Interaction between Glucose-regulated Destruction Domain of DNA Topoisomerase II (cid:1) and MPN Domain of Jab1/CSN5*

DNA topoisomerase (topo) II (cid:1) , an essential enzyme for cell proliferation, is targeted to a proteasome-dependent degradation pathway when human tumor cells are glu-cose-starved. Here we show that the topo II (cid:1) destabilization depends on the newly identified domain, GRDD (glu- cose-regulated destruction domain), which was mapped to the N-terminal 70–170 amino acid sequence. Indeed, the deletion of GRDD conferred a stable feature on topo II (cid:1) , whereas the fusion of GRDD rendered green fluorescent protein unstable under glucose starvation conditions. Nuclear localization was a prerequisite for GRDD function, because the inhibition of nuclear translocation resulted in the suppression of GRDD-mediated topo II (cid:1) degradation. Further, GRDD was identified as an interactive domain for Jab1/CSN5, which promoted the degradation of topo II (cid:1) in a manner dependent on the MPN (Mpr1p/ Prd1p N terminus) domain. Depleting Jab1/CSN5 by antisense oligonucleotide and treating cells with the CSN-associated

DNA topoisomerase II (topo II) 1 is an essential enzyme for eukaryotic cell proliferation and plays important roles in many aspects of the DNA process through modulating the topological states (1). In humans, topo II exists in two closely related isoforms, ␣ and ␤, which share a high degree of structural homology with a ϳ70% sequence identity (2,3). Three discrete functional domains are embedded in the topo II enzymes (1,4). The N-terminal and the central domains are responsible for ATPase and catalytic activities, respectively, and the C-terminal domain, the least conserved region, contains the nuclear localization signals (NLSs). Although the structural and the biochemical features are closely related, the topo II isoforms show a great difference in their expression characteristics (4).
The expression of topo II␣ is regulated by the state of cellular proliferation, high in proliferation and low in quiescence with a higher cell density or a lower serum concentration. The levels of topo II␣ also change within a single cell cycle, peaking in the G 2 /M phase and declining to a minimal level after M phase completion. However, the levels of topo II␤ are relatively constant throughout the cell cycle and in quiescent and proliferating states (5).
The topo II isoforms have been shown to be the molecular target for such clinically important antitumor drugs such as etoposide and doxorubicin (6,7). The topo II-directed drugs convert this enzyme into a cellular poison by stabilizing the covalent DNA-enzyme intermediates, the so-called cleavable complex. Interestingly, the increase in topo II-cleavable complexes also has been observed in the presence of a number of physiological stressors such as acidic pH, oxidation, and thiol (8,9). DNA damage resulting from the cleavable complexes is thought to lead to eventual cell death. Thus, any processes leading to a decrease in the number of cleavable complexes may have a positive impact on cell survival. In the case of topo II-directed drugs, cellular resistance can be achieved by both qualitative and quantitative changes in topo II much more frequently with the ␣-isoform. Indeed, the resistance often correlates with a decrease in topo II␣ expression levels or with mutations in the protein that reduce enzymatic activity (10). C-terminal truncations of topo II␣, which result in the loss of functional NLS and thus cytoplasmic localization of the enzyme, also have been found to correlate with resistance to topo II-directed drugs (11,12).
High levels of topo II␣ expression have been shown in a wide variety of tumors, partly due to the increased growth fraction (13). The high topo II␣ expression may provide a rationale for the use of topo II-directed drugs in clinical settings. However, most of the common solid tumors exhibit resistance to chemotherapy. Malignant cells within solid tumors are often surrounded by conditions such as glucose deprivation, hypoxia, low pH, and other forms of nutrient deprivation (8,9,14). These physiological conditions in culture can down-regulate the expression of topo II␣ with growth arrest or delay at the G 1 phase of the cell cycle (15). What we found to be consistent with the decreased topo II␣ levels was tumor cells becoming resistant to topo II-directed drugs when stressed by glucose starvation and related culture conditions (16,17). These observations suggest that the stress-mediated down-regulation of topo II␣ may be an obstacle to successful topo II-directed chemotherapy.
Recently, we have shown that inhibition of proteasome attenuates stress-induced resistance by inhibiting topo II␣ depletion (18). Topo II␣ restoration is seen only at the protein levels, indicating that topo II␣ protein depletion occurs through a proteasome-mediated degradation mechanism. In general, pro-teasome-mediated proteolysis is targeted by ubiquitylation of the substrate proteins (19). Consistently, topo II␣ can be conjugated with polyubiquitin in a cell-free system with extracts of cancer cells (20). Thus, the ubiquitin-proteasome system appears responsible for topo II␣ degradation under conditions of glucose starvation, but the regulatory mechanisms leading to topo II␣ degradation are largely unknown.
In this study, we have attempted to identify the domain of topo II␣ responsible for degradation under glucose starvation. We report herein a novel degradation signal designated glucose-regulated destruction domain (GRDD), which presents within the N-terminal ATPase domain. Our current results show that GRDD functions in the nucleus and can lead to proteasome-mediated degradation of heterologous proteins under stress conditions. We also demonstrate that Jab1/CSN5 binds to and promotes degradation of topo II␣ in a GRDD-dependent manner. Consistent with this finding was that specific inhibition of Jab1/CSN5 leads to the stabilization of endogenous topo II␣ under stress conditions.

EXPERIMENTAL PROCEDURES
Cell Culture and Treatments-HT1080 and MCF-7 cells were maintained in RPMI 1640 medium (Nissui, Tokyo, Japan), and 293T cells were maintained in Dulbecco's modified Eagle's medium (Nissui), each supplemented with 10% heat-inactivated fetal bovine serum and 100 g of kanamycin/ml. The cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2 . Glucose deprivation was achieved by substituting glucose-free RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum. PSI (carbobenzoxyl-L-isoleucyl-␥-t-butyl-L-glutamyl-L-alanyl-L-leucinal) and MG132 were purchased from the Peptide Institute Inc. (Osaka, Japan). Lactacystin and curcumin were from Kyowa Medex (Tokyo, Japan) and Sigma, respectively. These compounds were dissolved in Me 2 SO and added to culture medium so that the final concentration of Me 2 SO was Ͻ0.5%. All of the experiments were repeated at least three times using exponentially growing cells.
Expression Plasmids-For expression plasmid construction of fulllength topo II␣ and its deletion mutants from N or C terminus, the pBluescript SK ϩ plasmid containing full-length human topo II␣ cDNA (a generous gift from Dr James C. Wang, Harvard University) (21) was used as a template for PCR amplification with PfuTurbo DNA polymerase (Stratagene, LA Jolla, CA). To facilitate subcloning, a ClaI and a SalI restriction sites were introduced at the 5Ј-and 3Ј-ends, respectively. The PCR products first were cloned into the pPCR-Script Amp vector (Stratagene) and subsequently subcloned into the ClaI/SalI site of the pFLAG-CMV2 expression vector (Sigma). For construction of a ⌬714 -1287 topo II␣ internal deletion mutant, the sequence corresponding to amino acids 714 -1532 was removed from pFLAG-WT topo II␣ by BglII/SalI digestion and replaced with a PCR fragment corresponding to amino acids 1288 -1532. Further internal deletions within amino acids 1-713 were performed by replacing the sequence between the ClaI and BglII sites of pFLAG-⌬714 -1287 topo II␣ with each corresponding PCR fragment. Similarly, the expression plasmids of GFP-NLS2 fusion proteins were constructed by either replacing with or by inserting a GFP gene derived from the pCMV/Myc/nuc/GFP vector (Invitrogen). pFLAG-ND70 -170 topo II␣ was constructed using two PCR fragments (corresponding to amino acids 1-69 and 171-1532) that were introduced into the NotI-ClaI and ClaI-SalI sites at the 5Ј-and 3Ј-ends, respectively. Site-directed mutagenesis including single amino acid substitution, double amino acid substitution, and deletion up to 10 amino acids was carried out using a QuikChange mutagenesis kit (Stratagene).
The pcHA expression vector derived from pcDNA3 (Invitrogen) was described previously (22). Full-length human Jab1/CSN5 cDNA was generated by PCR from a cDNA library of HT29 cells. The PCR fragment was cloned into the pcHA expression vector at the BamHI/NotI site. A PCR fragment corresponding to amino acids 1-190 of Jab1/CSN5 also was cloned into the pcHA vector at the BamHI/NotI site. The plasmid construction of ⌬MPN Jab1/CSN5 was carried out using two PCR fragments (corresponding to amino acids 1-54 and 191-344) that were introduced to BamHI-NotI and NotI-XbaI sites at the 5Ј-and 3Ј-ends, respectively. The pcDNA3-GFP vector was described previously (23). The proper construction of all of the plasmids was confirmed by DNA sequencing. All of the plasmids for transfection were prepared using an EndoFree Plasmid Maxi Kit (Qiagen, Tokyo, Japan).
Transfection-Transfections were performed using FuGENE 6 trans-fection reagent (Roche Applied Science) according to the manufacturer's protocol with minor modifications. HT1080 cells were seeded at 5 ϫ 10 5 cells/well in a six-well plate 16 h before transfection. After changing to 2 ml of fresh culture medium, 200 l of transfection mixture containing 2 g of plasmid DNA and 12 l of FuGENE 6 reagent in 188 l of Opti-MEM (Invitrogen) was added to each well and the cells were incubated for 8 h. The cells were washed with phosphate-buffered saline and further incubated for 6 h in fresh medium. After transfection, the cells were cultivated in normal or glucose-free culture medium for the indicated periods of time.
Preparation of Nuclear and Cytoplasmic Extracts-Nuclear and cytoplasmic extracts of HT1080 cells were prepared using NE-PER TM nuclear and cytoplasmic extraction reagents (Pierce) according to the manufacturer's protocol.
Immunofluorescence Microscopy-Cells grown on a 35-mm glassbottom culture dish with a polylysine-coated glass (MatTek, Ashland, MA) were fixed in 4% formaldehyde for 10 min and permeabilized with methanol for 2 min. The fixed cells were processed for immunostaining with anti-FLAG M2 antibody (1:100 dilution) followed by a fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (1:1000 dilution). The nuclei were counterstained using 0.1 g/ml 4,6-diamino-2phenylindole. The cells were observed using a fluorescence microscope (Olympus IX-70) equipped with a CCD camera.
Yeast Two-hybrid System-We used a yeast two-hybrid system commercially available from Clontech. The coding region of the N-terminal (aa 1-200 and 1-584) of topo II␣ protein was fused in-frame with the GAL4 DNA-binding domain of the pGBKT7 vector (Clontech). The resulting bait plasmids were used to screen a human fetal brain cDNA library (Clontech) according to the manufacturer's protocol. From 1.5 ϫ 10 6 transfectants, 11 positive clones were isolated. DNA sequence analysis identified two Jab1 clones from the 11 positive clones obtained.
In Vitro Protein-binding Assay-The full-length and deletion mutants cDNAs of Jab1/CSN5 were amplified by PCR and inserted into a pGEX-5 bacterial expression vector (Amersham Biosciences) in-frame with GST. After DNA sequencing, GST-fused Jab1/CSN5 proteins were purified with a GSH-Sepharose (Amersham Biosciences) according to the manufacturer ' s instructions. FLAG-tagged topo II␣ proteins were expressed in 293T cells by transfection with SuperFect ® reagent (Qiagen). The FLAG-tagged topo II␣ proteins were immunoprecipitated using anti-FLAG M2 affinity gel (Sigma) and eluted from the resin with a FLAG peptide according to the manufacturer ' s instructions. The immunopurified FLAG-tagged topo II␣ proteins (100 ng) were added directly to GST-Jab1/CSN5 fusion proteins immobilized on the GSH beads (100 ng). The mixtures were incubated overnight at 4°C in a total volume of 1.5 ml of a binding buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100. After washing three times with the binding buffer, the protein complex was analyzed by SDS-PAGE and subsequent Western blot using anti-FLAG M2 and anti-GST antibodies (Amersham Biosciences).
Antisense Morpholino Oligomers-A morpholino antisense oligomer specific for human Jab1/CSN5 as well as a standard control oligomer was purchased from Gene Tools (Philomath, OR). The sequences were 5Ј-CGGACGCCGCCATCGCCGAGGAAG-3Ј for Jab1/CSN5 and 5Ј-CCTCTTACCTCAGTTACAATTTATA-3Ј for the control. Antisense treatments were performed according to the manufacturer's "special delivery protocol." HT1080 and MCF-7 cells were seeded at 6 ϫ 10 5 and 9 ϫ 10 5 cells/well, respectively, in a six-well plate 16 h before antisense treatment. Antisense mixtures with the ethoxylated polyethylenizamine special delivery solution were added to the cultures, and cells were incubated for 3 (HT1080) or 6 h (MCF-7). The cells were washed once with phosphate-buffered saline and cultured in normal or glucose-free medium for 18 (HT1080) or 24 h (MCF-7), and the whole cell lysates were prepared for analyzing expression of Jab1/CSN5 and topo II␣.

ATPase Domain and NLS Are Indispensable for Stress-induced
Degradation of Topo II␣-When cells are cultured under the stress condition of glucose starvation, topo II␣ is degraded in a proteasome-dependent manner. Indeed, degradation of topo II␣, but not topo II␤, was observed in glucose-starved HT1080 cells (Fig. 1A). Degradation of topo II␣ was suppressed by proteasome inhibitor PSI at 3 M but not by calpain inhibitor, carbobenzoxyl-L-leucyl-L-leucinal (Z-LL-H), at 50 M. Likewise, wild-type topo II␣ tagged with the FLAG epitope at the N terminus was degraded during glucose starvation when it was expressed transiently in HT1080 cells by transfection (Fig. 1C).
To determine which regions of topo II␣ were required for the degradation, we constructed a panel of expression plasmids of topo II␣ deletion mutants (Fig. 1B) and transfected them into HT1080 cells. Glucose starvation-induced degradation was monitored by Western blot analysis using the anti-topo II␣ C terminus and anti-FLAG M2 antibodies (Fig. 1C). Deletion mutants from the N terminus (⌬1-675, ⌬1-1007, and ⌬1-1287) revealed that the deletion of the ATPase domain led to degradation resistance under glucose starvation conditions. Deletion of the NLS2 region (⌬NLS2) from the C terminus also resulted in stabilization under the same conditions. In contrast, the ⌬714 -1287 mutant containing the ATPase domain and the NLS2 region was destabilized by glucose starvation, as was wild-type topo II␣. We also found that deletion mutants showed some differences in expression under the normal growth conditions. Although the exact reason is not known, we speculate that these differences are probably a result of alterations in the basal stability of each mutant topo II␣ protein.
Subcellular Localization of Topo II␣ Deletion Mutants-Immunostaining with anti-FLAG antibody demonstrated that wild-type topo II␣ localized in the nuclei of HT1080 cells in a predominantly diffusing manner ( Fig. 2A). The ⌬714 -1287 mutant topo II␣, which was sensitive to the stress-induced degradation, also localized in the nucleus, and the pattern of immunostaining was similar between wild-type and ⌬714 -1287 mutant topo II␣. In contrast, ⌬NLS2 topo II␣, which was resistant to the stress-induced degradation, accumulated mainly in the cytoplasm. A cell fractionation experiment revealed that ⌬NLS topo II␣ was more abundant in the cytoplasmic fractions than the WT topo II␣ (Fig. 2B). The action was in agreement with the fact that the NLS2 sequence is the major functional motif for nuclear translocation of topo II␣ (24, 25). Taken The cellular protein levels of topo II␣ and ␤ were determined by immunoblotting. B, schematic representation of WT and deletion mutants of FLAG-tagged topo II␣ proteins. Degradation of each protein under glucose starvation was analyzed by immunoblot and is summarized on the right (open circle, degradation and; crisscross, no or little degradation). C, HT1080 cells were co-transfected with the indicated plasmids of FLAG-tagged topo II␣ (1.5 g) and GFP proteins (0.5 g). After a 20-h treatment with glucose starvation (Ϫ), the expression of each topo II␣ protein was examined by immunoblot analysis using anti-topo II␣ C terminus and anti-FLAG and anti-GFP antibodies. The GFP expression levels were determined as a transfection efficiency and loading control. Note: FLAG-tagged topo II␣ (WT) co-migrated with endogenous topo II␣ (upper).
together, these results indicated that nuclear localization of topo II␣ was a prerequisite for degradation of this enzyme under glucose starvation conditions. Consistent with these findings, endogenous topo II␣ remained in the nucleus of stressed cells when degradation of topo II␣ was inhibited by proteasome inhibitors (Fig. 2C).
Analysis of Topo II␣ ATPase Domain for Stress-induced Degradation-The above results suggested that the sequence within the ATPase domain could affect topo II␣ stability under glucose starvation stress conditions. To determine the critical region, we constructed deletion mutants within the ATPase domain that were fused with the topo II␣ NLS2 domain for nuclear localization. Stress-induced degradation of the fusion proteins was monitored as described above. As shown in Fig.  3A, aa 1-584, 1-201, 1-170, and 1-140 of ATPase-NLS2 fusion proteins were destabilized by glucose starvation but the aa 1-130 fusion protein remained stable compared with the others. We further examined the N-terminal deletions of the aa In B, HT1080 cells were co-transfected with the expression plasmid of N-terminal deletion mutants of aa 1-170 ATPase-NLS2 fusion and GFP proteins. After a 20-h culture under glucose-free conditions (Ϫ), the expression of each topo II␣ protein was examined by immunoblot analysis using anti-topo II␣ C terminus and anti-FLAG antibodies (right) as indicated. In B, GFP expression levels also were determined by anti-GFP antibody for loading control.

FIG. 2. Subcellular localization of wild-type and mutant topo II␣ proteins.
A, HT1080 cells were transfected with expression plasmids of FLAG-tagged topo II␣ proteins as indicated. Twenty h after transfection, the immunofluorescence staining was carried out with anti-FLAG antibody (upper) and 4,6-diamino-2-phenylindole (DAPI, lower). Merged images (middle) are shown. B, HT1080 cells were transfected with expression plasmids of FLAG-tagged topo II␣ proteins as indicated and were cultured for 20 h under normal (ϩ) or glucose starvation conditions (Ϫ). Nuclear (Nu) and cytoplasmic (Cy) extracts from the transfected cells were isolated, and the protein levels were determined by immunoblot analysis using anti-FLAG, anti-actin (c-2) horseradish peroxidase, and anti-GRP78 horseradish peroxidase antibodies. Actin and GRP78 protein levels also were determined for a loading control. C, HT1080 cells were cultured for 20 h under normal (ϩ) or glucose starvation conditions (Ϫ) and were treated with MG132 or lactacystin during the last 6 h. The protein levels of topo II␣ of nuclear (Nu) and cytoplasmic (Cy) extracts were determined by immunoblot analysis using anti-topo II␣ C terminus antibody.
Interestingly, the 1-69 deletion, both in full-length topo II␣ and in the aa 1-170 ATPase-NLS2 fusion protein, decreased its expression level under normal growth conditions as compared with the 1-42 deletion (Fig. 3, B and C). The 1-42 deletion itself had little effect on the expression levels under normal conditions (data not shown). Conversely, the 1-124 deletion greatly increased the expression levels in the aa 1-170 ATPase-NLS2 fusion protein, although it had only a marginal effect on the full-length topo II␣ expression under normal conditions (Fig. 3, B  and C). These results indicate that the N-terminal regions can affect the stability of topo II␣ under normal growth conditions.

Identification of GRDD, a Novel Transposable Degradation
Signal-This deletion analysis implied that the minimal region necessary for the stress-induced degradation was located between 70 and 140 amino acid residues of topo II␣ ATPase domain. Indeed, topo II␣ became resistant to the stress-induced degradation when the 70 -170 residues were deleted (Fig. 4A). It is important to note that ND70 -170 topo II␣ localized in the nucleus (see Fig. 2). We designated the 70 -170 region of topo II␣ as GRDD. To evaluate the GRDD of topo II␣ as a stress-specific degradation signal, we constructed GFP fusion proteins that were fused with topo II␣ 1-170 or 70 -170 residues at the N terminus and topo II␣ NLS2 domain at the C terminus for nuclear localization (Fig. 4B). The GFP-NLS2 fusion protein (not containing GRDD) was expressed at similar levels under normal and glucose starvation conditions, showing that this fusion protein was insensitive to the mechanism (or mechanisms) of the stress-induced topo II␣ degradation. By adding GRDD to GFP-NLS2 (aa 1-170 and 70 -170), we found that the protein became vulnerable to degradation under glucose starvation conditions as was observed for topo II␣. Thus, the GRDD of topo II␣ can act as a transposable stress-specific degradation signal. , the expression of each protein was examined by immunoblot analysis using anti-topo II␣ C terminus and anti-GFP and anti-FLAG antibodies as indicated. A, analysis of WT and ND70 -170 topo II␣ that lacks the GRDD region (amino acids 70 -170). Expression plasmids of FLAG-tagged topo II␣ and GFP protein were co-transfected into HT1080 cells, and GFP protein levels also were determined for a transfection efficiency and loading control. B, analysis of FLAG-tagged GFP chimera proteins that were fused with no residues or topo II␣-(1-170) or topo II␣-(70 -170) residues at the N terminus and topo II␣ NLS2 domain at the C terminus. C, analysis of destruction box-like sequence in GRDD using ⌬714 -1287 topo II␣ proteins, which were introduced the deletion mutation of amino acids 112-120 (⌬DB).

FIG. 5. GRDD-dependent topo II␣ degradation by Jab1/CSN5.
A, WT, ND70 -170, and ⌬NLS2 topo II␣, as well as aa 1-584 and aa 125-170 ATPase-NLS2 fusion proteins, were immunopurified and were pulled-down by GST or GST-Jab1/CSN5 immobilized on GSH beads. Bound topo II␣ proteins were detected by immunoblot analysis using anti-FLAG antibody. The input lanes were loaded with one-tenth the amount of FLAG-tagged topo II␣ protein used in the binding reactions. B, HT1080 cells were co-transfected with expression plasmids encoding full-length (WT) or ND70 -170 of FLAG-topo II␣ (1 g) together with HA-Jab1/CSN5 or no insert (1 g). The cells were cultured under normal (ϩ) or glucose-free conditions (Ϫ) for 20 h, and the whole cell lysates were examined by immunoblot analysis using anti-FLAG and anti-HA antibodies. C, HT1080 cells were co-transfected with expression plasmids encoding aa 1-584 ATPase-NLS2 topo II␣ (1 g) and HA-Jab1/CSN5 or no insert (1 g). The cells were cultured under normal growth conditions for 24 h and were treated with the indicated inhibitors or Me 2 SO (no inhibitor) during the last 8 h. As a control, the cells also were cultured for 20 h under glucose-free conditions. The whole cell lysates were examined as in B.
Topo II␣ has a destruction box-like sequence that is located within GRDD (26). In fact, the amino acid sequence, ENNLISIWN, located between 112 and 120 of topo II␣ is similar to the consensus sequence of the cyclin B-type destruction box, RXXLXXIXN. To examine the contribution of the destruction box-like sequence, we used ⌬714 -1287 topo II␣, which consisted of the entire ATPase domain and the NLS2 region (Fig. 1). The deletion of amino acids 112-120 (⌬DB) within the GRDD of topo II␣ had no effect on stress-induced degradation of ⌬714 -1287 topo II␣ (Fig. 4C). We concluded that the destruction box-like sequence in GRDD is dispensable for topo II␣ degradation under glucose starvation conditions. GRDD-dependent Topo II␣ Degradation by Jab1/CSN5-Jab1/CSN5 was identified as a candidate protein that interacts with GRDD by a yeast two-hybrid screen using the topo II␣ ATPase domain (aa 1-200 and 1-584) as bait. To confirm a physical interaction between topo II␣ and Jab1/CSN5, we tested the ability of the GST-Jab1/CSN5 fusion protein to bind to immunopurified FLAG-tagged topo II␣. Full-length topo II␣ was captured by the GST-Jab1/CSN5 but not by the GST polypeptide alone (Fig. 5A). The in vitro binding between topo II␣ and Jab1/CSN5 was dependent on GRDD of topo II␣. Indeed, aa 1-584 ATPase-NLS2 and ⌬NLS2 topo II␣, which contained GRDD, were captured by the GST-Jab1/CSN5. However, ND70 -170 topo II␣ and aa 125-170 ATPase-NLS2, which lacked the complete GRDD, were not captured by the GST-Jab1/CSN5 (Fig. 5A).
To determine whether Jab1/CSN5 modulates stability of topo II␣ in vivo, we examined the expression levels of FLAGtagged topo II␣ and its deletion mutants in HT1080 cells coexpressing HA-tagged Jab1/CSN5 (Fig. 5, B and C). Similar to glucose starvation, co-expression of Jab1/CSN5 induced the degradation of wild-type topo II␣ and aa 1-584 ATPase-NLS2, which interacted physically with Jab1/CSN5 in vitro. However, Jab1/CSN5 as well as glucose starvation did not affect the expression levels of ND70 -170 topo II␣, which lacked Jab1/ CSN5 binding ability. The combination of glucose starvation and Jab1/CSN5 co-expression had no additive or synergistic effect on the topo II␣ expression, and the two treatments did not seem to affect each other under the experimental conditions. The Jab1/CSN5-induced decrease in aa 1-584 ATPase-NLS2 expression was suppressed by proteasome inhibitors PSI, MG132, and lactacystin (Fig. 5C). Thus, similar to glucose starvation, Jab1/CSN5 can stimulate proteasome-mediated degradation of topo II␣, and this stimulation occurs in a Jab1/ CSN5-topo II␣ binding-dependent manner. The Jab1/CSN5stimulated degradation of topo II␣ also was suppressed by the CSN kinase inhibitor, curcumin (Fig. 5C).
We further examined deletion mutants of Jab1/CSN5 to establish the relationship between degradation and Jab1/CSN5 binding of topo II␣. Jab1/CSN5 has a functional domain known as the MPN domain. Co-expression of ⌬MPN Jab1/CSN5 had little effect on degradation of the aa 1-584 ATPase-NLS2 of topo II␣, whereas co-expression of ⌬C Jab1/CSN5 containing the entire MPN domain down-regulated it similar to full-length Jab1/CSN5 (Fig. 6A). Consistent with this finding was that GST-⌬C Jab1/CSN5, but not ⌬MPN, interacted physically with full-length topo II␣ in vitro (Fig. 6B). These results suggested that Jab1/CSN5 stimulated the degradation of topo II␣ via direct binding in a GRDD-and MPN-dependent manner.
Involvement of Jab1/CSN5 in Stress-induced Topo II␣ Degradation-To address whether Jab1/CSN5 mediates degradation of endogenous topo II␣ under glucose starvation, we examined the effects of curcumin and Jab1/CSN5 antisense morpholino oligomers. As shown in Fig. 7A, curcumin inhibited stress-induced degradation of topo II␣ in HT1080 cells as effectively as the proteasome inhibitors, PSI, MG132, and lactacystin. Curcumin also inhibited topo II␣ degradation in MCF-7 cells, and interestingly, it caused a decreased expression of Jab1/CSN5 (Fig. 7C). Applying a Jab1/CSN5 antisense morpholino oligomer, but not scramble control, decreased cellular contents of Jab1/CSN5 by ϳ65% in both HT1080 and MCF-7 cells without affecting those of a different signalosome subunit, CSN1 or CSN8 (Fig. 7, B and C). The Jab1/CSN5 declined as a result of antisense suppressed stress-induced topo II␣ degradation. These results indicated that Jab1/CSN5 is involved in topo II␣ degradation under glucose starvation conditions. DISCUSSION Under the physiological cell conditions of glucose starvation, topo II␣ is degraded by the proteasome system. In this study, we identified GRDD as the degradation signal that was located between 70 and 170 amino acid residues in topo II␣. In the presence of the topo II␣ NLS2 region for nuclear translocation, GRDD gave the vulnerability to degradation under glucose starvation to GFP as well as various deletion mutants of topo II␣. On the other hand, ⌬NLS2 topo II␣ did not localize in the nucleus and was not subjected to stress-induced degradation, even though it contained the entire GRDD region. These results demonstrated that nuclear localization is a prerequisite for GRDD to act as a degradation signal. In addition, biochemical fractionation analysis showed that topo II␣ still existed in the nucleus when the stress-induced degradation was inhibited by a proteasome inhibitor (Fig. 2C). Therefore, it is probable that the stress-induced topo II␣ degradation occurs in the nucleus. Supporting this notion are previous observations that glucose starvation causes the nuclear accumulation of proteasomes, which can affect the efficiency of decreasing topo II␣ expression under the stress conditions (27). Our present results suggest that GRDD could be protected in a normal conformation of topo II␣, possibly by the N-terminal region adjacent to GRDD, because the deletion decreases stability of topo II␣ under normal growth conditions (Fig. 3C). Taken together, we concluded that GRDD of topo II␣ is a stress-activated degron acting in the nucleus.
We also demonstrated that Jab1/CSN5 mediates topo II␣ degradation under glucose starvation. So far, Jab1/CSN5 has been shown to interact with a number of proteins (28). The consequence of the Jab1/CSN5 interaction varies but can be generalized to change protein stability depending on the binding partner proteins. In the case of topo II␣, Jab1/CSN5 can promote proteasome-mediated degradation in a bindingdependent manner. Indeed, the ectopic expression of Jab1/ CSN5 was sufficient to induce degradation of exogenously coexpressed topo II␣. This Jab1/CSN5-induced degradation of topo II␣ was dependent on GRDD and was inhibitable by proteasome inhibitors as we observed for topo II␣ degradation under glucose starvation conditions. In addition, we identified GRDD as an element necessary for physical interaction with Jab1/CSN5 in vitro. However, the binding between Jab1/CSN5 and topo II␣ in vivo has not been detected by immunoprecipitation and subsequent immunoblotting (data not shown). Presumably, this was attributed to a transient feature of the in vivo interaction, leading to degradation of topo II␣. At present, the regulatory mechanisms of topo II␣ degradation mediated by Jab1/CSN5 remain to be determined. Two major mechanisms of Jab1/CSN5 leading to proteolysis have been described: 1) inducing intracellular redistribution (e.g. cyclin-dependentkinase inhibitor p27 Kip1 ) and 2) phosphorylation (e.g. tumor suppressor p53).
In the case of p27 Kip1 , the interaction with Jab1/CSN5 promotes the export of p27 Kip1 from the nucleus to the cytoplasm and enhances its degradation by a proteasome (29). The cytoplasmic shuttling of p27 Kip1 was shown recently to occur through the nuclear export signal located between 233-242 amino acids of Jab1/CSN5 (30). We noted with great interest that a Jab1/CSN5 deletion mutant that lacked the C-terminal region (amino acids 199 -334) containing the nuclear export signal lost the ability to induce p27 Kip1 degradation. This observation is quite different from our present finding that ⌬C Jab1/CSN5, which lacks amino acids 191-334, retained the ability to promote topo II␣ degradation (Fig. 6). Therefore, it is unlikely that the nuclear export activity of Jab1/CSN5 is involved in topo II␣ degradation, further supporting the notion that topo II␣ degradation occurs in the nucleus.
Phosphorylation via interaction with Jab1/CSN5 can be mediated by a Ser/Thr kinase known as the CSN-associated kinase that is co-purified with the CSN complex localizing in the nucleus (31)(32)(33)(34). In the case of p53, which also binds to Jab1/ CSN5 in vitro, CSN-mediated phosphorylation promotes its proteasome-dependent degradation (35). Indeed, the inhibition of the CSN-associated kinase by curcumin or a competitor peptide derived from p53 results in the accumulation of endogenous p53. Consistent with this finding is that topo II␣ degradation also is inhibited by curcumin (Figs. 5C and 7), suggesting that the CSN-mediated phosphorylation plays a role in targeting topo II␣ for degradation. In fact, topo II␣ is a phosphoprotein, and the phosphorylation is involved in the regulation of the activity of this enzyme (4). So far, multiple Ser/Thr phosphorylation sites have been identified, although most of them exist in the C-terminal domain far from the GRDD region. Only one site, Ser-29, has been identified as phosphoryl- ation site present near GRDD in N-terminal ATPase domain (36). However, the substitution of Ser-29 to Ala had no impact on topo II␣ stability under either normal or glucose starvation conditions (data not shown). Further studies will be needed to determine the significance of CSN-mediated phosphorylation in targeting topo II␣ for degradation.
The present data demonstrated clearly that the ability of Jab1/CSN5 to interact with and to promote degradation of topo II␣ depends on its MPN domain. Recently, it has been shown that Jab1/CSN5 can regulate SCF ubiquitin ligase activity through the JAMM motif identified in the MPN domain (37). The JAMM motif was essential for the CSN complex to cleave the ubiquitin-like protein, Nedd8, from the Cul1 subunit of SCF ubiquitin ligases. The Nedd8 modification (neddylation) of Cul1 is known to enhance the recruitment of the E2 enzyme to SCF ubiquitin ligase and thereby stimulate protein polyubiquitylation (38). Thus, the deneddylation activity by the JAMM motif can regulate SCF ubiquitin ligase negatively. In fact, it is reported that a microinjection of purified CSN complex inhibits p27 Kip1 degradation through deneddylation of Cul1 of SCF ubiquitin ligase complexes (39). These reports indicate that the MPN domain of Jab1/CSN5 can modulate the ubiquitin system directly. Therefore, it is possible that Jab1/CSN5 functions as an interface between the topo II␣ GRDD and the ubiquitin system through the MPN domain. Further studies, especially those that identify E3 ligase complexes for topo II␣, will be needed to clarify the precise mechanisms of targeting topo II␣ for proteasome-dependent degradation by Jab1/CSN5.
In addition to glucose starvation, other conditions may lead to proteasome-mediated degradation of topo II␣. Indeed, topo II␣ also is degraded by proteasome during adenovirus E1Ainduced apoptosis (20,26). Proteasome-mediated degradation also appears to be involved in cell cycle-dependent expression of this enzyme, especially in the transition from M to G 1 cell cycle phase (40). Although not determined so far, the proteasome might be involved in decreased expression of topo II␣ in the quiescent cell state. Our present findings would be useful in elucidating the mechanisms of topo II␣ degradation in these situations. It should then be noted that topo II␣ often escapes the quiescence-and cell cycle-dependent regulation in tumor cells (15,41,42), suggesting that deregulated proteolysis of topo II␣ may be involved in tumor development by analogy with p27 Kip1 (43). Nevertheless, topo II␣ degradation under glucose starvation has been observed in a variety of solid tumor lines, even in cells where the cell cycle-dependent regulation is abrogated (15,18). Thus, the GRDD-and MPN-dependent degradation of topo II␣ might be particularly important for cellular adaptation to severe stress conditions. Meanwhile, topo II␤ isozyme is degraded preferentially over ␣-isozyme through a ubiquitin-proteasome pathway when the intermediate-cleavable complex is trapped by the antitumor drug, VM-26 (44). Interestingly, the GRDD region identified in the present study is conserved highly in ␤-isozyme, and Ͼ90% of the amino acids are identical. It will be interesting to see whether the GRDDand MPN-dependent mechanisms are involved in topo II␤ degradation as well. We now are investigating the possibility that the GRDD (-like sequence) of topo II␤ acts as a degron.
High-level expression of topo II␣ is essential for cell death induced by topo II-directed drugs such as etoposide, VM-26, and doxorubicin. Therefore, development of methods restoring topo II␣ expression under stress conditions would be important for topo II-directed chemotherapy against solid tumors. Actually, we have shown previously that the proteasome inhibitor lactacystin effectively enhances the antitumor activity of etoposide in a solid tumor model (18). The findings described here indicate that Jab1/CSN5 could be a new selective target for restoring topo II␣ expression in solid tumors. Indeed, microenvironmental stressors, such as glucose starvation and hypoxia, are not observed in normal tissues, and the Jab1/CSN5-mediated topo II␣ degradation pathway appears to be selective in stressed cells. Further studies on the mechanisms of how Jab1/ CSN5 promotes topo II␣ degradation would provide selective strategies to enhance topoII-directed chemotherapy against solid tumors.