Interaction between Glucose-regulated Destruction Domain of DNA Topoisomerase IIα and MPN Domain of Jab1/CSN5

DNA topoisomerase (topo) IIα, an essential enzyme for cell proliferation, is targeted to a proteasome-dependent degradation pathway when human tumor cells are glucose-starved. Here we show that the topo IIα destabilization depends on the newly identified domain, GRDD (glucose-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 IIα, 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 IIα degradation. Further, GRDD was identified as an interactive domain for Jab1/CSN5, which promoted the degradation of topo IIα 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 kinase inhibitor, curcumin, inhibited topo IIα degradation induced by glucose starvation. These findings demonstrate that GRDD can act as a stress-activated degron for regulating topo IIα stability, possibly through interaction with the MPN domain of Jab1/CSN5. DNA topoisomerase (topo) IIα, an essential enzyme for cell proliferation, is targeted to a proteasome-dependent degradation pathway when human tumor cells are glucose-starved. Here we show that the topo IIα destabilization depends on the newly identified domain, GRDD (glucose-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 IIα, 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 IIα degradation. Further, GRDD was identified as an interactive domain for Jab1/CSN5, which promoted the degradation of topo IIα 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 kinase inhibitor, curcumin, inhibited topo IIα degradation induced by glucose starvation. These findings demonstrate that GRDD can act as a stress-activated degron for regulating topo IIα stability, possibly through interaction with the MPN domain of Jab1/CSN5. DNA topoisomerase II (topo II) 1The abbreviations used are: topo IIα, DNA topoisomerase IIα; NLS, nuclear localization signal; GRDD, glucose-regulated destruction domain; HA, hemagglutinin; CSN5, COP9 signalosome subunit5; WT, wild-type; aa, amino acid; GST, glutathione S-transferase; E2, ubiquitin carrier protein; MPN, Mpr1p/Prd1p N terminus; CSN, COP9 signalosome; SCF, SKP1, CUL1/CDC53, F-box protein. is an essential enzyme for eukaryotic cell proliferation and plays important roles in many aspects of the DNA process through modulating the topological states (1Berger J.M. Gamblin S.J. Harrison S.C. Wang J.C. Nature. 1996; 379: 225-232Google Scholar). In humans, topo II exists in two closely related isoforms, α and β, which share a high degree of structural homology with a ∼70% sequence identity (2Austin C.A. Marsh K.L. BioEssays. 1998; 20: 215-226Google Scholar, 3Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Google Scholar). Three discrete functional domains are embedded in the topo II enzymes (1Berger J.M. Gamblin S.J. Harrison S.C. Wang J.C. Nature. 1996; 379: 225-232Google Scholar, 4Isaacs R.J. Davies S.L. Sandri M.I. Redwood C. Wells N.J. Hickson I.D. Biochim. Biophys. Acta. 1998; 1400: 121-137Google Scholar). 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 (4Isaacs R.J. Davies S.L. Sandri M.I. Redwood C. Wells N.J. Hickson I.D. Biochim. Biophys. Acta. 1998; 1400: 121-137Google Scholar). The expression of topo IIα 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 IIα also change within a single cell cycle, peaking in the G2/M phase and declining to a minimal level after M phase completion. However, the levels of topo IIβ are relatively constant throughout the cell cycle and in quiescent and proliferating states (5Kroll D.J. Rowe T.C. J. Biol. Chem. 1991; 266: 7957-7961Google Scholar). The topo II isoforms have been shown to be the molecular target for such clinically important antitumor drugs such as etoposide and doxorubicin (6Burden D.A. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Google Scholar, 7Li T.K. Liu L.F. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 53-77Google Scholar). 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 (8Brown J.M. Giaccia A.J. Cancer Res. 1998; 58: 1408-1416Google Scholar, 9Tomida A. Tsuruo T. Anticancer Drug Des. 1999; 14: 169-177Google Scholar). 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 α-isoform. Indeed, the resistance often correlates with a decrease in topo IIα expression levels or with mutations in the protein that reduce enzymatic activity (10Vassetzky Y.S. Alghisi G.C. Gasser S.M. BioEssays. 1995; 17: 767-774Google Scholar). C-terminal truncations of topo IIα, 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 (11Harker W.G. Slade D.L. Parr R.L. Feldhoff P.W. Sullivan D.M. Holguin M.H. Cancer Res. 1995; 55: 1707-1716Google Scholar, 12Yu Q. Mirski S.E. Sparks K.E. Cole S.P. Biochemistry. 1997; 36: 5868-5877Google Scholar). High levels of topo IIα expression have been shown in a wide variety of tumors, partly due to the increased growth fraction (13Dingemans A.M. Pinedo H.M. Giaccone G. Biochim. Biophys. Acta. 1998; 1400: 275-288Google Scholar). The high topo IIα 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 (8Brown J.M. Giaccia A.J. Cancer Res. 1998; 58: 1408-1416Google Scholar, 9Tomida A. Tsuruo T. Anticancer Drug Des. 1999; 14: 169-177Google Scholar, 14Sutherland R.M. Science. 1988; 240: 177-184Google Scholar). These physiological conditions in culture can down-regulate the expression of topo IIα with growth arrest or delay at the G1 phase of the cell cycle (15Kim H.D. Tomida A. Ogiso Y. Tsuruo T. J. Cell. Physiol. 1999; 180: 97-104Google Scholar). What we found to be consistent with the decreased topo IIα levels was tumor cells becoming resistant to topo II-directed drugs when stressed by glucose starvation and related culture conditions (16Shen J. Hughes C. Chao C. Cai J. Bartels C. Gessner T. Subjeck J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3278-3282Google Scholar, 17Yun J. Tomida A. Nagata K. Tsuruo T. Oncol. Res. 1995; 7: 583-590Google Scholar). These observations suggest that the stress-mediated down-regulation of topo IIα 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 IIα depletion (18Ogiso Y. Tomida A. Lei S. Omura S. Tsuruo T. Cancer Res. 2000; 60: 2429-2434Google Scholar). Topo IIα restoration is seen only at the protein levels, indicating that topo IIα protein depletion occurs through a proteasome-mediated degradation mechanism. In general, proteasome-mediated proteolysis is targeted by ubiquitylation of the substrate proteins (19Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Google Scholar). Consistently, topo IIα can be conjugated with polyubiquitin in a cell-free system with extracts of cancer cells (20Nakajima T. Morita K. Ohi N. Arai T. Nozaki N. Kikuchi A. Osaka F. Yamao F. Oda K. J. Biol. Chem. 1996; 271: 24842-24849Google Scholar). Thus, the ubiquitin-proteasome system appears responsible for topo IIα degradation under conditions of glucose starvation, but the regulatory mechanisms leading to topo IIα degradation are largely unknown. In this study, we have attempted to identify the domain of topo IIα 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 IIα in a GRDD-dependent manner. Consistent with this finding was that specific inhibition of Jab1/CSN5 leads to the stabilization of endogenous topo IIα under stress conditions. 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 μg of kanamycin/ml. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Glucose deprivation was achieved by substituting RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine and were from the and were from and These were in and to culture medium that the of was of the were at least expression of topo IIα and deletion from N or the containing human topo IIα from C. Liu L.F. Liu J. T. K. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; was used as a for with DNA a and a were at the and The were into the and into the of the expression of a topo IIα deletion the sequence to amino was from topo IIα by and with a to amino within amino were by the sequence the and of topo IIα with each the expression of fusion proteins were by with or by a from the topo IIα was two to amino and that were into the and at the and single amino acid amino acid and deletion to amino was a The expression from was K. Tomida A. Y. T. Y. Tsuruo T. Scholar). human Jab1/CSN5 was by from a of The was into the expression at the to amino of Jab1/CSN5 also was into the at the The of Jab1/CSN5 was two to amino and that were to and at the and The was T. Tsuruo T. 1999; Scholar). The of of the was by DNA of the for were an Tokyo, were to the with cells were at in a to of culture of containing μg of DNA and of in of was to each and the cells were for The cells were with and for in the cells were in or culture medium for the of of Nuclear and and cytoplasmic extracts of cells were nuclear and cytoplasmic to the were with and in 5% as J. Tomida A. Nagata K. Tsuruo T. Oncol. Res. 1995; 7: 583-590Google Scholar). of the was a protein of were by and a were with topo IIα and The specific signals were an system on a culture with a were in for and with for The cells were for with by a The were The cells were observed a with a used a system from The of the N-terminal and of topo IIα protein was with the domain of the The resulting were used to a human fetal to the positive were DNA sequence identified two from the positive In and deletion of Jab1/CSN5 were by and into a expression with DNA Jab1/CSN5 proteins were with a to the topo IIα proteins were in 293T cells by with The topo IIα proteins were and from the with a to the The topo IIα proteins were to fusion proteins on the The were at °C in a of of a containing and with the the protein was by and and antisense specific for human Jab1/CSN5 as as a was from The were for Jab1/CSN5 and for the were to the and MCF-7 cells were at and respectively, in a antisense with the were to the and cells were for or The cells were with and cultured in or medium for or and the cell were for expression of Jab1/CSN5 and topo ATPase and NLS for of Topo cells are cultured under the stress of glucose starvation, topo IIα is in a proteasome-dependent manner. Indeed, degradation of topo IIα, but topo was observed in cells of topo IIα was by proteasome at but by inhibitor, at topo IIα with the at the N was glucose starvation when was in cells by which of topo IIα were for the we a of expression of topo IIα deletion and into Glucose degradation was by the IIα and from the N and that the deletion of the ATPase domain to degradation resistance under glucose starvation conditions. of the from the also resulted in stabilization under the conditions. In the containing the ATPase domain and the was by glucose starvation, as was topo We also found that deletion in expression under the growth conditions. Although the is we that are a result of in the of each topo IIα protein. of Topo IIα with that topo IIα in the of cells in a manner The topo IIα, which was to the stress-induced also in the and the of was and topo In topo IIα, which was resistant to the stress-induced in the cell that topo IIα was more in the cytoplasmic the topo IIα The was in with the that the sequence is the functional for nuclear translocation of topo IIα S.E. Cole S.P. Cancer Res. 1995; 55: Scholar, S.E. Cole S.P. Cell Res. 1997; Scholar). results that nuclear localization of topo IIα was a prerequisite for degradation of this enzyme under glucose starvation conditions. Consistent with endogenous topo IIα in the nucleus of stressed cells when degradation of topo IIα was inhibited by proteasome of Topo IIα ATPase for results that the sequence within the ATPase domain topo IIα under glucose starvation stress conditions. the region, we deletion within the ATPase domain that were with the topo IIα domain for nuclear degradation of the fusion proteins was as shown in and of fusion proteins were by glucose starvation but the fusion protein stable with the We the N-terminal of the fusion protein by with the protein Although stress-induced destabilization when and were the deletion to degradation The N-terminal also were to topo IIα and and the results were with to the under glucose starvation conditions Interestingly, the both in topo IIα and in the fusion decreased expression level under growth conditions as with the deletion and The deletion on the expression levels under conditions the deletion increased the expression levels in the fusion only a on the topo IIα expression under conditions and These results that the N-terminal can the of topo IIα under growth conditions. of GRDD, a deletion that the minimal for the stress-induced degradation was and amino acid of topo IIα ATPase domain. Indeed, topo IIα resistant to the stress-induced degradation when the 70–170 were is important to that topo IIα in the nucleus We designated the 70–170 of topo IIα as the GRDD of topo IIα as a degradation we fusion proteins that were with topo IIα or 70–170 at the N and topo IIα domain at the for nuclear localization The fusion protein containing was at levels under and glucose starvation that this fusion protein was to the of the stress-induced topo IIα degradation. GRDD to and we found that the protein to degradation under glucose starvation conditions as was observed for topo Thus, the GRDD of topo IIα can act as a degradation Topo IIα has a destruction sequence that is within GRDD T. Ohi N. Arai T. Nozaki N. Kikuchi A. Oda K. 1995; Scholar). In the amino acid and of topo IIα is to the sequence of the destruction the of the destruction we used topo IIα, which of the ATPase domain and the The deletion of amino within the GRDD of topo IIα on stress-induced degradation of topo IIα We that the destruction sequence in GRDD is for topo IIα degradation under glucose starvation conditions. GRDD-dependent Topo IIα by Jab1/CSN5 was identified as a protein that with GRDD by a the topo IIα ATPase domain and as a interaction topo IIα and Jab1/CSN5, we the of the fusion protein to to topo topo IIα was by the but by the The in topo IIα and Jab1/CSN5 was dependent on GRDD of topo Indeed, and topo IIα, which GRDD, were by the However, topo IIα and which the GRDD, were by the Jab1/CSN5 of topo IIα in we the expression levels of topo IIα and deletion in cells Jab1/CSN5 and to glucose starvation, of Jab1/CSN5 induced the degradation of topo IIα and which with Jab1/CSN5 in However, Jab1/CSN5 as as glucose starvation the expression levels of topo IIα, which Jab1/CSN5 The of glucose starvation and Jab1/CSN5 or on the topo IIα and the two to each other under the conditions. The decrease in expression was by proteasome and Thus, to glucose starvation, Jab1/CSN5 can proteasome-mediated degradation of topo IIα, and this occurs in a IIα manner. The degradation of topo IIα also was by the kinase inhibitor, We deletion of Jab1/CSN5 to the degradation and Jab1/CSN5 of topo Jab1/CSN5 has a functional domain as the MPN domain. of Jab1/CSN5 on degradation of the of topo IIα, whereas of Jab1/CSN5 containing the MPN domain to Jab1/CSN5 Consistent with this finding was that Jab1/CSN5, but with topo IIα in These results that Jab1/CSN5 the degradation of topo IIα in a and manner. of Jab1/CSN5 in Topo IIα Jab1/CSN5 degradation of endogenous topo IIα under glucose starvation, we the of and Jab1/CSN5 antisense shown in inhibited stress-induced degradation of topo IIα in cells as as the proteasome and also inhibited topo IIα degradation in MCF-7 and a decreased expression of Jab1/CSN5 a Jab1/CSN5 antisense but decreased cellular of Jab1/CSN5 by in both and MCF-7 cells of a signalosome or and The Jab1/CSN5 as a result of antisense stress-induced topo IIα degradation. These results that Jab1/CSN5 is in topo IIα degradation under glucose starvation conditions. the physiological cell conditions of glucose starvation, topo IIα is by the proteasome In this study, we identified GRDD as the degradation signal that was and amino acid in topo In the presence of the topo IIα for nuclear GRDD the to degradation under glucose starvation to as as deletion of topo the other topo IIα in the nucleus and was to stress-induced the GRDD These results that nuclear localization is a prerequisite for GRDD to act as a degradation In biochemical that topo IIα in the nucleus when the stress-induced degradation was inhibited by a proteasome is that the stress-induced topo IIα degradation occurs in the this are observations that glucose starvation the nuclear of which can the of topo IIα expression under the stress conditions Y. Tomida A. Tsuruo T. Cancer Res. Scholar). Our results suggest that GRDD be in a of topo IIα, possibly by the N-terminal to GRDD, because the deletion of topo IIα under growth conditions we that GRDD of topo IIα is a stress-activated degron in the We also that Jab1/CSN5 topo IIα degradation under glucose starvation. Jab1/CSN5 has been shown to with a number of proteins C. Cell Biol. 2001; Scholar). The of the Jab1/CSN5 interaction but can be to change protein on the In the case of topo IIα, Jab1/CSN5 can proteasome-mediated degradation in a manner. Indeed, the expression of Jab1/CSN5 was to degradation of topo degradation of topo IIα was dependent on GRDD and was by proteasome as we observed for topo IIα degradation under glucose starvation conditions. In we identified GRDD as an for interaction with Jab1/CSN5 in However, the Jab1/CSN5 and topo IIα in has been by and this was to a feature of the in leading to degradation of topo the regulatory mechanisms of topo IIα degradation by Jab1/CSN5 to be mechanisms of Jab1/CSN5 leading to proteolysis have been and tumor In the case of the interaction with Jab1/CSN5 promotes the of from the nucleus to the and degradation by a proteasome K. Y. J. Nature. 1999; Scholar). The cytoplasmic of was shown to through the nuclear signal amino of Jab1/CSN5 K. Y. Y. S. T. N. J. Biol. Chem. Scholar). We with great that a Jab1/CSN5 deletion that the C-terminal containing the nuclear signal the to degradation. is from finding that Jab1/CSN5, which amino the to topo IIα degradation is that the nuclear activity of Jab1/CSN5 is in topo IIα the that topo IIα degradation occurs in the interaction with Jab1/CSN5 can be by a kinase as the CSN-associated kinase that is with the in the nucleus N. K. K. J. J. A. D.A. 2000; Scholar, K. C. J. 1998; Scholar, S. J. J. Scholar, N. T. G. N. K. Biol. 1998; Scholar). In the case of which also binds to Jab1/CSN5 in promotes proteasome-dependent degradation C. J. 2001; 20: Scholar). Indeed, the inhibition of the CSN-associated kinase by or a from results in the of endogenous Consistent with this finding is that topo IIα degradation also is inhibited by and that the plays a in topo IIα for degradation. In topo IIα is a and the is in the of the activity of this enzyme (4Isaacs R.J. Davies S.L. Sandri M.I. Redwood C. Wells N.J. Hickson I.D. Biochim. Biophys. Acta. 1998; 1400: 121-137Google Scholar). have been most of in the C-terminal domain from the GRDD has been identified as GRDD in N-terminal ATPase domain N.J. A.M. F. C. Hickson I.D. J. Biol. Chem. 1995; Scholar). However, the of to impact on topo IIα under or glucose starvation conditions be to the of in topo IIα for degradation. The that the of Jab1/CSN5 to with and to degradation of topo IIα depends on MPN domain. Recently, has been shown that Jab1/CSN5 can ubiquitin activity through the identified in the MPN domain S.E. S.L. R.J. Science. Scholar). The was essential for the to the from the of ubiquitin The of is to the of the enzyme to ubiquitin and protein T. T. T. K. K. N. N. Y. Osaka F. K. J. 2001; 20: Scholar). Thus, the activity by the can ubiquitin In is that a of degradation through of of ubiquitin complexes S. K. T. Wang R.J. N. Biol. Scholar). These that the MPN domain of Jab1/CSN5 can the ubiquitin system is that Jab1/CSN5 functions as an the topo IIα GRDD and the ubiquitin system through the MPN domain. that identify complexes for topo IIα, be to the mechanisms of topo IIα for proteasome-dependent degradation by Jab1/CSN5. In to glucose starvation, other conditions may lead to proteasome-mediated degradation of topo Indeed, topo IIα also is by proteasome (20Nakajima T. Morita K. Ohi N. Arai T. Nozaki N. Kikuchi A. Osaka F. Yamao F. Oda K. J. Biol. Chem. 1996; 271: 24842-24849Google Scholar, T. Ohi N. Arai T. Nozaki N. Kikuchi A. Oda K. 1995; Scholar). degradation also appears to be in cell expression of this enzyme, in the from M to G1 cell cycle phase Biochem. Pharmacol. 2001; Scholar). Although the proteasome be in decreased expression of topo IIα in the quiescent cell Our findings be in the mechanisms of topo IIα degradation in be that topo IIα often the and cell in tumor cells (15Kim H.D. Tomida A. Ogiso Y. Tsuruo T. J. Cell. Physiol. 1999; 180: 97-104Google Scholar, Liu L.F. Cancer Res. 1988; Scholar, D.M. Cancer Res. 1987; that proteolysis of topo IIα may be in tumor by with A. S. S.L. Scholar). topo IIα degradation under glucose starvation has been observed in a variety of solid tumor in cells the cell is (15Kim H.D. Tomida A. Ogiso Y. Tsuruo T. J. Cell. Physiol. 1999; 180: 97-104Google Scholar, Y. Tomida A. Lei S. Omura S. Tsuruo T. Cancer Res. 2000; 60: 2429-2434Google Scholar). Thus, the and degradation of topo IIα be important for cellular to stress conditions. topo IIβ is through a ubiquitin-proteasome pathway when the is by the antitumor Y. J. Liu L.F. J. Biol. Chem. 2001; Scholar). Interestingly, the GRDD identified in the is conserved in and of the amino are be to the and mechanisms are in topo IIβ degradation as We are the that the GRDD of topo IIβ as a expression of topo IIα is essential for cell induced by topo II-directed drugs such as and of topo IIα expression under stress conditions be important for topo II-directed solid we have shown that the proteasome the antitumor activity of etoposide in a solid tumor (18Ogiso Y. Tomida A. Lei S. Omura S. Tsuruo T. Cancer Res. 2000; 60: 2429-2434Google Scholar). The findings that Jab1/CSN5 be a target for topo IIα expression in solid Indeed, such as glucose starvation and hypoxia, are observed in and the topo IIα degradation pathway appears to be in stressed on the mechanisms of Jab1/CSN5 promotes topo IIα degradation provide to solid We and for