Involvement of Valosin-containing Protein, an ATPase Co-purified with Ikappa Balpha and 26 S Proteasome, in Ubiquitin-Proteasome-mediated Degradation of Ikappa Balpha *

Ren-Ming DaiDagger , Eying ChenDagger , Dan L. Longo§, Carlos M. Gorbea, and Chou-Chi H. LiDagger par

From the Dagger  Intramural Research Support Program, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, § NIA, National Institutes of Health, Gerontology Research Center, Baltimore, Maryland 21224, and the  Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The inactivation of the prototype NF-kappa B inhibitor, Ikappa Balpha , occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. We identify valosin-containing protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co-precipitates with Ikappa Balpha immune complexes. The ubiquitinated Ikappa Balpha conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-kappa B. In ultracentrifugation analysis, physically associated VCP and ubiquitinated Ikappa Balpha complexes sediment in the 19 S fractions, while the unmodified Ikappa Balpha sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of Ikappa Balpha are critical for VCP binding, which in turn is necessary but not sufficient for Ikappa Balpha degradation; while the N-terminal domain of Ikappa Balpha is required in all three reactions, both N- and C-terminal domains are required in degradation. Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physical and functional link between Ikappa Balpha and the 26 S proteasome and play an important role in the proteasome-mediated degradation of Ikappa Balpha .

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Transcription factor NF-kappa B is involved in a large variety of processes, such as cell growth, transcriptional regulation, immune, inflammatory, and stress responses (reviewed in Refs. 1-4). NF-kappa B is a homo- or heterodimer consisting of various combinations of the family members, including NFkappa B1 (p50 and precursor p105), c-Rel, RelA, NFkappa B2 (p52 and precursor p100), RelB, and Drosophila proteins Dorsal and Dif. Unlike many other transcription factors that are localized in the nucleus, the NF-kappa B dimeric factor is sequestered in the cytoplasm of most cells through binding to a family of inhibitor proteins, Ikappa B. In response to extracellular stimuli, the inhibitors are partially or entirely degraded, thus liberating the DNA-binding dimer for translocation to the nucleus. The I6B family contains Ikappa Balpha , Ikappa Bbeta , Ikappa Bgamma , Bcl-3, the precursor proteins p105 and p100, and the Drosophila protein Cactus. All members of the Ikappa B family contain 5-8 ankyrin motifs, thought to be involved in protein-protein interactions. It has been shown that when the precursor protein p105 is involved as the inhibitor, the processing from p105 to the active p50 occurs through the ubiquitin-dependent proteasome (Ub-Pr)1 pathway, which degrades the C-terminal ankyrin-containing domain of p105 (5, 6).2 For the prototype complex that contains p50, p65, and Ikappa Balpha , upon stimulation the entire NF-kappa B complex becomes hyperphosphorylated. The induced phosphorylation of Ikappa Balpha does not lead to its immediate dissociation from the complex; rather, it signals for rapid Ikappa Balpha degradation, thus liberating the active p50·p65 dimer for translocation to the nucleus (7-15). We and others showed that the degradation of Ikappa Balpha is sensitive to proteasome inhibitors and is ubiquitin-dependent. Recently, it was further shown that signal-induced phosphorylation precedes Ikappa Balpha degradation and targets Ikappa Balpha to the Ub-Pr pathway (7-15). It was proposed that the inactivation of Ikappa Balpha occurs through a series of ordered processes including phosphorylation, ATP-dependent multiple ubiquitin conjugation, and proteasome-mediated proteolysis. However, the link between ubiquitination and proteasome-mediated degradation remains unclear.

The extralysosomal, energy-dependent Ub-Pr pathway is a major mechanism used to regulate many critical cellular proteins that must be rapidly destroyed for normal growth and metabolism (reviewed in Refs. 16-21). The rapidly growing list of the substrates for the Ub-Pr pathway includes mitotic cyclins, G1 cyclins, cyclin-dependent kinase inhibitors p27, Sic1 protein, proto-oncogene products p53, c-Myc, c-Jun, and c-Mos, NF-kappa B inhibitors, yeast MATalpha 2 transcription factor, major histocompatibility complex molecules, and others. The Ub-Pr proteolytic pathway is ATP-dependent and present in both cytoplasm and nucleus. The pathway consists of two distinct, sequential steps. The target protein is first conjugated with multiple ubiquitin (Ub) molecules that mark the substrate for destruction. The Ub-tagged target is then translocated to (probably with the help of molecular chaperones) and degraded by a large protease complex with an apparent sedimentation coefficient of 26 S. The 26 S proteasome is a multisubunit complex, consisting of a central cylinder-shaped 20 S multicatalytic proteinase core and a 19 S cap-like regulatory complex attached to each end of the cylinder. The terminal cap structure consists of at least 18 distinct subunits with molecular masses of 35-110 kDa and has ATPase and ubiquitin conjugate binding activities. It is presumed that ATP hydrolysis by the 19 S complex provides the energy needed for the chaperoning and unfolding of substrates degraded within the proteasome cylinder.

VCP (22-24), the mammalian homolog of the cell division cycle protein Cdc48p in yeast and p97 in Xenopus, is a member of a recently identified AAA family (reviewed in Ref. 25). The family members are characterized by having ATPase domain(s) with striking sequence similarities and having ring structures consisting of homooligomers. Despite the high sequence and structural homology, these proteins unexpectedly are implicated in a large variety of seemingly unrelated biological functions. These functions reviewed in Ref. 25 include control of cell division cycle, T cell activation (23, 24), membrane fusion (26-29), and vesicle-mediated transport, peroxisome assembly and biogenesis, gene expressions, and the Ub-Pr degradation pathways (30). Furthermore, at least six family members have been identified as subunits of the 26 S proteasome (subunits 4, 6, 7, 8, 10, and TBP-1) (31-36), and it is likely that other family proteins will fill this function as well (31, 37). The apparent paradox between the striking sequence homology and the large diversity of functions in this family suggests that these proteins have a basic and critical role in cellular function, and the involvement in many other functions may be indirect.

During the course of studying the molecular mechanisms involved in Ikappa Balpha degradation, we detected VCP physically associated with Ikappa Balpha complexes both in vivo and in vitro. In this report, we demonstrate that VCP is involved in the proteasome-mediated degradation of Ikappa Balpha , and VCP is co-purified with the 26 S proteasome. Consistent with this hypothesis, VCP indeed has in vitro ATPase activity and an apparent sedimentation coefficient of 19 S (Ref. 24 and this study), the same as that of the regulatory complex of the 26 S proteasome. We propose that physical association of VCP with Ub-tagged Ikappa Balpha targets Ikappa Balpha to the Ub-Pr pathway.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Lines and Culture Conditions-- Two interchangeably used human B cell lines, DB (38) and CA46 (39), were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin.

Immunoprecipitation (IP) and Western (Immuno-) Blot Analyses-- Both analyses were performed as described previously (9, 40, 41) with minor modifications. Cells were labeled with 0.1 mCi/ml (1000 Ci/mmol) [35S]methionine/cysteine (ICN) at a density of 5 × 106/ml for approximately 16 h, washed twice with phosphate-buffered saline, and lysed in RIPA buffer (20 mM Tris/HCl, pH 7.6, 2 mM EDTA, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100) containing protease inhibitors (1% aprotinin, 70 µg/ml phenylmethanesulfonyl fluoride, 40 µg/ml Tos-Phe-CH2Cl, 5 µg/ml Tos-Lys-CH2Cl, 5 µg/ml leupeptin). The lysates were clarified by centrifugation at 12,000 × g for 30 min and incubated with antisera. The immune complexes were collected with Protein A-Sepharose beads, washed with RIPA buffer, boiled, resolved by SDS-polyacrylamide gel electrophoresis (PAGE), electrophoretically transferred onto polyvinylpyrrolidone membranes, and analyzed by autoradiography. IPs shown in Figs. 1A and 2B were conducted in Buffer I (1% Nonidet P-40, 1% bovine serum albumin in phosphate-buffered saline) containing protease inhibitors, 100 µM calpain inhibitor I, and 10 mM iodoacetamide, and the precipitates were washed with Buffer I without bovine serum albumin. For reimmunoprecipitation experiments, the immune complexes isolated from the first IP were boiled in RIPA containing 1% SDS, diluted, and subjected to the second IP. For immunoblot analysis, equal amounts of protein or immune complexes were resolved by SDS-PAGE and transferred onto membranes. The membrane was blocked, washed, and incubated with antiserum (typically at 1:3,000), followed by reaction with peroxidase-conjugated anti-rabbit immunoglobulin serum (Boehringer Mannheim), and developed by the enhanced chemiluminescence detection system (ECL) (Amersham Corp.). When serial blotting analyses were performed, previous reactivity was stripped off from the membrane.

Antisera-- All antisera used in this study are polyclonal rabbit sera. Anti-Ikappa Balpha -1 and anti-Ikappa Balpha -2 (40, 9) were independently generated against synthetic peptide corresponding to residues 300-317 of the human Ikappa Balpha sequence (42). Anti-Ikappa Balpha -3 (SC-847) and -5 (SC-371) were purchased from Santa Cruz Biotechnology, Inc., and anti-Ikappa Balpha -4 (06-494) was purchased from Upstate Biotechnology, Inc. Anti-VCP-1 and anti-VCP-2, raised against peptides of residues 792-806 and 20-40 of murine VCP (23), respectively, were kindly provided by L. Samelson (NIH). Anti-VCP-3, -4, -5, -6, -7, and -8 were generated against glutathione S-transferase (GST)-VCP (full-length) and residues 792-806, 721-734, 184-197, 240-253, and 167-180 of murine VCP (23), respectively. Anti-Ub polyclonal antisera were purchased from either Sigma or Biogenesis or obtained from M. Rechsteiner (University of Utah, Salt Lake City, Utah) (43). Antisera to subunits 4 and 5 and to the 26 S proteasome were kindly provided by M. Rechsteiner (43, 44).

Protein Sequencing-- Well resolved protein bands were sliced from Coomassie Blue-stained gel and subjected to an in-gel partial V8 digestion (45). The proteolytic fragments were resolved by 15% SDS-PAGE, transferred onto a polyvinylpyrrolidone membrane, and stained with Coomassie Blue. The well separated bands were sliced and subjected to N-terminal peptide sequencing using an ABI 494A sequencer.

In Vitro Association-- Ikappa Balpha (7) or VCP (23) was synthesized in a coupled in vitro transcription and translation reaction from a rabbit reticulocyte lysate system (Promega) in the presence of [35S]cysteine (for Ikappa Balpha ) or [35S]methionine (for VCP). GST-Ikappa Balpha expression plasmid was constructed by inserting the EcoRI fragment of Ikappa Balpha (7) into pGEX-4T-2 vector (Pharmacia Biotech Inc.) (9). The GST-VCP (24) or GST-Ikappa Balpha fusion proteins were prepared according to the manufacturer (Pharmacia). Glutathione-Sepharose beads containing GST or GST fusion proteins were mixed with 35S-labeled in vitro translated proteins or unlabeled B cell lysates in a total volume of 225 µl of binding buffer (35 mM Tris, pH 7.6, 50 mM NaCl, 0.1% Nonidet P-40, 0.5 mM dithiothreitol including protease inhibitors). After incubation for 2 h at 4 °C and three washes with binding buffer, the bound products were analyzed by SDS-PAGE followed by Western transfer, autoradiography, and immunoblotting.

Glycerol Gradient Sedimentation Centrifugation-- Velocity sedimentation centrifugation was carried out in 10-40% glycerol gradients in a total volume of 13 ml of 12 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2 mM CaCl2, 1 mM MgCl2. Freshly prepared CA46 cell lysate in a volume of 200 µl was loaded onto the gradient and centrifuged at 4 °C, in an SW41 rotor at 36,000 rpm for 16 h. Fractions of 0.5 ml were collected and analyzed by immunoblotting or IP. Protein markers were centrifuged in separate tubes and included thyroglobulin (19 S), immunoglobulin G (7 S), and bovine serum globulin (4.5 S).

Proteasome Purification-- Cytosolic fractions were prepared by lysing cells in a hypotonic buffer (5 mM Hepes, pH 8.0, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol) on ice for 10 min. The lysates were clarified by low speed centrifugation for 10 min. The supernatant was further centrifuged at 100,000 × g for 1-2 h, and the supernatant, S100, was stored in aliquots at -70 °C. Proteasome-enriched (Pr+), and proteasome-depleted (Pr-) fractions were extracted from CA46 cells as described by Palombella et al. (5). The highly purified (26 S-1) and the partially purified (26 S-2) proteasomes were isolated from human red blood cells as described (46).

Ikappa Balpha Mutants-- Deletion mutants of Ikappa Balpha encoding amino acids 37-317, and 1-242 were constructed as described by Brockman et al. (10). The deletion mutant encoding amino acids 23-317 was constructed by polymerase chain reaction using specific oligonucleotide primers (5'-GGGAAACTTCTCGTCCGCGCCATGCGGCTACTGGACGACCGC-3' and 5'-GGTCTAGATCATAACGTCAGACGCTGGCCT-3') and the wild-type Ikappa Balpha cDNA (7) as a template. Amplified product was purified and cloned into pCRII vector (TA cloning kit, Invitrogen). The site-specific mutants, S32A/S36A and K21R/K22R, were kindly provided by D. Ballard (12).

In Vitro Assays-- The wild-type and mutants of Ikappa Balpha were synthesized in reticulocyte lysate-based in vitro transcription/translation system (Promega) in the presence of [35S]cysteine and used as the substrates in the Ub-Pr degradation assay (9). S100 was extracted from CA46 cells (8) and used as enzyme source. Master reaction mixture containing 5 µl of substrate, 8 µg of dialyzed ubiquitin (Sigma), 12 mM Tris-HCl, pH 7.5, 60 mM KCl, 3.5 mM MgCl2, 5 mM CaCl2, 1 mM dithiothreitol, and 1 mM ATP was prepared and aliquoted into four tubes on ice. At various time points, 50 µg of S100 was added to individual tubes to start the reaction at 37 °C. The final volume in each reaction was adjusted to 50 µl. All of the reactions were simultaneously terminated by boiling and analyzed by SDS-PAGE followed by Western transfer and autoradiography. The ubiquitinated Ikappa Balpha (Ub-Ikappa Balpha ) conjugates were generated by slightly modified Ub-conjugation assays (6, 8), which were essentially the same as the degradation assay except that 1 µg/ml okadaic acid, 1 mM ATPgamma S, and 100 µM calpain inhibitor I were included in the reaction, and the reactions were carried out for 90 min.

Two-dimensional Gel Electrophoresis-- Two-dimensional gel electrophoresis analysis was carried out as described previously (44). The highly purified 26 S proteasome isolated from human red blood cells was resolved by two-dimensional isoelectric focusing followed by SDS-gel electrophoresis.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

VCP and High Mr Ub-Ikappa Balpha Are Co-immunoprecipitated in Vivo-- IP performed on cell lysates containing active NF-kappa B detected Ikappa Balpha , p50, p65, and an unidentified 90-kDa cellular protein (p90) (Refs. 9 and 40; Fig. 1A, top, lanes 1-4). p90 was detected in complexes precipitated by Ikappa Balpha antisera from various sources and was not detected by the preimmune serum (Ref. 9; Fig. 1C, top, lanes 1 and 6) or the anti-Ikappa Balpha immune serum preincubated with the immunogenic peptide (40). When the p90-containing Ikappa Balpha complex was subjected to the in vitro Ub-Pr degradation assay, unlike Ikappa Balpha molecules, p90 was resistant to degradation (9). To identify p90, Ikappa Balpha immune complexes were resolved on SDS-polyacrylamide gel, and the p90 band was visualized by Coomassie Blue staining, excised, and subjected to N-terminal peptide sequencing. Although the N terminus of p90 was blocked, peptide sequencing of two of the V8 proteolytic fragments of p90 revealed a total of 20 residues identical to those of the murine valosin-containing protein, VCP (23) (Fig. 1B). IP followed by immunoblotting with either C terminus-specific anti-VCP-1 or N terminus-specific anti-VCP-2, kindly provided by L. Samelson (National Institutes of Health, Bethesda, MD), confirmed that the p90 was VCP (Fig. 1A, bottom). VCP was co-precipitated with Ikappa Balpha in nondenatured lysate (Fig. 1C, lanes 2), but not in boiled lysate (lanes 7), indicating that VCP physically associates with an Ikappa Balpha -containing complex. When the reciprocal IP was performed, however, virtually no Ikappa Balpha was detected in VCP immune complexes (lanes 3, 5, 8, and 10). Although a protein with a similar size to that of Ikappa Balpha was detected in VCP-2 immune complexes (Fig. 1C, top, lanes 5 and 10), it was not Ikappa Balpha because it did not react with the antiserum specific to the C terminus of Ikappa Balpha (bottom, lanes 5 and 10). The lack of 36-kDa Ikappa Balpha in the anti-VCP IP was initially unexpected. However, since VCP is resistant to degradation in Ub-Pr assays (9) and is an ATPase, a common characteristic of molecular chaperones, we hypothesize that VCP is a molecular chaperone that only associates with Ikappa Balpha molecules that have been modified by ubiquitination. Therefore, the 36-kDa Ikappa Balpha would not be detected in VCP complexes. Based on this hypothesis, two predictions were made: 1) in addition to 36-kDa Ikappa Balpha and VCP, anti-Ikappa Balpha antiserum should precipitate high Mr Ub-Ikappa Balpha , and 2) anti-VCP IP should also detect high Mr Ub-Ikappa Balpha molecules. With respect to the first prediction, we previously demonstrated that anti-Ikappa Balpha -1 and -2 detected high Mr Ub-Ikappa Balpha proteins in a ladderlike pattern (9). To further substantiate the specificity, we subjected the Ikappa Balpha immune complexes (Fig. 1A, lanes 1-4) to second IPs using various antisera specific to Ikappa Balpha (lanes 5-8) or to Ub (lane 10). As predicted, both IPs detected abundant high Mr Ub-Ikappa Balpha molecules but not the c-Rel proteins co-precipitated with Ikappa Balpha (the 80-kDa band in lanes 1-4, indicated by a small arrow on the left). By contrast, Ikappa Balpha IP followed by VCP reimmunoprecipitation did not detect similar high Mr proteins (lane 9). Predominant species with molecular masses of 90 and 85 kDa were noted. Although the majority of p90 precipitated by anti-Ikappa Balpha was indeed VCP, as shown in the second IP with anti-VCP (lane 9), a minor portion of p90 co-migrating with VCP was Ub-Ikappa Balpha , because it was reprecipitated by both anti-Ikappa Balpha (lanes 5-8) and anti-Ub (lane 10) but not reactive to anti-VCP in the subsequent immunoblot (bottom panel).


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Fig. 1.   Identification of VCP and high Mr Ikappa Balpha in Ikappa Balpha immune complexes. A, DB cells were metabollically labeled with [35S]methionine/cysteine, and the cell lysates were subjected to single IPs (lanes 1-4) or sequential double IPs (lanes 5-10) with the specified antisera. Anti-VCP-4 immune serum was used in the second IP in lane 9. Three times as much lysates were used in each of lanes 5-10 as in lanes 1-4. The immune complexes were separated by SDS-PAGE, transferred onto membrane, and visualized by autoradiography (top; the exposure time for lanes 5-10 is longer than that for lanes 1-4). The membrane was subsequently immunoblotted (IB) with anti-VCP-4 (bottom). The gel mobilities of Mr standards (in kDa), VCP, and unmodified Ikappa Balpha are indicated. The small arrow above the 68-kDa standard indicates the c-Rel protein co-immunoprecipitated with Ikappa Balpha , and the line above the arrow indicates the 85-kDa protein. B, amino acid identity between the murine VCP sequence and two polypeptides generated from V8 proteolysis of purified human p90 are shown. C, DB cells were metabolically labeled with [35S]methionine/cysteine and separated into two parts; one half was lysed in RIPA buffer without SDS (lanes 1-5), and the other half was lysed by boiling for 5 min in RIPA buffer containing 0.5% SDS and then diluted to a final SDS concentration of 0.1%. Aliquots of the lysates were immunoprecipitated with the indicated sera (anti-Ikappa Balpha -1 was used for Ikappa Balpha ). The washed precipitates were separated by SDS-PAGE, transferred to membranes, and detected by autoradiography (top). The mobilities of the molecular mass standards (in kDa), VCP, and Ikappa Balpha are indicated. The upper portion of the membrane was subsequently immunoblotted with anti-VCP-2 (middle). The faint bands with molecular mass larger than 90 kDa detected in lanes 6 and 7 are nonspecific. The lower portion of the membrane was immunoblotted with anti-Ikappa Balpha -1, and the 36-kDa Ikappa Balpha is indicated (bottom).

To test the second prediction, we generated six polyclonal antisera against VCP (Fig. 2A) and examined whether high Mr Ikappa Balpha was co-precipitated with VCP. All six antisera specifically reacted with VCP in IPs (even-numbered lanes) and immunoblots (data not shown), while the corresponding preimmune sera did not (odd-numbered lanes). The VCP-containing complexes were disrupted by boiling and then subjected to a second cycle of IPs with various Ikappa Balpha antisera (Fig. 2B, lanes 5, 6, 8, 9, 11, and 12) and Ub antiserum (lanes 7, 10, and 13). Although anti-VCP-3 precipitated a lesser amount of high Mr proteins than the other antisera (lanes 4 and 11-13), as predicted, high Mr forms of Ikappa Balpha , but not the 36-kDa Ikappa Balpha , were reprecipitated (lanes 5-13). By contrast, second IPs using control preimmune serum did not detect an appreciable amount of high Mr proteins (data not shown). Predominant Ub-Ikappa Balpha species with molecular mass of 90 and 85 kDa were again detected. The detection of 90-kDa Ub-Ikappa Balpha was not a result of cross-reactivity of antiserum to VCP, because it was not reactive to anti-VCP in a subsequent immunoblot (bottom panel). Taken together, these results indicate that, in cell lysates, VCP and high Mr Ub-Ikappa Balpha are present in the same complexes.


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Fig. 2.   Generation of VCP antisera and co-immunoprecipitation of high Mr Ub-Ikappa Balpha in VCP immune complexes. A, DB cells were metabolically labeled with [35S]methionine/cysteine and lysed in RIPA buffer containing 0.1% SDS. The cell lysates were immunoprecipitated in the same buffer with the specified preimmune sera (P) or the corresponding anti-VCP immune sera (I). Immune complexes were separated by SDS-PAGE, transferred to membrane, and visualized by autoradiography. B, 35S-labeled DB cell lysates were subjected to single IPs (lanes 1-4) or sequential double IPs (lanes 5-13) with the specified antisera. Three times as much lysates were used in each of lanes 5-13 as in lanes 1-4. After SDS-PAGE, Western transfer, and autoradiography (top; lanes 1-4 are from a 2-day exposure, and lanes 5-13 are from a 20-day exposure), the membrane was immunoblotted (IB) with anti-VCP-3 (bottom).

Ub-Ikappa Balpha Physically Associates and Co-sediments with VCP in the 19 S Fractions, while the Unmodified Ikappa Balpha Sediments in Fractions Deficient in VCP-- To further characterize the association of VCP with Ub-Ikappa Balpha , ultracentrifugation-fractionation analyses were performed (Fig. 3, A and B). Human CA46 lymphoma cells were lysed, and the lysate was separated by 10-40% glycerol density gradient centrifugation and fractionated. The odd-numbered fractions were analyzed by SDS-PAGE followed by immunoblotting with anti-VCP-2 immune serum (Fig. 3A, part a). The majority of VCP (fractions 7-15) migrated similarly to the marker thyroglobulin (data not shown) with an apparent sedimentation coefficient of 19 S, consistent with the previous report (24). Since thyroglobulin has a molecular mass of 650 kDa, it is compatible with the previous findings that VCP and other ATPase family proteins consist of homohexamers, reminiscent of the oligomeric nature of molecular chaperones. When the same fractions were analyzed by blotting with preimmune (part b) or anti-Ikappa Balpha -1 immune serum (part c), only the high Mr Ikappa Balpha and not the unmodified Ikappa Balpha co-sedimented with VCP in the 19 S fractions (fractions 7-13). As in the previous experiments, we also detected a prominent 85-kDa (and less reproducibly, a 90-kDa) anti-Ikappa Balpha -reactive protein, representing a major species of Ub-Ikappa Balpha (also see Fig. 3B) in CA46 cells. It was noted that the peak fractions of VCP (fractions 7-9) did not exactly correspond to the peak fractions of the high Mr Ikappa Balpha (fractions 11-13). However, it is likely that VCP may exist in different populations, some of which can bind substrates other than Ub-Ikappa Balpha ; thus, the peak fractions of VCP and Ikappa Balpha may not be identical. Contrary to the high Mr Ikappa Balpha , the unmodified 36-kDa Ikappa Balpha was detected only in the later fractions (fractions 17-19) virtually lacking VCP. Furthermore, anti-Ikappa Balpha -1 IP followed by anti-VCP-2 immunoblotting (part d) showed that VCP physically associated with the high Mr Ikappa Balpha , and these complexes sedimented in the 19 S fractions (fractions 7-13), whereas the unmodified Ikappa Balpha did not associate with VCP and sedimented in fractions deficient in VCP.


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Fig. 3.   Co-sedimentation and physical association of Ub-Ikappa Balpha with VCP, and no binding of VCP to the NF-kappa B p50·p65 complexes. CA46 cells were lysed with RIPA buffer without SDS, and the cleared lysate was centrifuged through a 10-40% glycerol gradient. Twenty-six 0.5-ml fractions, with fraction 1 being the bottom and fraction 26 being the top, were collected. A, an aliquot of 40 µg of protein from each odd-numbered fraction was resolved by SDS-PAGE, electrophoretically transferred onto a membrane, and immunoblotted (IB) with anti-VCP-2 (part a). An identical membrane was immunoblotted first with preimmune (part b) and then with immune anti-Ikappa Balpha -1 serum (panel c). The gel mobilities of molecular size standards (in kDa), high Mr Ub-Ikappa Balpha , and the unmodified 36-kDa Ikappa Balpha are indicated. The open triangles indicate a nonspecific reactivity also detected by the preimmune serum, and the dot to the left of part c indicates the 85-kDa Ub-Ikappa Balpha . Half of each odd-numbered fraction was immunoprecipitated (IP) with anti-Ikappa Balpha -1, and the immune complex was analyzed by SDS-PAGE followed by immunoblotting with anti-VCP-2 (part d). B, equal amounts from fractions 7-13 and 17-20 were combined and named 19 and 4.5 S pools, respectively. Half of each pool was immunoprecipitated with anti-Ikappa Balpha -1 or anti-VCP-1 as indicated at the top of part a. The immune complexes were subjected to SDS-PAGE and immunoblot analysis with anti-VCP-2 (part a), anti-Ikappa Balpha -1 (part b), or anti-Ub (part c). Only the upper portions of the blots are shown in parts a and c. The open triangles mark nonspecific reactivities that could be detected with control sera. Gel mobilities of molecular mass standards (in kDa), VCP, Ub-Ikappa Balpha conjugates, Ig, and unmodified Ikappa Balpha are indicated. C, human CA46 cells, which contain constitutively activated NF-kappa B, were used in IPs with antisera specific to p50 (40, 41) (lanes 1-3), p65 (40) (lanes 4-6), or anti-Ikappa Balpha -1 (lanes 7-9). Half of each precipitate was used in SDS-gel analysis (lane 1, p50; lane 4, p65; lane 7, Ikappa Balpha ). The other half was incubated with an equal amount of in vitro-translated VCP (lane 10) at room temperature for 1 h, and the precipitates (lanes 2, 5, and 8) were removed from the supernatant (lanes 3, 6, and 9), and analyzed separately. The samples were analyzed by SDS-PAGE, transferred to membrane, and immunoblotted with anti-VCP2. The heavy bands detected at the bottom of the filter represent Ig heavy chain reactivity.

To further characterize the high Mr Ikappa Balpha molecules, 19 S fractions (fractions 7-13) and 4.5 S fractions (fractions 17-20) were pooled and analyzed by IP followed by immunoblotting (Fig. 3B). In the 19 S pool, only high Mr Ikappa Balpha , but not 36-kDa Ikappa Balpha , was detected in the Ikappa Balpha IP (part b, lane 1), and a significant level of VCP was co-precipitated with high Mr Ikappa Balpha (part a, lane 1). The prominent 85-kDa high Mr Ikappa Balpha was also reactive to Ub antiserum (part c, lane 1), confirming that it is a Ub-Ikappa Balpha conjugate. As in Fig. 1C, anti-VCP-1 did not co-precipitate detectable amounts of Ikappa Balpha (part b, lane 2). This was partially a result of competition between the antibody and the Ikappa Balpha molecules for binding to the same C terminus of VCP (data not shown). In the 4.5 S pool, the majority of Ikappa Balpha was unmodified (part b, lane 3, marked as Ikappa Balpha ). Although a Ub-Ikappa Balpha conjugate with a molecular mass slightly smaller than 85 kDa was also detected, no VCP was co-precipitated (part a, lane 3). Actually, these conjugates were present in fractions 15-17, which were deficient in VCP (Fig. 3A, part c). A low level of VCP was detected in IPs of the 4.5 S pool (part a, lane 4). This probably represents monomeric or dimeric VCP, which does not readily associate with Ikappa Balpha . In summary, these results indicate that, in vivo, VCP preferentially binds Ub-Ikappa Balpha over the unmodified 36-kDa Ikappa Balpha , in agreement with a chaperone role proposed for VCP.

NF-kappa B p50·p65 Complexes Are Not Associated with VCP-- It has been reported that Ub-Ikappa Balpha remained bound to NF-kappa B in vitro (8) but dissociated from NF-kappa B in vivo (14). Thus, it is possible that ubiquitination per se is not sufficient to dissociate Ikappa Balpha from NF-kappa B complexes, whereas VCP, which is lacking in that specific in vitro assay, may serve as a molecular chaperone that releases Ub-Ikappa Balpha from NF-kappa B in vivo. We examined whether the VCP-bound Ub-Ikappa Balpha conjugates were free from or bound to NF-kappa B in vivo and whether exogenously added VCP could bind to the purified NF-kappa B complexes (Fig. 3C). Immune complexes precipitated by various antisera reactive to different regions of p50 and p65 did not contain detectable levels of VCP (one such example was shown in lanes 1 and 4 for p50 and p65, respectively) or Ub-Ikappa Balpha (data not shown). Further incubation of NF-kappa B precipitates with VCP did not result in detectable binding of VCP to NF-kappa B (lanes 2 and 5) or diminution (lanes 3 and 6) of the input free VCP (lane 10). By contrast, Ikappa Balpha immune complexes contained significant amounts of VCP (lane 7), and almost all of the added VCP could further bind to the complexes (lanes 8 and 9). These results suggest that NF-kappa B p50·p65 immune complexes do not contain detectable levels of VCP (Fig. 3C) or Ub-Ikappa Balpha conjugates (Ref. 14; data not shown) in vivo. The NF-kappa B·Ub-Ikappa Balpha complex detected in vitro by Chen et al. (8) is probably an unstable intermediate that is readily dissociated by VCP in vivo (see model in Fig. 9).

Ubiquitinated Ikappa Balpha Binds to VCP in Vitro-- To further characterize the VCP-Ikappa Balpha association, we performed in vitro binding assays using GST-Ikappa Balpha and GST-VCP full-length fusion proteins. VCP (Fig. 4, lane 1) and Ikappa Balpha (lane 4) were in vitro translated in the presence of [35S]methionine/cysteine in rabbit reticulocyte lysates and incubated with glutathione-Sepharose beads containing either GST or GST fusion proteins. Translated VCP and Ikappa Balpha bound to GST-Ikappa Balpha (lane 3) and GST-VCP (lane 7; also see "Discussion"), respectively, but not to the control GST (lanes 2 and 6). PhosphorImager (Molecular Dynamics) scanning showed that about 5-10% of the input VCP was bound to GST-Ikappa Balpha beads, and 15% of the input Ikappa Balpha bound to GST-VCP beads. A longer exposure showed a smeary pattern at the high Mr region in lanes 4 and 7 (data not shown), suggesting that high Mr species of Ikappa Balpha , presumably the Ub-Ikappa Balpha conjugates, were present in the in vitro translated product and could associate with GST-VCP. The reticulocyte lysate is known to contain enzymes required for ubiquitin conjugation; therefore, it is capable of supporting ubiquitination of Ikappa Balpha in the in vitro translation reaction. To demonstrate the binding between the high Mr Ub-Ikappa Balpha and VCP, we performed Ub conjugation reactions (8, 16) on the translated Ikappa Balpha to increase the yield of Ub-Ikappa Balpha conjugates (lane 5) and then used the products in binding assays (lanes 8 and 9). Similar to the long exposure of lane 7, a smeary pattern above and including Ikappa Balpha was detected in VCP-bound Ikappa Balpha (lane 9), and scanning analysis showed an approximately 40% binding efficiency, significantly higher than that of the non-Ub-enriched preparations. Subsequent anti-Ikappa Balpha (lanes 10-13) and anti-Ub (lanes 14-17) immunoblotting confirmed that these high Mr proteins were Ub-Ikappa Balpha conjugates. The dissimilar patterns observed in lanes 13 and 17 could result from the difference in the number of epitopes recognized by different antibodies. The high Mr Ub-Ikappa Balpha molecules have many more Ub-specific epitopes than Ikappa Balpha -specific epitopes and therefore should have a stronger anti-Ub reactivity. More importantly, VCP probably also bound to ubiquitinated proteins other than Ub-Ikappa Balpha in the assay mixture, resulting in a higher reactivity in lane 17 than in lane 13. The immunoreactivity was specific to Ub-Ikappa Balpha , because GST-VCP alone was not reactive to either antiserum (data not shown). Furthermore, when B cell lysates were used in the binding assays (lanes 18-21), abundant high Mr Ub-Ikappa Balpha conjugates also specifically bound to VCP (lanes 19 and 21). Notably, among the VCP bound Ub-Ikappa Balpha proteins, a major 85-kDa species was again detected in cell lysates and in in vitro translated Ikappa Balpha (marked by dots in lanes 13, 17, 19, and 21; also see Figs. 1-3), representing a prominent species of Ub-Ikappa Balpha that bound to VCP.


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Fig. 4.   Association of ubiquitinated Ikappa Balpha with VCP in vitro. VCP (lane 1) and Ikappa Balpha (lane 4) were in vitro translated in the presence of 35S (designated as *) and assayed for binding with glutathione-Sepharose beads containing GST (lanes 2 and 6), GST-Ikappa Balpha (lane 3), or GST-VCP (lane 7) fusion proteins. The in vitro translated Ikappa Balpha was further treated in a Ub conjugation reaction to enrich Ub-Ikappa Balpha (lane 5) and assayed for VCP-binding (lanes 8 and 9; Ub (superior type) indicates the Ub conjugation reaction). After binding and three washes, the bound products and the input proteins were analyzed by SDS-PAGE, Western transfer, and autoradiography (lanes 1-9). Identical filters containing lanes 6-9 were subsequently immunoblotted (IB) with anti-Ikappa Balpha -1 (lanes 10-13), or anti-Ub (lanes 14-17). Glutathione-Sepharose beads containing GST or GST-VCP were incubated with unlabeled CA46 lysates (in RIPA buffer without SDS), and the washed beads were analyzed by SDS-PAGE and transferred onto membrane. Identical membranes were immunoblotted with anti-Ikappa Balpha -1 (lanes 18 and 19), or anti-Ub (lanes 20 and 21) antiserum. Gel mobilities of Mr standards, VCP, and Ikappa Balpha are indicated. The multiple proteins smaller than VCP (lane 1) are proteolytic products. The prominent protein marked by a dot in lanes 13, 17, 19, and 21 has a molecular mass of 85 kDa.

Phosphorylation and Ubiquitination of Ikappa Balpha Are Critical for VCP Association, and both N- and C-terminal Domains of Ikappa Balpha Are Required for Ikappa Balpha Degradation-- The N-terminal serines 32 and 36 (7, 10, 11, 15) and lysines 21 and 22 (12, 13, 15) have been shown to be the major signal-induced phosphorylation and ubiquitination sites, respectively. To demonstrate the physiological significance of VCP in the Ikappa Balpha degradation pathway, we studied how Ikappa Balpha mutations that blocked the upstream events, specifically phosphorylation and ubiquitination, affected the VCP association (Fig. 5A) and the subsequent degradation (Fig. 5B; summarized in Fig. 5C). We subjected wild-type and mutant Ikappa Balpha to Ub conjugation reactions (Fig. 5A, lanes 2-7) before performing VCP binding assays (lanes 8-21). In agreement with the previous reports, the constructs with intact phosphorylation and ubiquitination sites were further ubiquitinated (lanes 2 and 5), and no significant ubiquitination was detected in Ikappa Balpha with mutated phosphorylation sites (S32A/S36A, lane 6), mutated ubiquitination sites (K21R/K22R, lane 7), or Ikappa Balpha lacking both sites (37-317, lane 4). Interestingly, the mutant Ikappa Balpha -(23-317), although missing the major ubiquitination sites Lys21 and Lys22, was detectably ubiquitinated (lane 3). This result agrees with a similar finding by DiDonato et al. (15) and suggests that other Lys residues in the molecule could accept Ub. The subsequent binding experiments revealed that while all of the constructs capable of ubiquitination could bind to VCP (lanes 9, 11, 13, and 17), the ubiquitination-defective mutants all failed to bind VCP (lanes 15, 19, and 21). Taken together, the data indicate that phosphorylation and ubiquitination of Ikappa Balpha are critical for VCP association, and Ikappa Balpha ubiquitination is necessary for VCP binding (see Fig. 5C).


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Fig. 5.   Requirement of Ikappa Balpha phosphorylation and ubiquitination for VCP association and requirement of both N- and C-terminal domains of Ikappa Balpha for degradation. A, in vitro translated, 35S-labeled wild-type (lane 1) and various mutants of Ikappa Balpha were subjected to Ub conjugation reactions (lanes 2-7). The reaction products were used in the binding assays with beads containing either GST control (even-numbered lanes from lane 8 to lane 21), or GST-VCP (odd-numbered lanes from lane 8 to lane 21). The samples were analyzed by SDS-PAGE, transferred onto membrane, and visualized by autoradiography. The closed circle, the open circle, and the arrowhead indicate the translation products of constructs 23-317, 37-317, and 1-242, respectively. The faint bands indicated by open triangles are nonspecific reactivities that are present in most reactions. B, in vitro translated, 35S-labeled wild-type (lanes 1-4) and deletion mutants (lanes 5-8 for mutant 23-317; lanes 9-12 for mutant 37-317, and lanes 13-16 for combination) of Ikappa Balpha were used in the in vitro Ub-Pr degradation assay. The discrete bands represent the undegraded substrates remained in the reactions at the indicated time points. C, the results from in vitro ubiquitination, VCP-binding, and degradation assays were summarized. The data on phosphorylation were based on published reports (Refs. 7-15; see Introduction).

Using the in vitro Ub-Pr degradation assays (9), we further examined how these Ikappa Balpha mutants affected the ultimate degradation (Fig. 5B). In this assay, in vitro translated [35S]cysteine-labeled Ikappa Balpha was used as the substrate, 50 µg of protein from the S100 fraction extracted from B cells was used as the enzyme source, and the disappearance/degradation of the substrate was assayed. The wild-type Ikappa Balpha was readily degraded (Fig. 5B, lanes 1-4), indicating that the assay system contained all of the needed ingredients to facilitate the degradation. Compatible with the model, the mutants that could not bind VCP all failed to be degraded (mutant 37-317 is shown in lanes 5-8 and 13-16; other mutants are summarized in Fig. 5C). However, mutants that could bind VCP were not necessarily degraded (mutant 1-242 is shown in lanes 9-16). These results suggest that VCP binding is necessary but not sufficient for Ikappa Balpha degradation. The failure in degradation is not a result of the lack of needed reagents in the in vitro assay but a result from the mutations in the substrates themselves. Consistent with a previous report (47) that both N- and C-terminal domains of Ikappa Balpha were required for degradation, we also found that both N- (mutant 23-317) and C- (mutant 1-242) terminally truncated mutants, although capable of VCP binding, were resistant to degradation. The assays also showed that the required N- and C-terminal domains have to be on the same molecule, because incubating the two mutants together did not restore the degradation (lanes 13-16).

The Level of VCP Correlates with the Proteolytic Activity in the Ub-Pr Assay-- To further demonstrate the functional involvement of VCP in Ikappa Balpha degradation at a biochemical level, we correlated the proteolytic activity (Fig. 6A) with the level of VCP (Fig. 6C) contained in purified cell lysates. Pr+ and Pr- fractions were isolated from the cytosolic S100 fraction of the human B cell line, and an equal amount of protein from each fraction was resolved by SDS-PAGE and analyzed by immunoblotting. Preimmune serum showed no reactivity (Fig. 6B), whereas anti-VCP-2 immune serum clearly detected VCP in Pr+, S100, and cytosolic fractions but not in the Pr- fraction (Fig. 6C). The in vitro Ub-Pr degradation assay was used to determine the Ub-dependent proteolytic activity of each fraction. The proteolytic activity measured by the disappearance of the substrate Ikappa Balpha (Fig. 6A) at 30 and 60 min correlated with the level of VCP (Fig. 6C), being the highest in Pr+ (lanes 1), the lowest in Pr- (lanes 2), and higher in the S100 fraction (lanes 3) than in the crude cytosol (lanes 4).


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Fig. 6.   Correlation of VCP level with Ikappa Balpha -degrading activity. A crude cytosolic fraction (Cyt) was prepared from CA46 cells. The S100, Pr-, and Pr+ fractions were further isolated from the cytosolic fraction. The Ub-Pr in vitro assay was performed using 35S-labeled Ikappa Balpha as the substrate and an aliquot of 50 µg of protein from each fraction as the enzyme source (panel A). After 30 (upper panel) or 60 (lower panel) min, the reaction was terminated by boiling in the SDS-gel buffer and analyzed by SDS-PAGE, electrophoretic transfer, and autoradiography. The starting level of Ikappa Balpha was the same as that in lane 2. To determine the level of VCP, an aliquot of 50 µg of protein from each fraction was separated on SDS-gel and transferred onto membrane, and the membrane was immunoblotted with preimmune (panel B) or anti-VCP-2 (panel C) serum. To deplete the VCP complexes, the S100 fraction was subjected to three rounds of anti-VCP-1 IP in RIPA buffer without SDS. One-fifth of each precipitate was used to determine the completeness of VCP depletion by SDS-PAGE and immunoblotting with anti-VCP-2 (panel D). The supernatant from the third IP was further precipitated with anti-VCP-1 (lane 5) to detect residual VCP. The amount of S100 analyzed in lane 1 was one-fifth of that used in the immunodepletion. Four-fifths of the precipitates were used to determine the proteolytic activity (panel E). The control S100 (lane 1), the washed VCP immune complexes (IPs 1-3) (lanes 2-4), and the final supernatant (number 3) (lane 5) were used as the enzyme sources to test their proteolytic activities in the in vitro Ub-Pr assays. After 1 h of assay, reactions were stopped, resolved by SDS-PAGE, transferred onto membrane, and visualized by autoradiography. Lane C shows the input level of 35S-labeled Ikappa Balpha used in the assay. An equivalent amount of the S100 as was used for immunodepletion was used in the assay shown in lane 1.

VCP Antibodies Deplete Proteasome Activity, While Adding Back Immunopurified VCP Complexes Restores the Proteolytic Activity-- We further determined if depletion of VCP (Fig. 6D) resulted in a loss of proteasome activity (Fig. 6E). VCP-containing complexes were removed from the S100 fraction by subjecting it to repeated cycles of anti-VCP-1 IPs. After the third IP, virtually no VCP could be further precipitated (Fig. 6D, lane 5). When the VCP-depleted lysate was used as the enzyme source in the in vitro degradation assay, no Ikappa Balpha -degrading activity was detected, as shown by the undiminished level of substrate that remained at the end of the assay (Fig. 6E, lane 5). In contrast, when the VCP complexes obtained from each of the first three IP cycles were added back to the VCP-depleted lysate, the Ikappa Balpha -degrading activity was restored, as shown by the loss of the input substrate at the end of the assay (Fig. 6E, lanes 2-4). These results demonstrate that VCP is necessary for Ikappa Balpha degradation and can be a functional component of the Ub-Pr pathway involved in Ikappa Balpha degradation.

VCP Co-migrates with Subunit 2 of the Proteasome in Biochemically Purified 26 S Proteasome and Is Co-immunoprecipitated with the 26 S Proteasome-- The 26 S proteasome can be resolved into many subunits by SDS-PAGE (Refs. 31, 32, and 48; Fig. 7B, part a; Fig. 8A, lane 6). Based on the observation that the molecular mass of VCP is similar to that of S2 of the purified 26 S proteasome, we determined the presence of VCP in 26 S proteasome preparations (Fig. 7). Partially purified (Fig. 7A, lanes 1 and 5) and highly purified (lanes 2 and 6) 26 S proteasomes isolated from human red blood cells (46) were compared with the Pr+ fraction isolated from a human B cell line (lanes 3 and 7). Coomassie Blue staining (lanes 1-3) and anti-VCP-1 immunoblotting (lanes 5-7) revealed that VCP was present in all three preparations (lanes 5-7), and VCP co-migrated with S2 of the purified proteasome (lanes 1-2; for the designation of S2, see Fig. 7B and Refs. 31, 32, and 48). To demonstrate the specificity of this analysis, Pr+ was immunoblotted with preimmune (lane 8), anti-VCP-2 (lane 9), and anti-VCP-1 (lane 10) sera and was shown to only be reactive to immune sera. To further resolve the proteins co-migrating with S2, highly purified 26 S proteasomes (as in Fig. 7A, lane 2) from human red blood cells were subjected to two-dimensional gel electrophoresis analysis (Fig. 7B). The stained filter showed a major protein with a pI around 5.1 (marked by an arrow in part a) and a more basic triplet that co-migrated to the S2 position; immunoblotting confirmed that VCP was the more acidic species (marked by an arrow in part b). The pI values of S2, cellular VCP, and in vitro translated VCP were all estimated to be between 5 and 6 (data not shown), consistent with the reported pI of 5.5 for the Xenopus counterpart, p97 (49).


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Fig. 7.   Co-purification of VCP with the 26 S proteasome. A, approximately 5 µg each of the purified 26 S proteasome (26 S-2) (lanes 1 and 5), the highly purified 26 S proteasome (26 S-1) (lanes 2 and 6), and the proteasome-enriched Pr+ fraction (lanes 3 and 7) was separated by SDS-PAGE and stained with Coomassie Blue (lanes 1-4; lane 4 shows the mobilities of the size standards of 68 and 43 kDa) or immunoblotted with anti-VCP-2 (lanes 5-7). Aliquots of 5 µg of Pr+ were analyzed by immunoblotting with preimmune serum (lane 8), anti-VCP-2 (lane 9), or anti-VCP-1 (lane 10). B, the highly purified 26 S proteasome (as 26 S-1 in A) isolated from human red blood cells was analyzed by two-dimensional isoelectric focusing and SDS-PAGE and transferred onto membrane. The membrane was stained by Ponceau S (part a) and then immunoblotted with anti-VCP-2 (part b). The sample on the left side of the gel is a one-dimensional analysis of the highly purified proteasome, and the assignments of individual subunits are indicated on the left. The stained VCP and the anti-VCP reactivity are indicated by arrows.


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Fig. 8.   Co-immunoprecipitation of VCP with subunits of the 26 S proteasome. DB cells were metabolically labeled with [35S]methionine/cysteine, and the lysates were immunoprecipitated (IP) with the specified antisera. The purified immune complexes were analyzed by SDS-PAGE, transferred onto membrane, and visualized by autoradiography (panel A). The membrane was subsequently immunoblotted (IB) with anti-VCP-3 (panel B) or anti-S4 (panel C, the lower part is a longer exposure of the upper part). The gel mobilities of the molecular mass standards, VCP, S4, and S5, are indicated. The bracket around 50 kDa marks for subunits 6-11, and the lower bracket marks for the smaller subunits. The heavy band detected in the bottom of panel C is Ig reactivity.

We further examined whether VCP was co-immunoprecipitated with the 26 S proteasome (Fig. 8). Labeled cell lysates were immunoprecipitated with antisera specific to VCP (lanes 1-3), subunit 4 (S4, lane 4), subunit 5 (S5, lane 5), or the 26 S proteasome (lane 6). Although the oligopeptide-specific anti-VCP antisera did not readily precipitate recognizable proteasome subunits (Fig. 8A, compare lanes 1 and 2 with lanes 4-6), anti-VCP-3, raised against GST-VCP full-length fusion protein, co-precipitated proteins of sizes similar to those of subunits 1-11 (lane 3) (32). Subsequent immunoblot (Fig. 8C) and reimmunoprecipitation (data not shown) analyses further confirmed the co-precipitation of VCP and S4. Correspondingly, IPs using antisera specific to S4, S5, and the 26 S proteasome all detected small amounts of VCP (Fig. 8, A and B, lanes 4-6). In conclusion, VCP is co-purified with 26 S proteasome biochemically and immunologically.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, we identified a cellular ATPase, VCP, that physically associated with Ub-Ikappa Balpha before the proteasome-mediated degradation of Ikappa Balpha . This association was detected by more than one type of Ikappa Balpha antiserum (Fig. 1A) in a variety of cells, including human B cell lines (this study), activated HeLa cells, activated T lymphocytes, and proteasome inhibitor-treated cell lines (data not shown). These results suggest that the physical complexing is a general phenomenon and not a cell line- or an antibody-specific observation. We demonstrated that VCP was physically associated with 26 S proteasome by biochemical and immunological analyses, thus providing a physical and functional link between Ikappa Balpha modification and proteolysis. We propose the following model shown in Fig. 9. In response to stimulation, the inhibitor Ikappa Balpha is hyperphosphorylated by a kinase and polyubiquitinated. The Ub-Ikappa Balpha conjugate physically associates with VCP, which displaces the NF-kappa B dimer, thus releasing the dimer for translocation into the nucleus. The Ub-Ikappa Balpha conjugate is then transferred to the 26 S proteasome, unfolded, transported into the proteolytic core, and degraded. Meanwhile, Ub and VCP molecules are released and recycled.


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Fig. 9.   A model for the Ub-Pr mediated degradation of Ikappa Balpha . In response to stimulation, Ikappa Balpha is phosphorylated and polyubiquitinated. The ubiquitinated Ikappa Balpha physically associates with VCP, which displaces the NF-kappa B dimer, thus releasing the dimer for translocation into the nucleus. The ubiquitinated Ikappa Balpha is then transferred to the 26 S proteasome, unfolded, transported into the proteolytic core, and degraded. The VCP and Ub molecules are released and recycled. The bracketed NF-kappa B·Ub-Ikappa Balpha complex is presumably an unstable intermediate that is not readily detected in vivo.

In this model, two non-mutually exclusive roles, as a molecular chaperone that transfers the Ub-Ikappa Balpha to the proteasome and/or as a physical component of the 26 S proteasome, are proposed for VCP. The hypothesis of VCP being a molecular chaperone was first suggested by our observation that when the p90(VCP)-containing Ikappa Balpha complexes were subjected to the in vitro degradation assay, only Ikappa Balpha and not p90(VCP) was degraded (9). This is consistent with the nature of a molecular chaperone, which should be recycled and not consumed after the degradation of the substrates. In addition, VCP is an ATPase and forms an oligomeric ring structure (24, 49); both are common properties of molecular chaperones. One important characteristic of a molecular chaperone is its ready association with specific substrates. If VCP is truly a molecular chaperone in Ub-Pr-mediated degradation of Ikappa Balpha , it should preferentially bind the Ub-tagged Ikappa Balpha . The in vivo IP and co-sedimentation experiments clearly showed such a substrate specificity, in that 1) Ikappa Balpha IP detected physically associated VCP and high Mr Ub-Ikappa Balpha in addition to the 36-kDa Ikappa Balpha ; 2) serial IPs using VCP followed by Ikappa Balpha antisera showed that VCP immune complexes contained high Mr Ub-Ikappa Balpha but not 36 kDa-Ikappa Balpha ; 3) VCP and the high Mr Ub-Ikappa Balpha co-sedimented in the 19 S fractions; 4) 36-kDa Ikappa Balpha sedimented in the 4.5 S fractions lacking VCP; 5) in the 19 S pool, VCP and Ub-Ikappa Balpha were co-precipitated in the same complex; and 6) in the 4.5 S pool, the majority of Ikappa Balpha was not ubiquitinated and was free from VCP. The in vitro binding experiments also showed a ready association of VCP with Ub-Ikappa Balpha . When cell lysates were used as the source of Ikappa Balpha , only Ub-Ikappa Balpha and not the 36-kDa Ikappa Balpha bound to VCP, consistent with the in vivo analysis. Furthermore, when in vitro translated Ikappa Balpha constructs with various mutations were used in the assays (Fig. 5, A and C), a perfect correlation between Ikappa Balpha ubiquitination and VCP binding was detected; all Ikappa Balpha variants capable of ubiquitination could bind VCP, and all constructs defective in ubiquitination could not. It was noted that when, and only when, the in vitro translated Ikappa Balpha was used in the in vitro binding assays, in addition to Ub-Ikappa Balpha , the 36-kDa Ikappa Balpha was also detected in VCP complexes (Figs. 4 and 5A). It is presently unclear whether the 36-kDa Ikappa Balpha genuinely binds to VCP in vitro or is detected as a consequence of rapid deubiquitination under the specific experimental condition. The recent identification of a Ub isopeptidase, a component of the 26 S proteasome, that quickly removes Ub chains to regenerate the substrate (50) supports the latter contention. In addition, the preference for the substrates with short Ub chains, commonly found in in vitro conditions, by this isopeptidase is consistent with our findings. It is also formally possible that this 36-kDa Ikappa Balpha is hyperphosphorylated but not ubiquitinated. Although not detected in vivo, this form of Ikappa Balpha may bind to VCP in vitro. Interestingly, we frequently detected binding of VCP to an 85-kDa Ub-Ikappa Balpha in cell lysates (Figs. 1-4) and in Ub-Ikappa Balpha generated from the Ub conjugation reaction (Fig. 4). In addition, we also detected prominent Ub-Ikappa Balpha species with Mr of 120 and 90 kDa (Figs. 1A, 2B, and 4). Consistently, Ub-Ikappa Balpha species with similar sizes were also detected in other studies (8, 14, 15). These Ub-Ikappa Balpha molecules probably represent major polyubiquitinated species that are preferentially bound by VCP. In summary, VCP readily binds Ub-Ikappa Balpha conjugates, requires Ikappa Balpha ubiquitination for binding, and clearly demonstrates a binding preference for them over the unmodified Ikappa Balpha in vivo, consistent with the property of a molecular chaperone.

Chen et al. (8) showed that Ub-Ikappa Balpha was still bound to NF-kappa B in vitro, while Roff et al. (14) showed that Ub-Ikappa Balpha was dissociated from p50-containing NF-kappa B in vivo. The discrepancy between the two findings suggests the existence of an in vivo-specific molecular chaperone that displaces the NF-kappa B from Ub-Ikappa Balpha . We identified VCP as the candidate chaperone to fulfill this function. Our data demonstrating VCP binding to Ikappa Balpha but not p50·p65 complexes is consistent with the model that following Ikappa Balpha ubiquitination, VCP binds the Ub-Ikappa Balpha conjugates and releases NF-kappa B to allow for its nuclear translocation. It is noted that, contradictory to the findings of Roff et al. (14), DiDonato et al. (15) reported that Ub-Ikappa Balpha remained bound to the p65-containing complexes in cells treated with proteasome inhibitor. At present, we do not have a definitive explanation for this discrepancy. However, both studies were performed in proteasome inhibitor-treated cells, in which unusually high amounts of Ub-Ikappa Balpha could be accumulated. It is conceivable that when the level of Ub-Ikappa Balpha exceeds the level of available VCP, presumably as in the latter but not in the former study, Ub-Ikappa Balpha -p65 complexes can be detected.

To demonstrate the involvement of VCP in the signaling pathway of Ikappa Balpha degradation, both mutant and biochemical studies were performed. We examined how the phosphorylation- and/or ubiquitination-defective Ikappa Balpha mutants affected the VCP binding and the ultimate degradation (summarized in Fig. 5C). A perfect correlation between ubiquitination and VCP binding was detected, supporting the model that VCP binding requires the upstream ubiquitination. Interestingly, the mutant 37-317, lacking both phosphorylation and ubiquitination sites, was defective in VCP binding; whereas the mutant 23-317, lacking the major ubiquitination sites, was capable of ubiquitin conjugation and VCP binding. This result suggested two points. First, other lysine residues in the Ikappa Balpha molecule could also accept Ub, which then signaled for VCP binding. This notion is consistent with another report (15) and is supported by studies reporting that the Ub-conjugating apparatus has the capacity to utilize alternative lysines when the primary acceptors are disrupted (51, 52). However, if alternative Lys residues can accept Ub molecules, it is unclear why the mutant K21R/K22R was defective in ubiquitination and VCP binding, although consistent with other reports. Second, the recognition of Ub by VCP is probably not highly site-specific. Ub attached to Lys residues other than the major sites Lys21 and Lys22 can also be efficiently recognized by VCP, at least in our in vitro binding assays. Presently, we do not know how VCP recognizes Ub-Ikappa Balpha conjugates and whether VCP can recognize Ub chains attached to all of the nine Lys residues in Ikappa Balpha . Nevertheless, this finding further substantiated the requirement of ubiquitination for VCP binding.

The in vitro degradation assays showed that not only were the mutants defective in VCP binding unable to be degraded, but several mutants capable of binding VCP were also resistant to degradation (Fig. 5C). These results support the model that while binding with VCP is necessary for degradation, it is not sufficient. In agreement with a previous study (47), we also found both N- and C-terminal domains of Ikappa Balpha were required for degradation. This is consistent with the findings that the C-terminal acidic PEST (Pro-Glu-Ser-Thr) sequences are often associated with rapid protein turnover and frequently found in Ub-Pr substrates. Moreover, the N- and the C-terminal domains must be present in a cis relationship, suggesting that a successful degradation requires the presence of a complete protein domain and that intramolecular conformation of the Ikappa Balpha protein may be important in the process.

All of our data are compatible with both roles proposed for VCP, one being a molecular chaperone with a distinct hexameric structure and the other being a component of the 26 S proteasome. Biochemically, VCP co-purifies with the 26 S proteasome; immunologically, VCP co-immunoprecipitates with subunits of the proteasome, all supporting the "component" role. Interestingly, the VCP antisera raised against synthetic oligopeptides co-precipitated the high Mr Ub-Ikappa Balpha but not the proteasome components. On the other hand, anti-VCP-3, which was raised against a full-length VCP fusion protein, readily co-precipitated the proteasome subunits, but only co-precipitated low levels of Ub-Ikappa Balpha . These data along with the serial IP experiments (data not shown) seem to suggest that there are at least two populations of VCP in the cell; while the peptide-specific VCP antisera may recognize the proteasome-free VCP, anti-VCP-3 may react more readily with the proteasome-bound VCP. It is conceivable that VCP may exist in a state of equilibrium between a mobile form, which presumably functions as a molecular chaperone, and a relatively immobile form that assembles on the top of the 26 S proteasome. Functionally, VCP may hydrolyze ATP (24) to provide the energy needed for the unfolding of the modified Ikappa Balpha molecule and its entry into the 20 S proteolytic core. Analogous dual activities have been demonstrated in VCP relatives, ClpA and ClpX in Escherichia coli. Both proteins appear to exhibit intrinsic ATP-dependent chaperone activities (53) and are supposed to be part of the regulatory structure on top of the cylinder-shaped Clp protease (54). These notions also bear similarities with a recent report that Ub chain-binding protein Mcb1, subunit 5a, exists in two populations, 26 S proteasome-bound and -free forms (55).

Although VCP co-purifies with the 26 S proteasome and co-migrates with S2 of the 19 S cap complex on SDS-gels, it is not identical to S2. Recently, an unrelated protein named TRAP-2 (56), corresponding to the more basic triplet of S2 on our two-dimensional gel (Fig. 7B), has been identified as S2 by protein sequencing. It is possible that there is more than one protein constituting S2. This biochemical heterogeneity observed in a protein resolved by SDS-PAGE to apparent homogeneity is precedented by the finding that S5 of the 26 S proteasome consists of two unrelated proteins, S5a and S5b (43, 44). Alternatively, the regulatory 19 S cap may not be a single homogeneous entity, and the VCP complex may represent an alternative version of the 19 S cap that confers specificity to certain substrates such as Ikappa Balpha . Thus, subunit composition and the functional capability of the 26 S proteasome may vary with physiological conditions.

VCP has been highly conserved during evolution and found in every tissue examined to date. The paradox present in the high sequence homology and the variety of functions suggests a basic and critical role for VCP/Cdc48p/p97. Recently, cell cycle progression, cytokine-induced signal transduction pathways, and endoplasmic reticulum degradation were reported to be regulated by the Ub-Pr pathways (16-30, 57, 58). By identifying the involvement of VCP in Ikappa Balpha degradation through the Ub-Pr pathway, we may provide a potential link between the 26 S proteasome and other Ub-Pr substrates, thus offering an explanation for the paradox. In conjunction with our findings, it would be interesting to examine whether VCP is also involved in other Ub-Pr-mediated processes in the context of proteasomes.

    ACKNOWLEDGEMENTS

We are grateful to Drs. L. Samelson and M. Rechsteiner for providing the critical reagents and making valuable suggestions about the manuscript. We thank Dr. D. Ballard for site-specific mutants; Dr. R. Fisher and Y. Kim for protein sequencing; Dr. J.-J. Lin for graphics; and Drs. M. Beckwith, H. F. Kung, H. Young, and J.-J. Lin for reviewing the manuscript.

    FOOTNOTES

* 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.

par To whom correspondence should be addressed. Tel. 301-846-1478; Fax: 301-846-6107; E-mail: licc{at}ncifcrf.gov.

1 The abbreviations used are: Ub-Pr, ubiquitin-dependent proteasome; Ub, ubiquitin; Ub-Ikappa beta alpha , ubiquitinated Ikappa Balpha ; IP, immunoprecipitation; RIPA, radioimmune precipitation; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; ATPgamma S, adenosine 5'-O-(thiotriphosphate); VCP, valosin-containing protein.

2 C.-C. H. Li, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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