkappaBalpha monoclonal antibody MAD 10B (c). The positions of molecular mass markers, IkappaBctag, IkappaBalpha phosphorylated derivatives, and ubiquitin adducts are indicated.




DISCUSSION

Peptide aldehyde inhibitors, which block the activity of the proteasome, inhibit signal induced degradation of IkappaBalpha and activation of NF-kappaB(20, 50) . Although it was demonstrated that ubiquitination was involved in processing of p105 to p50(20) , ubiquitination of IkappaBalpha was not reported. However a recent report (63) has shown that IkappaBalpha is ubiquitinated, and in vitro studies demonstrated that this process required residues Ser-32 and Ser-36. Furthermore it was shown that ubiquitinated IkappaBalpha was a substrate for the 26 S proteasome in vitro. Here we demonstrate that signal induction with TNFalpha results in phosphorylation-dependent ubiquitination of IkappaBalpha and release of NF-kappaB. This is in contrast with the situation observed in vitro where ubiquitinated IkappaBalpha remains bound to NF-kappaB(63) . Thus it is unlikely that ubiquitination per se is responsible for the dissociation of IkappaBalpha from NF-kappaB, but one possibility is that a protein with chaperonin activity could release ubiquitinated IkappaBalpha from NF-kappaB in vivo. Ubiquitinated IkappaBalpha must represent a short lived intermediate in the degradative pathway, as ubiquitinated IkappaBalpha is only readily detected when proteasomal degradation is inhibited. The results support a model for signal-induced activation of NF-kappaB in which IkappaBalpha is first phosphorylated on residues Ser-32 and Ser-36 (36, 42, 43) by an as yet unidentified kinase. Phosphorylated IkappaBalpha remains bound to NF-kappaB but is recognized by proteins involved in ubiquitin addition. Enzymes responsible for ligation of ubiquitin to IkappaBalpha have not been identified, but it is likely that this process is mediated by a specific E1-E2-E3 type thioester cascade of the type involved in the E6-induced ubiquitination of p53(64, 65) . Although IkappaBalpha contains nine lysines, five of which are located in the amino-terminal region, it is clear that residues Lys-21 and Lys-22 are the primary sites of phosphorylation dependent ubiquitination. (^2)Given the specificity of the peptide aldehyde inhibitors (54) and the recent in vitro experiments(63) , it is then likely that the ubiquitinated IkappaBalpha is degraded by the multicatalytic protease or proteasome(66) . Although ubiquitinated IkappaBalpha is detected when proteasomal degradation is blocked, only a small proportion of the IkappaBalpha accumulates in the ubiquitinated form. This is probably due to the activity of ubiquitin carboxyl-terminal hydrolases, which remove ubiquitin from ubiquitin-protein conjugates and process the primary products of polyubiquitin gene mRNA translation(67) . Although IkappaBalpha-ubiquitin conjugates do not constitute a large proportion of the total IkappaBalpha pool, the observation that Z-LLL-H only partially blocks translocation of NF-kappaB to the nucleus (Fig. 1) but efficiently blocks degradation of IkappaBalpha (Fig. 2) is thus explained by release of NF-kappaB from ubiquitinated IkappaBalpha ( Fig. 3and Fig. 5).

Mutational analysis has indicated that both the amino and carboxyl termini of IkappaBalpha are required for signal-induced degradation(36, 42, 43, 57) . The requirement for the amino terminus can be explained by the location of sites for signal-induced phosphorylation and ubiquitination, while the carboxyl terminus contains PEST sequences, which are thought to destabilize proteins. A role for ubiquitin conjugating enzymes in the degradation of S and M phase cyclins has been established in vivo(68) , and in vitro studies have demonstrated that like IkappaBalpha, ubiquitination of the G(1) cyclin Cln 2p is preceded by phosphorylation of the protein(69) . Our observations emphasize the importance of signal-induced protein degradation in activation of NF-kappaB and demonstrate a crucial role for the ubiquitin-proteasome pathway (66) in this process.


FOOTNOTES

*
This work was supported by the Medical Research Council, Agence Nationale pour la Recherche sur le Syndrome d'Immuno Deficience Acquise, and the European Communities Concerted Action (Project ROCIO). 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.

§
Supported by the Leukaemia Research Fund.

Supported by a fellowship from Consejo Nacional de Ciencia et Tecnologia (Mexico).

**
To whom correspondence should be addressed. Tel.: 44-1334-463396; Fax: 44-1334-463400; rth{at}st-and.ac.uk.

(^1)
The abbreviations used are: TNFalpha, tumor necrosis factor alpha; HA, hemagglutinin; HA-ubi, HA-tagged ubiquitin; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

(^2)
Rodriquez, M. S., Wright, J., Thomson, J., Thomas, D., Baleux, F., Virelizier, J. L., Hay, R. T., and Arenzana-Seisdedos, F.,(1996) Oncogene, in press.


ACKNOWLEDGEMENTS

We thank R. J. Mayer for anti-ubiquitin antibodies, L. Guarino for the 4F3 monoclonal antibody, R. E. Randall for the SV5 Pk tag monoclonal antibody, M. Bownes for the strain expressing ubiquitin carboxyl-terminal hydrolase, D. Bohmann for the HA-tagged ubiquitin plasmid, and B. Blyth for photography. Comments on the manuscript by A. Webster and J.L. Virelizier were greatly appreciated.


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