Tài liệu Báo cáo Y học: Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity pot

Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity Shilpa Gandre, Zippi Bercovich and Chaim Kahana Department of Molecular Genetics, Weizmann Institute of Science, Israel Antizyme is a polyamine-induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin-independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells. In contrast with previous studies demonstrating stability in vitro in a r eticulocyte lysate-based degradation system, in cells antizyme is rapidly degraded and this degradation is inhibited by specific proteasome inhibitors. While the deg- radation of ODC is stimulated by the presence of cotrans- fected antizyme, degradation of antizyme seems to be independent of ODC, suggesting t hat antizyme degradation does not occur while presenting ODC to the 26S protea- some. Interestingly, both species of antizyme, which repre- sent initiation at two in-frame initiation c odons, are rapidly degraded. The degradation of both antizyme proteins is inhibited i n ts20 cells containing a thermosensitive ubiquitin- activating enzyme, E1. Therefore we conclude that in contrast with ubiquitin-independent degradation of ODC, degradation of antizyme requires a functional ubiq uitin system. Keywords: antizyme; ornithine decarboxylase; protein deg- radation; proteasome; polyamines. The polyamines spermidine and spermine and t heir precur- sor putrescine are ubiquitous aliphatic polycation s wi th multiple cellular functions. Polyamines were demonstrated to be essential for fundamental cellular processes such as growth, differentiation, transformation and apoptosis [1–5], although their explicit role in these cellular processes is mostly unknown. Nevertheless, due to the critical role of polyamines i n various cellular functions, multiple pathways such as biosynthesis, catabo lism, uptake, and excretion tightly r egulate their intracellular concentration. One of t he major sources of ce llular polyamines c omes from t heir synthesis from amino acid precursors. In this biosynthesis pathway ornithine is decarboxylated to form putrescine by the action of ornithine decarboxylase (ODC, EC 4.1.1.17). Next an aminopropyl group generated by the action of S-adenosylmethionine decarboxylase (EC 4.1.1.50) on S-adenosylmethionine, is attached to putrescine and sper- midine to form spermidine and spermine, respectively. Both enzymes a re highly regulated and are subjected t o feedback control by c ellular polyamines. Control of cellular polyam- ines by rapid r egulated degradation of ODC constitutes an important feedback regulatory mechanism. ODC is one of the most rapidly degraded proteins in eukaryotic ce lls. Interestingly it is degraded with out req ui- ring ubiquitination [6,7]. Instead, ODC is targeted to degradation due to its i nteraction with a u nique poly- amine-induced protein termed antizyme [8]. Although not requiring ubiquitination, the degradation of ODC also occurs by the action of the 26S proteasome [8–10]. Synthesis of antizyme requires translational frameshifting, which results in bypassing a stop codon located shortly down- stream of the initiation codon (ORF1) [11,12]. High concentration of polyamines subverts the ribosome from its original reading frame to the +1 frame to encode a second ORF and synthesize complete functional antizyme protein. A n tizyme binds to ODC s ubunit to f orm e n zymat- ically inactive heterodimers [13]. The affinity of antizyme to ODC subunits is higher than the affinity that ODC subunits have to each other. Interaction between antizyme and ODC subunits has two outcomes: ODC is inactivated [13], and the ODC s ubunits are t argeted t o degradation [8,13–15]. It w as suggested that binding of antizyme to ODC results in the exposure of the C-terminal destabilizing signal of ODC [16]. Antizyme was a lso d emonstrated to negatively regulate the process of polyamine transport by a yet unresolved mechanism [17,18]. Mammalian cells contain another relevant regulatory protein, antizyme inhibitor, a protein that displays homology t o ODC, but lacks d ecarb oxylating activity [19]. It binds to antizyme with higher affinity than ODC thus it may release active ODC from the inactive antizyme–ODC heterodimer [20]. While it is clear that interaction with antizyme is absolutely required for marking ODC for rapid degrada- tion, it is not clear what happens to antizyme during this proteolytic process. Some studies performed in vitro in degradation extracts s uggested that while targeting ODC to degradation, antizyme remains stable and is released to Correspondence to C. Kahana, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, 76100, Israel. Fax: + 972 8 9344199, Tel.: + 972 8 9342745, E-mail: chaim.kahana@weizmann.ac.il Abbreviations: ODC: ornithine decarboxylase; DMEM, Dulbecco’s modified Eagle’s medium. Enzymes: ornithine decarboxylase (EC 4.1.1.17); S-adenosylmethio- nine decarboxylase (EC 4.1.1.50). (Received 23 October 2001, revised 20 December 2001, accepted 9 January 2002) Eur. J. Biochem. 269, 1316–1322 (2002) Ó FEBS 2002 participate in subsequent cycles of ODC degradation [2,13,21]. In contrast, other studies demonstrated rapid degradation of antizyme in rat hepatoma (HTC) and HTC- derived ODC overproducing cells under basal conditions and after hypo-osmotic shock [22,23]. Additional studies provided further support to the notion that antizyme is rapidly degraded [24,25]. In the present study we have further investigated the metabolic fate of antizyme in mammalian cells. We show here that like ODC, antizyme is rapidly degraded by the proteasome. However, in contrast with the degradation of ODC that requires interaction with antizyme, the degrada- tion of antizyme may occur without in teracting with ODC. We also demonstrate that in c ontrast with the degradation of ODC that occurs in a mutant cell line with a temperature- sensitive ubiquitin-activating enzyme, E1, the degradation of antizyme is imp aired at the restric tive temperature. EXPERIMENTAL PROCEDURES Materials Proteasome inhibitors MG115 (Z-Leu-Leu-Norvalinal) and MG132 (Z-Leu-Leu-Leucinal) were from C albiochem. Tissue culture reagents and chemicals were from Sigma. Constructs Z1 rat antizyme DNA [26] was cloned into the pSVL vector (Pharmacia) as a SalI(5¢)– ClaI(3¢) fragment. FLFS wild- type rat antizyme DNA (containing the two initiation codons) was cloned into t he pCI-neo e xpression vector (Promega) as an EcoRI fragment or into the bicistronic vector, pEFIRES-p [27], as a XhoI(5¢)-NotI(3¢)fragment. FLFS DNA lacking the first initiation codon was cloned into pCI-neo as a XbaI(5¢)–SalI(3¢) fragment. DNAs encoding wild-type mouse ODC or the stable Del-6 mutant [28] were cloned into pCI-neo as EcoRI(5¢)–XbaI(3¢) fragments or into pEFIRES-p as XhoI(5¢)–NotI(3¢) fragments. Cells and cell culture conditions The ODC overproducing mouse myeloma cell line, 653-1 was selected as described previously [29]. 653-1 and the parental 653 cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% bovine calf serum. a-Difluoromethylornithine (20 m M ) was added to the growth medium of 653-1 cells. The human embryonic kidney epithelial 293 cells and monkey cos-7 cells were cultured at 37 °C in DMEM containing 10% bovine c alf serum and transfected with the indicated constructs, using the calcium phosphate precipitation method [30]. To determine the degradation rate of the test proteins, cycloheximide (20 lgÆmL )1 ) was added to t he growth medium 48 h post-transfection and cellular extracts were prepared at various times thereafter. The tested proteins were then detected by Western blot analysis. A31N-ts20 (containing thermosensitive ubiquitin-activating enzyme, E1), and A31N (parental cells) [31] were cultured at 32 °C in DMEM containing 10% foetal bovine serum. They were transfected with the indicated constructs using the X-tremeGENE Q2 Transfection reagent (Roche) as recommended by the manufacturer. At the indicated times the cells were transferred to the nonpermissive temperature (39 °C) and cellular extracts were prepared 16 h thereafter. The level of the indicated proteins was determined by Western blot an alysis. Western blot analysis Cells were harvested at the indicated times, lysed in lysis buffer (150 m M NaCl 50 m M Tris/HCl, pH 7.2, 0.5% NP40, 1% Triton-X 100, 1% sodium deoxycholate) in the presence of protease inhibitor cocktail. Protein concentra- tion in the cellular extracts was determined u sing Bradford’s method. Samples containing equal amounts of protein were denatured in Laemmli buffer, fractionated by SDS/PAGE and b lotted onto nitrocellulose membrane. The blots were then probed with t he indicated antibodies, and the protein signals were detected using the Supersignal chemilumines- cence detection system (Pierce). These experiments were repeated at least three times and representative data are presented. The primary antibodies used were monoclonal antimouse ODC (Sigma, clone ODC-29 originally devel- oped by us), monoclonal antirat antizyme made by us which recognizes amino acids 36–69 (with the second initiation codon being amino acid number 1) and monoclonal antimouse p53 (kindly provided by M. Oren). Polyamine determination Polyamines were determined essentially as described by Seiler [32]. Ce lls were collected, resuspended i n 500 lL NaCl/Pi and the material precipitating in 3% perchloric acid was removed by centrifugation. Four-hundred microlitres of dansyl c hloride (30 mgÆmL )1 prepared in acetone) was mixed with a 200 lL aliquot of the supernatant; 20 mg of sodium carbonate was then added and the mixture was incubated in the dark. After 12 h of incubation 100 lL of proline (100 mgÆmL )1 )wasadded and the mixture was incubated for an additional 1 h. Dansylated derivatives were then extrac ted into 0.5 mL toluene. Portions (50–100 lL) were spotted on silica G-50 plates and the dansylated derivatives were resolved using ethyl acetate/cyclohexane (2 : 3) as a solvent, with dansylated derivatives of known polyamines serving as markers. The individual polyamines were visualized by UV illumination. RESULTS Antizyme is rapidly degraded by the action of the 26S proteasome While it is clear that interaction with antizyme is required for the degradation of ODC, the cellular fate of antizyme during this proteolytic process is unclear. The prominent notion which is based predominantly on studies performed in vitro in degradation extracts assumes that a ntizyme is a stable protein that is recycled during the degradation of ODC. We use here two cellular systems to determine whether antizyme is a stable or a rapidly degraded protein. We have noted that the ODC overproducing 653-1 mouse myeloma cells [29,33–35] also show detectable levels of antizyme (Fig. 1A, compare lanes 1 and 2) when grown Ó FEBS 2002 Characterization of antizyme degradation (Eur. J. Biochem. 269) 1317 without the ODC inhibitor a-difluoromethylornithine. The induction of antizyme expression can be attributed to the accumulation of putrescine and cadaverine (Fig. 1B). As in the case of O DC, the overexpressed a ntizyme was also rapidly degraded and the specific proteasome inhibitor, MG132 (Fig. 1A, lanes 2, 3 and 4) effectively inhibited this degradation. The second system is based on monkey cos-7 cells that were transiently transfected with constructs expressing rat a ntizyme. In order to uncouple between antizyme expression and the requirement for polyamines, we have utilized the Z1 antizyme clone to which an initiation codon was appended in the +1 frame [26]. As a result, frameshifting is not required for its expression [12,26]. In both cases cycloheximide was added t o the growth medium of the cells and cellular extracts were prepared at various times thereafter. The extracts were resolved by SDS/PAGE and a ntizyme was detected by Western b lot analysis using specific antiantizyme monoclonal antibodies. As noted in 653-1 cells, also in transfected Cos-7 cells antizyme was rapidly d egraded i n a proteasome-dependent manner (Fig. 1 C). We therefore conclude that in contrast with the observations made in the in vitro degradation systems, in cells antizyme is a rapidly degraded protein and that as with ODC, the degradation of antizyme is also carried out by the proteasome. Antizyme mRNA contains two variably used in-frame initiation codons giving rise to two antizyme forms of 24 and 29.5 kDa [12,23]. Studies performed in vitro in a reticulocyte lysate-based translation mix demonstrated that the second initiation codon is utilized preferentially [11,12]. Utilization of the two initiation codons with clear preference towards the second one was also inferred in HTC cells [24]. The above used Z1 antizyme represents initiation at the second ATG thus enco ding the shorter form of antizyme. To test for the cellular stability of the long form of antizyme we used a full-length frame-shifted antizyme cDNA denoted FLFS, which like the Z1 clone encodes antizyme without requiring frameshifting [12]. As observed in the in vitro translation system [12], also in transfected 293 cells initiation of translation occurred predominantly at the second ATG (Fig. 1 D). Both forms of antizyme were rapidly degraded (Fig. 1 D). Fig. 1. Antizyme is rapidly degraded in mammalian cells through the action of the proteasome. (A) 653-1 ODC overproducing cells were grown for 10 days without a-DFMO. Under these conditions ODC inhibition is relieved and a ntizyme is induced. Cycloheximide (CHX 20 lgÆmL )1 )was then added to the growth medium of 653-1 cells alone or together with the proteasome inhibitor, MG132 (50 l M ). Cellular extracts were prepared at the indicated times and aliquots containing 50 lg total protein were resolved by SDS/PAGES using a 12% polyacrylamide gel. The fractionated material was transferred onto nitrocellulose membrane, which was then probed with anti-ODC and antiantizyme monoclonal antibodies. Signals were detected using th e Supersignal Chemiluminescence detection system. Lane 1 contains equal amount of protein extracted from the p arental 653 cells. (B) Polyamines were extracted from 653 and 653-1 cells and analysed as described in Experimental procedures. (C) Cos-7 cells were transfected with the pSVL-Z1 co nstruct. Forty-eight hours post-transfection cycloheximide was added to the growth medium alone o r together with the proteasome inhibitor, MG115. Cellular extracts were prepared and analysed as described in A. (D) 293 cells were transfected with expression constructs containing wild-type antizyme (FLFS, see Experimental procedures) DNA. Cellular extracts were prepared and analysed as described in A. The molecular weight and position of the two forms of antizyme is indicated on the right. *NS indicates the position of nonspecific proteins recognized by the antibodies. 1318 S. Gandre et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The degradation of antizyme is independent of the degradation of ODC As demonstrated above, antizyme, the mediator of ODC degradation is itself rapidly degraded. Therefore, we set out to determine whether this degradation occurs together with ODC while presenting ODC to the proteasome or whether the degradation of antizyme is independent of that of ODC. 293 cells were transfected with constructs encoding ODC, antizyme or both. As demonstrated before [8,13,36], coex- pression with antizyme stimulated the degradation of ODC (Fig. 2D). In contrast, antizyme was rapidly degraded in both the presence and the absence of coexpressed ODC (Figs 2C and D). Similarly, we have noted that antizyme that was induced by the addition of spermidine to the growth medium was degraded with similar kinetics (half-life % 1 h) in 653-1 cells that massively overproduce O DC and in their pare ntal 653 cells in which ODC is prac tically undetected (Fig. 2 A and B). Moreover, in 653-1 cells the degradation rate of antizyme was different from that of ODC. Although our results do not necessarily negate the notion that antizyme may be degraded together with ODC, they suggest that interaction with ODC is not essential f or the degradation of antizyme. This conclusion is supported mainly by the experiment like that presented in Fig. 2A as in 653 cells antizyme is present in vast excess whereas ODC is practically undetected. Testing for antizyme degradation in cells completely lacking ODC protein will allow d rawing of a definite conclusion. Interestingly, coexpression of anti- zyme together with the stable ODC variant, Del6, which lacks the C-terminal degradation signal [28], stabilized antizyme (Fig. 3). Similar stabilization of antizyme was observed when it was coexpressed with a stable ODC variant in which Cys441 was converted to Trp [22]. The degradation of antizyme depends on the p resence of an active ubiquitin-dependent proteolytic system. The observation that antizyme is capable of being d egraded independently of ODC prompted us to characterize this degradation process. As degradation through the ubiquitin system is a prominent possibility we have investigated antizyme degradation in ts20 cells, containing a thermosen- sitive ubiquitin-activating enzyme E1 [31]. It was demon- strated that in these cells, p53, a typical substrate of the ubiquitin system, accumulates upon their shift to the nonpermissive temperature [31]. E xpression constructs encoding ODC and antizyme were transfected into ts20 cells and into their parental wild-type c ells and the cells were kept at the permissive temperature (32 °C). Twenty-four hours post-transfection half of the cells were transferred to the restrictive temperature (39 °C) and incubated for additional 16 h. Cellular extracts were then prepared and the relevan t prote ins were detected by Western blot analysis. Fig. 2. Antizyme degradation can occur independently of the degradation of ODC. (A) 653 and 653-1 c ells were treated with 5 m M spermidine in the presence of 1 m M aminoguanidine (an inhibitor of serum amine oxidases) for 1 h in order to induce antizyme. Cycloheximide (20 lgÆmL )1 )was added to the growth medium and cellular extracts were prepared and analysed as described in Fig. 1. The lanes r epresenting 653 cells contain twice the amount of protein as those representing 653-1 cells. (B) The blot presen ted in A was scanned u sing a UmaxIII scanner and PHOTOSHOP -4.0. The relative intensities of the antizyme bands were determined by using IMAGE GAUGE V3.41 (C and D). Cos-7 cells were transfected with expression constructs encoding antizyme (the short form) o r ODC (C), or were cotransfected with both exp ression constructs (D). Forty-eight hours post- transfection th e cells were treated with cycloheximide and MG132 for the indicated times. Cellular extracts were prepared, fractionated and antizyme and ODC detected as described in Fig. 1. Ó FEBS 2002 Characterization of antizyme degradation (Eur. J. Biochem. 269) 1319 The two control proteins, p53 and ODC, behaved as expected: en dogenous p53 accumulated only in the ts20 cells grown at the restrictive temperature but not in wild-type cells (Fig. 4). In contrast, not only did ODC not accumulate in either cell line but its concentration was actually slightly reduced at the restrictive t emperature (Fig. 4), probably due to accelerated degradation at 39 °C. As shown in the figure, both forms of antizyme accumulated in ts20 cells at the nonpermissive temperature. We therefore conclude that antizyme, the mediator of the ubiquitin-independent degra- dation of ODC, requires a functional ubiquitin system for its degradation. DISCUSSION Antizyme is a unique cellular regulatory protein that is both regulated by polyamines and regulates polyamine metabo- lism in a feedback loop. Antizyme expression is regulated translationally by a polyamine-stimulated ribosomal frame- shifting [2,11,12]. In turn, antizyme contributes to reducing the intracellular concentration of polyamines, both by marking ODC for rapid degradation [2,8,13,37], and by reducing polyamine uptake via a yet unknown mechanism [17,38–40]. In this sense antizyme expression by frameshift- ing serves a s the cellular sensor for polyamines. While the molecular mechanisms by which antizyme directs ODC to degradation is partially revealed (reviewed in [2,37]), there is ambiguity about the fate of antizyme during this proteolytic process. Previous studies performed in vitro in degradation extracts suggested that while taking ODC to the protea- some, antizyme remains stable and is recycled to participate in subsequent rounds of ODC degradation [2,13,21]. In contrast, other studies suggested that antizyme is also a rapidly degraded protein [8,23–25]. We demonstrate here that endogenous antizyme in the ODC overproducing 653-1 cells and antizyme that is expressed in 293 cells from an expression vector is rapidly degraded. Like the degradation of ODC, the degradation of antizyme is also mediated by the proteasome as specific inhibitors of this protease effectively inhibit this proteolytic process. This result refutes the long-standing idea that antizyme is a stable protein [2,13,21]. The rapid degradation of antizyme is compatible with the critical role this protein plays in regulating cellular polyamine levels. A key protein such as antizyme can not function as an effective r egulator unless it is rapidly degraded or its activity is tightly modulated. Rapid degradation of antizyme may suggest that it is degraded together with ODC while taking the latter to the 26S proteasome. In such a case it could be expected that stoichiometric relationship would be required for the degradation of these two proteins. Indeed, as demonstrated previously [36], cotransfection with antizyme accelerated ODC degradation. In contrast, the degradation of antizyme was not affected by the simultaneous expression of ODC. Similarly, the rate of antizyme degradation in 653-1 cells which contain large amount of ODC is identical to that observed in 653 cells in which ODC is practically undetec- ted. These results strongly support the alternative possibility that the degradation of antizyme is independent from that of ODC. Further in supporting this possibility is the observation that ODC a nd antizyme differ in their rate of degradation in 653-1 cells (Fig. 2B). Therefore, w e can conclude that even if antizyme is degraded together with ODC, its degradation can also occur independently of ODC. As demonstrated here (Fig. 3) antizyme degradation was inhibited when it was expressed together with the stable ODC variant. A similar observation was made recently and in a previous study [22]. The interpretation in that study was Fig. 4. Degradation of antizyme depends on the integrity of the ubiqui- tination machinery. Wild-type (A31N) and ts20 cells containing ther- mosensitive ubiquitin-activating enzyme E1 were transfected with expression constructs encoding ODC and antizyme (FLFS antizyme). The transfected cells were incubated for 24 h at 32 °C (permissive temperature) and then half of the cells were transferred to 39 °C (restrictive temperature) for an additional 16 h. Cellular extracts were prepared, fractionated and the proteins of i nterest were detected by immunoblotting. Fig. 3. Co-expression with the stable ODC mutant, Del-6, inhibits antizyme degradation. 293 cells were cotransfected with expression constructs encoding antizyme and the stable ODC mutant, Del6 that lacks the C-terminal destabilizing segment encompassing amino acids 423–461. The transfected cells were treated and antizyme and ODC were detected as described in Fig. 1. 1320 S. Gandre et al. (Eur. J. Biochem. 269) Ó FEBS 2002 that as part of a complex that is a poor substrate for the 26S proteasome, antizyme is protected from degradation [22]. We propose here an a lternative possibility, that antizyme is protected from degradation not because it is trapped in a poorly degradable complex but because it should be free in order to be recognized and marked for rapid degradation. It will be of great interest in this respect to determine how interaction with antizyme inhibitor [19,20,41–43] affects antizyme degradation. The observation that antizyme may be degraded inde- pendently of ODC raised the possibility t hat antizyme may be degraded via different recognition machinery. The major cellular proteolytic system responsible for marking the destiny of a protein to rapid degradation is the u biquitin- dependent proteolytic system [44–47]. We have used the Balb/C 3T3-derived cell line ts20 that contains a thermo- sensitive ubiquitin-activating enzyme E1 [31] to determine whether the ubiquitin system is involved in t he degradation of antizyme. Two control proteins were used in this experiment: p53 a substrate of the ubiquitin system that was demonstrated to accumulate in t he mutant cells at the nonpermissive temperature; and ODC whose degradation is independent of ubiquitination. As expected p53 accumu- lated at the restrictive temperature while ODC did not. In fact, p robably due to increased degradation activity at the restrictive temperature (39 °C) the level of ODC actually dropped. Both forms of antizyme, which represent initia- tions at two altern ative translation start sites, accumulated in the mutant cells at the nonpermissive temperature. We therefore conclude that a f unctional ubiquitin system is required for the degradation of antizyme. This is an interesting situation in which antizyme, the protein that marks ODC to rapid ubiquitin-independent degradation may be itself degraded through the ubiquitin system. It must be emphasized however, that while our results demonstrate that a functional ubiquitin system is required for the degradation of antizyme, we cannot state that antizyme is directly targeted for the degradation by ubiquitination as we could not demonstrate the presence of ubiquinated anti- zyme (in cells cotransfected with antizyme and HA-ubiqui- tin). Such demonstration may require the identification of the components of the ubiquitin sytem (E2 and E3) involved in mediating antizyme d egradation as such components a re likely to be limiting. Indeed, ubiquinated forms of p53 were noted only when p53 and HA-ubiquitin were complemented by MDM2 (E3 for p53) in the t ransfection assay. The revelation of their potential relationships to the cellular polyamine metabolism will be of great interest. ACKNOWLEDGEMENTS We thank S. Hobbs for the pEFIRES-p vector and H. L. Ozer for the A31N wild type and ts20 cells. 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