Ornithine decarboxylase-antizyme is rapidly degraded througha mechanism that requires functional ubiquitin-dependent proteolytic activity Shilpa Gandre, Zippi Bercovich and Chaim Kahana
Trang 1Ornithine 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 vitroin a reticulocyte 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 that 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 codons, are rapidly degraded The degradation of both antizyme proteins is inhibited in 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 ubiquitin system
Keywords: antizyme; ornithine decarboxylase; protein deg-radation; proteasome; polyamines
The polyamines spermidine and spermine and their
precur-sor putrescine are ubiquitous aliphatic polycations with
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 in various cellular functions, multiple pathways
such as biosynthesis, catabolism, uptake, and excretion
tightly regulate their intracellular concentration One of the
major sources of cellular polyamines comes from their
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 spersper-midine and spermine, respectively Both
enzymes are highly regulated and are subjected to feedback
control by cellular polyamines Control of cellular
polyam-ines by rapid regulated degradation of ODC constitutes an
important feedback regulatory mechanism
ODC is one of the most rapidly degraded proteins in eukaryotic cells Interestingly it is degraded without requi-ring ubiquitination [6,7] Instead, ODC is targeted to degradation due to its interaction with a unique 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 Antizyme binds to ODC subunit to form enzymat-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 subunits are targeted to degradation [8,13–15] It was suggested that binding of antizyme to ODC results in the exposure of the C-terminal destabilizing signal of ODC [16] Antizyme was also demonstrated 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 to ODC, but lacks decarboxylating 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 suggested 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)
Trang 2participate 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 interacting with ODC
We also demonstrate that in contrast 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 impaired at the restrictive temperature
E X P E R I M E N T A L P R O C E D U R E S
Materials
Proteasome inhibitors MG115 (Z-Leu-Leu-Norvalinal) and
MG132 (Z-Leu-Leu-Leucinal) were from Calbiochem
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 the pCI-neo expression 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 mM)
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 calf 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 the
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 analysis
Western blot analysis Cells were harvested at the indicated times, lysed in lysis buffer (150 mM NaCl 50 mM 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 using Bradford’s method Samples containing equal amounts of protein were denatured in Laemmli buffer, fractionated by SDS/PAGE and blotted onto nitrocellulose membrane The blots were then probed with the 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] Cells were collected, resuspended in 500 lL NaCl/Pi and the material precipitating in 3% perchloric acid was removed by centrifugation Four-hundred microlitres of dansyl chloride (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) was added and the mixture was incubated for an additional 1 h Dansylated derivatives were then extracted 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
R E S U L T S 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 vitroin degradation extracts assumes that antizyme 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
Trang 3without 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 ODC, the overexpressed antizyme 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 antizyme 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 to the growth medium
of the cells and cellular extracts were prepared at various
times thereafter The extracts were resolved by SDS/PAGE
and antizyme was detected by Western blot analysis using
specific antiantizyme monoclonal antibodies As noted in
653-1 cells, also in transfected Cos-7 cells antizyme was
rapidly degraded in a proteasome-dependent manner
(Fig 1C) 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 encoding 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 1D) Both forms of antizyme were rapidly degraded (Fig 1D)
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 antizyme 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 the Supersignal Chemiluminescence detection system Lane 1 contains equal amount of protein extracted from the parental 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 construct Forty-eight hours post-transfection cycloheximide was added to the growth medium alone or 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.
Trang 4The 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 ODC and
in their parental 653 cells in which ODC is practically
undetected (Fig 2A 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 for
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 drawing 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 presence of
an active ubiquitin-dependent proteolytic system The observation that antizyme is capable of being degraded 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] Expression constructs encoding ODC and antizyme were transfected into ts20 cells and into their parental wild-type cells 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 relevant proteins were detected by Western blot analysis
Fig 2 Antizyme degradation can occur independently of the degradation of ODC (A) 653 and 653-1 cells 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 representing 653 cells contain twice the amount of protein as those representing 653-1 cells (B) The blot presented in A was scanned using 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) or ODC (C), or were cotransfected with both expression constructs (D) Forty-eight hours post-transfection the 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.
Trang 5The two control proteins, p53 and ODC, behaved as
expected: endogenous 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 temperature (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
D I S C U S S I O N 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 as 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 regulator 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 and antizyme differ in their rate of degradation in 653-1 cells (Fig 2B) Therefore, we 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 interest 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.
Trang 6that as part of a complex that is a poor substrate for the 26S
proteasome, antizyme is protected from degradation [22]
We propose here an alternative 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 that 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
ubiquitin-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 the degradation
of antizyme Two control proteins were used in this
experiment: p53 a substrate of the ubiquitin system that
was demonstrated to accumulate in the 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, probably 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 alternative translation start sites, accumulated
in the mutant cells at the nonpermissive temperature We
therefore conclude that a functional 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 antianti-zyme and
HA-ubiqui-tin) Such demonstration may require the identification of
the components of the ubiquitin sytem (E2 and E3) involved
in mediating antizyme degradation as such components are
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 transfection assay The
revelation of their potential relationships to the cellular
polyamine metabolism will be of great interest
A C K N O W L E D G E M E N T S
We thank S Hobbs for the pEFIRES-p vector and H L Ozer for the
A31N wild type and ts20 cells This study was supported by a grant from
the Leo and Julia Forchheimer Center for Molecular Genetics at the
Weizmann Institute of Science and by a research grant from the Israeli
science foundation and the Jean-Jacques Brunschwig memorial fund.
R E F E R E N C E S
1 Coffino, P & Poznanski, A (1991) Killer polyamines? J Cell
Biochem 45, 54–58.
2 Coffino, P (2001) Regulation of cellular polyamines by antizyme.
Nature Rev Mol Cell Biol 2, 188–194.
3 Marton, L.J & Pegg, A.E (1995) Polyamines as targets for therapeutic intervention Annu Rev Pharmacol Toxicol 35, 55–91.
4 Tabor, C.W & Tabor, H (1984) Polyamines Annu Rev Biochem.
53, 749–790.
5 Tabor, C.W & Tabor, H (1985) Polyamines in microorganisms Microbiol Rev 49, 81–99.
6 Bercovich, Z., Rosenberg-Hasson, Y., Ciechanover, A & Kahana, C (1989) Degradation of ornithine decarboxylase in reticulocyte lysate is ATP-dependent but ubiquitin-independent.
J Biol Chem 264, 15949–15952.
7 Rosenberg-Hasson, Y., Bercovich, Z., Ciechanover, A & Kahana, C (1989) Degradation of ornithine decarboxylase in mammalian cells is ATP dependent but ubiquitin independent Eur J Biochem 185, 469–474.
8 Murakami, Y., Matsufuji, S., Kameji, T., Hayashi, S., Igarashi, K., Tamura, T., Tanaka, K & Ichihara, A (1992) Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination Nature 360, 597–599.
9 Mamroud-Kidron, E & Kahana, C (1994) The 26S proteasome degrades mouse and yeast ornithine decarboxylase in yeast cells FEBS Lett 356, 162–164.
10 Mamroud-Kidron, E., Rosenberg-Hasson, Y., Rom, E & Kahana, C (1994) The 20S proteasome mediates the degradation
of mouse and yeast ornithine decarboxylase in yeast cells FEBS Lett 337, 239–242.
11 Matsufuji, S., Matsufuji, T., Miyazaki, Y., Murakami, Y., Atkins, J.F., Gesteland, R.F & Hayashi, S (1995) Autoregulatory frame-shifting in decoding mammalian ornithine decarboxylase anti-zyme Cell 80, 51–60.
12 Rom, E & Kahana, C (1994) Polyamines regulate the expression
of ornithine decarboxylase antizyme in vitro by inducing ribo-somal frame-shifting Proc Natl Acad Sci USA 91, 3959–3963.
13 Mamroud-Kidron, E., Omer-Itsicovich, M., Bercovich, Z., Tobias, K.E., Rom, E & Kahana, C (1994) A unified pathway for the degradation of ornithine decarboxylase in reticulocyte lysate requires interaction with the polyamine-induced protein, ornithine decarboxylase antizyme Eur J Biochem 226, 547–554.
14 Li, X & Coffino, P (1992) Regulated degradation of ornithine decarboxylase requires interaction with the polyamine-inducible protein antizyme Mol Cell Biol 12, 3556–3562.
15 Murakami, Y., Tanaka, K., Matsufuji, S., Miyazaki, Y & Hayashi, S (1992) Antizyme, a protein induced by polyamines, accelerates the degradation of ornithine decarboxylase in Chinese-hamster ovary-cell extracts Biochem J 283, 661–664.
16 Li, X & Coffino, P (1993) Degradation of ornithine decar-boxylase: exposure of the C-terminal target by a polyamine-inducible inhibitory protein Mol Cell Biol 13, 2377–2383.
17 Mitchell, J.L., Judd, G.G., Bareyal-Leyser, A & Ling, S.Y (1994) Feedback repression of polyamine transport is mediated by anti-zyme in mammalian tissue-culture cells Biochem J 299, 19–22.
18 Suzuki, T., He, Y., Kashiwagi, K., Murakami, Y., Hayashi, S & Igarashi, K (1994) Antizyme protects against abnormal accu-mulation and toxicity of polyamines in ornithine decarboxylase-overproducing cells Proc Natl Acad Sci USA 91, 8930–8934.
19 Murakami, Y., Ichiba, T., Matsufuji, S & Hayashi, S (1996) Cloning of antizyme inhibitor, a highly homologous protein to ornithine decarboxylase, J Biol Chem 271, 3340–3342.
20 Nilsson, J., Grahn, B & Heby, O (2000) Antizyme inhibitor is rapidly induced in growth-stimulated mouse fibroblasts and releases ornithine decarboxylase from antizyme suppression Biochem J 346, 699–704.
21 Tokunaga, F., Goto, T., Koide, T., Murakami, Y., Hayashi, S., Tamura, T., Tanaka, K & Ichihara, A (1994) ATP- and anti-zyme-dependent endoproteolysis of ornithine decarboxylase to oligopeptides by the 26S proteasome J Biol Chem 269, 17382– 17385.
Trang 722 Mitchell, J.L., Choe, C.Y., Judd, G.G., Daghfal, D.J., Kurzeja,
R.J & Leyser, A (1996) Overproduction of stable ornithine
decarboxylase and antizyme in the
difluoromethylornithine-resistant cell line DH23b Biochem J 317, 811–816.
23 Mitchell, J.L., Judd, G.G., Leyser, A & Choe, C (1998) Osmotic
stress induces variation in cellular levels of ornithine
decar-boxylase-antizyme Biochem J 329, 453–459.
24 Hayashi, S & Murakami, Y (1995) Rapid and regulated
degradation of ornithine decarboxylase Biochem J 306, 1–10.
25 Murakami, Y., Fujita, K., Kameji, T & Hayashi, S (1985)
Accumulation of ornithine decarboxylase-antizyme complex in
HMOA cells Biochem J 225, 689–697.
26 Matsufuji, S., Miyazaki, Y., Kanamoto, R., Kameji, T.,
Murakami, Y., Baby, T.G., Fujita, K., Ohno, T & Hayashi, S.
(1990) Analyses of ornithine decarboxylase antizyme mRNA with
a cDNA cloned from rat liver J Biochem (Tokyo) 108, 365–371.
27 Hobbs, S., Jitrapakdee, S & Wallace, J.C (1998) Development of
a bicistronic vector driven by the human polypeptide chain
elon-gation factor 1alpha promoter for creation of stable mammalian
cell lines that express very high levels of recombinant proteins.
Biochem Biophys Res Commun 252, 368–372.
28 Rosenberg-Hasson, Y., Bercovich, Z & Kahana, C (1991)
Characterization of sequences involved in mediating degradation
of ornithine decarboxylase in cells and in reticulocyte lysate Eur.
J Biochem 196, 647–651.
29 Kahana, C & Nathans, D (1984) Isolation of cloned cDNA
encoding mammalian ornithine decarboxylase Proc Natl Acad.
Sci USA 81, 3645–3649.
30 Graham, F.L & van der Eb, A.J (1973) A new technique for the
assay of infectivity of human adenovirus 5 DNA Virology 52,
456–467.
31 Chowdary, D.R., Dermody, J.J., Jha, K.K & Ozer, H.L (1994)
Accumulation of p53 in a mutant cell line defective in the ubiquitin
pathway Mol Cell Biol 14, 1997–2003.
32 Tobias, K.E & Kahana, C (1995) Exposure to ornithine results in
excessive accumulation of putrescine and apoptotic cell death in
ornithine decarboxylase overproducing mouse myeloma cells Cell
Growth Differ 6, 1279–1285.
33 Kahana, C & Nathans, D (1985) Translational regulation of
mammalian ornithine decarboxylase by polyamines J Biol.
Chem 260, 15390–15393.
34 Kahana, C & Nathans, D (1985) Nucleotide sequence of murine
ornithine decarboxylase mRNA Proc Natl Acad Sci USA 82,
1673–1677.
35 Katz, A & Kahana, C (1989) Rearrangement between ornithine decarboxylase and the switch region of the gamma 1 immunoglobulin gene in alpha-difluoromethylornithine resistant mouse myeloma cells EMBO J 8, 1163–1167.
36 Murakami, Y., Matsufuji, S., Miyazaki, Y & Hayashi, S (1992) Destabilization of ornithine decarboxylase by transfected anti-zyme gene expression in hepatoma tissue culture cells Antianti-zyme2 is
a negative regulator of ornithine decarboxylase and polyamine transport J Biol Chem 267, 13138–13141.
37 Coffino, P (2001) Antizyme, a mediator of ubiquitin-independent proteasomal degradation Biochimie 83, 319–323.
38 Sakata, K., Kashiwagi, K & Igarashi, K (2000) Properties of a polyamine transporter regulated by antizyme Biochem J 347, 297–303.
39 Zhu, C., Lang, D.W & Coffino, P (1999) Antizyme2 is a negative regulator of ornithine decarboxylase and polyamine transport.
J Biol Chem 274, 26425–26430.
40 Sakata, K., Fukuchi-Shimogori, T., Kashiwagi, K & Igarashi, K (1997) Identification of regulatory region of antizyme necessary for the negative regulation of polyamine transport Biochem Biophys Res Commun 238, 415–419.
41 Koguchi, K., Kobayashi, S., Hayashi, T., Matsufuji, S., Murakami, Y & Hayashi, S (1997) Cloning and sequencing of a human cDNA encoding ornithine decarboxylase antizyme inhibitor Biochim Biophys Acta 1353, 209–216.
42 Kitani, T & Fujisawa, H (1989) Purification and characterization
of antizyme inhibitor of ornithine decarboxylase from rat liver Biochim Biophys Acta 991, 44–49.
43 Fujita, K., Murakami, Y & Hayashi, S (1982) A macromolecular inhibitor of the antizyme to ornithine decarboxylase Biochem.
J 204, 647–652.
44 Ciechanover, A., Orian, A & Schwartz, A.L (2000) Ubiquitin-mediated proteolysis: biological regulation via destruction Bioessays 22, 442–451.
45 Ciechanover, A., Orian, A & Schwartz, A.L (2000) The ubiqui-tin-mediated proteolytic pathway: mode of action and clinical implications J Cell Biochem 34 (Suppl.), 40–51.
46 Hershko, A & Ciechanover, A (1992) The ubiquitin system for protein degradation Annu Rev Biochem 61, 761–807.
47 Kornitzer, D & Ciechanover, A (2000) Modes of regulation
of ubiquitin-mediated protein degradation J Cell Physiol 182, 1–11.