(Received for publication, April 17, 1995; and in revised form, June 28, 1995)
From the
Iron regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that bind to specific structures, termed iron-responsive elements (IREs), that are located in the 5`- or 3`-untranslated regions of mRNAs that encode proteins involved in iron homeostasis. IRP1 and IRP2 RNA binding activities are regulated by iron; IRP1 and IRP2 bind IREs with high affinity in iron-depleted cells and with low affinity in iron-repleted cells. The decrease in IRP1 RNA binding activity occurs by a switch between apoprotein and 4Fe-4S forms, without changes in IRP1 levels, whereas the decrease in IRP2 RNA binding activity reflects a reduction in IRP2 levels. To determine the mechanism by which iron decreases IRP2 levels, we studied IRP2 regulation by iron in rat hepatoma and human HeLa cells. The iron-dependent decrease in IRP2 levels was not due to a decrease in the amount of IRP2 mRNA or to a decrease in the rate of IRP2 synthesis. Pulse-chase experiments demonstrated that iron resulted in a 3-fold increase in the degradation rate of IRP2. IRP2 degradation depends on protein synthesis, but not transcription, suggesting a requirement for a labile protein. IRP2 degradation is not prevented by lysosomal inhibitors or calpain II inhibitors, but is prevented by inhibitors that block proteasome function. These data suggest the involvement of the proteasome in iron-mediated IRP2 proteolysis.
Iron regulatory proteins (IRPs) ()are cytosolic
RNA-binding proteins that regulate the post-transcriptional expression
of genes that are involved in iron
homeostasis(1, 2, 3, 4) . IRPs were
formerly known as the iron-responsive element-binding protein (IRE-BP),
the ferritin repressor protein (FRP), and the iron regulatory factor
(IRF). IRPs bind with high affinity to RNA stem-loops known as
iron-responsive elements (IREs). IREs are located in the 5`-
untranslated regions of ferritin and erythroid
-aminolevulinic
acid synthase mRNAs where binding causes translational repression (5, 6, 7) . Five IREs are located in the
3`-untranslated region of transferrin receptor
mRNA(8, 9) , and binding of the IRP stabilizes
transferrin receptor mRNA(9, 10) .
Two distinct IRPs have been cloned and characterized in mammalian cells and have been designated as IRP1 and IRP2. IRP1 has been cloned from a variety of mammalian species(5, 11, 12) . IRP1 has a molecular mass of 98,000 Da and shares 30% amino acid identity with the 4Fe-4S enzyme, mitochondrial aconitase(13) . The 18 active site residues in mitochondrial aconitase, including the 3 cysteines that serve as ligands for the 4Fe-4S cluster are conserved in IRP1(13) . In addition, IRP1 is an active cytosolic aconitase(14, 15) . In iron-repleted cells, IRP1 exhibits aconitase activity and contains iron, but binds the IRE with low affinity. In contrast, in iron-depleted cells, IRP1 lacks aconitase activity and iron, but binds the IRE with high affinity. UV cross-linking studies have shown overlap between RNA binding and the aconitase active sites, indicating that RNA binding and aconitase activities are mutually exclusive(16, 17) . Recent data indicated that aconitase activity is not necessary for iron regulation of IRP1, since substitution of an alanine for an active site serine does not prevent assembly and disassembly of the 4Fe-4S cluster(18) .
IRP2 has been characterized in rat tissues by RNA band shift assays (19, 20, 21, 22) and has been purified from rat liver and rat hepatoma cells (20) . The partial amino acid sequence of rat IRP2 is similar to the predicted protein sequence encoded by a cDNA isolated from a human T cell library(11, 23) , suggesting that this is the rat version of the human protein. A second IRP has been characterized from mouse tissues by RNA band shift analysis(24) , which is presumed to be homologous to the rat and the human IRP2.
IRP2 contains similar biochemical properties to IRP1 in that it binds IREs with similar affinity (20, 24) and represses translation of IRE-containing mRNAs in vitro(20, 25) . IRP1 and IRP2 differ in two aspects: first, unlike IRP1, IRP2 does not exhibit aconitase activity, indicating that aconitase activity is not necessary for regulation by iron(20) , and second, that although iron results in a decrease in IRP1 and IRP2 RNA-binding activities, the amount of IRP1 remains constant(12, 26) , whereas the amount of IRP2 protein is substantially reduced (20, 27
These novel properties of IRP2 raised questions as to how intracellular iron regulates IRP2 RNA binding activity. To answer this question, we analyzed the regulation of IRP2 by iron in rat hepatoma cells and human HeLa cells. We found that the marked reduction in IRP2 RNA binding activity and protein levels in iron-treated cells is due to increased turnover of IRP2. IRP2 synthesis and IRP2 mRNAs levels are unaffected by iron treatment. The iron-mediated degradation of IRP2 requires protein synthesis, but not transcription, suggesting that the synthesis of a labile protein is required. We also demonstrate that the proteasome complex is required for iron-mediated degradation of IRP2. Regulation of IRP2 protein levels by iron occurs in a variety of cell types, indicating that iron-mediated degradation is a common pathway for regulating IRP2 RNA binding activity.
For immunoblot analysis, 50 µg of protein from cell lysates was fractionated on 8% SDS-PAGE. The protein was transferred to nitrocellulose membranes, and the membranes were incubated with a chicken anti-IRP1 antibody generated against the entire IRP1 protein (20) or a rabbit anti-IRP2 antibody(20) . After 1 h, the membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-chicken or goat anti-rabbit IgG for 1 h. The protein was visualized using the enhanced chemiluminescence Western blotting detection system (Amersham Corp.) according to the manufacturer.
Figure 1:
Effect of iron treatment on RNA binding
activity and levels of IRP1 and IRP2 in rat hepatoma cells. A,
FTO2B cells were grown in the presence (lanes 2-7) or
the absence (lanes 1 and 8) of 50 µg/ml FAC for
1-24 h after which lysates were prepared as described under
``Experimental Procedures.'' C24, untreated cells
harvested at 24 h. Equal amounts of protein (10 µg) were incubated
with a P-labeled IRE RNA followed by electrophoresis of
the RNA-protein complexes by a 5% native polyacrylamide gel. The
positions of IRP1
IRE and IRP2
IRE complexes and free IRE RNA
are indicated. B, equal amounts of protein (50 µg) from A were subjected to 8% SDS-PAGE followed by immunoblot
analysis using anti-rat IRP1 or anti-IRP2 antisera. Molecular weight
standards are indicated.
To determine if the iron-mediated decrease
in IRP2 levels occurs in other cell types, we measured RNA binding
activity and protein levels for IRP1 and IRP2 in human HeLa cells
treated with FAC for 1-24 h (Fig. 2). Since human
IRP1IRE and IRP2
IRE complexes comigrate on native
polyacrylamide gels, we carried out supershift assays using anti-IRP2
antisera in RNA band shift assays. We have previously demonstrated that
anti-IRP2 antisera does not interfere with RNA binding and results in a
supershifted IRP2
IRE complex(20) . Treatment of cells
with FAC caused RNA-binding activity of IRP1 and IRP2 to decrease 2-
and 5-fold, respectively (Fig. 2, A and C).
Immunoblot analysis indicated that the amount of IRP1 remained constant
during iron treatment, whereas the amount of IRP2 decreased 5-fold (Fig. 2, B and C). We have observed reductions
in the amount of IRP2 in mouse 3T3 fibroblasts and transformed human
primary embryonal 293 kidney cells treated with FAC for the same time
course (data not shown). These data indicated that the decrease in IRP1
and IRP2 RNA binding activities induced by iron are mediated by
different cellular processes and occurs in a variety of cell types.
Figure 2:
Effect of iron treatment on RNA binding
activity and protein levels of IRP1 and IRP2 in human HeLa cells. A, HeLa cells were grown in the presence (lanes
2-7) or absence (lane 1) of 50 µg/ml FAC. Equal
amounts of protein (10 µg) were incubated with (lanes
9-15) or without (lanes 1-8) anti-IRP2
antisera for 5 min followed by the addition of P-labeled
IRE RNA. As a control, rabbit preimmune antisera was added to an
extract from untreated cells (C/PI, lane 8). IRP2
IRE
complexes are indicated by an asterisk. B, equal
amounts of protein (50 µg) from extracts in A were
subjected to immunoblot analysis using anti-IRP1 or anti-IRP2 antisera. C, the data in A and B were quantified by
densitometry and plotted using untreated control as
100%.
Figure 3:
Effect
of iron treatment on IRP2 mRNA levels in rat hepatoma cells. FTO2B
cells were grown in the presence of 50 µg/ml FAC (Iron)
for 1-24 h or 200 µM desferrioxamine (Df)
for 16 h. Total RNA was isolated and analyzed on an 1%
formaldehyde-agarose gel. C0 and C24, untreated cells
harvested at 0 and 24 h, respectively. The RNA was transferred to a
membrane and sequentially hybridized with a P-labeled IRP2
cDNA (A) or a
P-labeled glyceraldehyde phosphate
dehydrogenase cDNA (B) to control for gel loading. The size of
the IRP2 transcripts are indicated by arrows. RNA molecular
weight standards are from Life Technologies,
Inc.
Figure 4:
Effect
of iron on the rate of IRP2 degradation. A, FTO2B cells were
pulse-labeled for 4 h with TranS-label and chased in
medium containing an excess of unlabeled methionine in the absence (lanes 1-11) or the presence (lanes
12-18) of 50 µg/ml FAC for 0-8 h. IRP2 was
immunoprecipitated using anti-IRP2 antisera (lanes
3-18). As a control, IRP2 was immunoprecipitated from
extracts from untreated cells using preimmune rabbit serum (PI) (lanes 1 and 2). Labeled
immunoprecipitated protein was analyzed by 8% SDS-PAGE. The positions
of the molecular weight standards and IRP2 are indicated. B,
the turnover data in A was quantified by densitometry, and the
intensity of the IRP2 bands were plotted relative to the percent of
radioactivity remaining after 0 h (lanes 3 and 4).
These experiment were carried out three times, and one representative
experiment is shown. Symbols:
, no addition;
,
iron.
It was possible that in addition to decreasing the half-life of IRP2, iron could also reduce its rate of synthesis. Measurements of synthesis rates in the presence of iron could be misleading, since it would not only measure synthesis, but would also measure degradation of newly synthesized protein. To determine if synthesis of IRP2 was affected by iron, we treated cells with FAC for 2.5 h, then quantified the amount of methionine incorporated into IRP2 during a short 1-h time course. Our turnover data indicated that in 1 h after iron treatment approximately 30% of labeled pre-existing IRP2 is degraded. Labeled IRP2 was immunoprecipitated using anti-IRP2 antibodies at 10, 20, 40, and 60 min followed by fractionation of the immunocomplexes by SDS-PAGE (Fig. 5A) and quantification of the radioactivity in the IRP2 bands by densitometry (Fig. 5B). At time points between 10 and 40 min, the rate of IRP2 synthesis was not significantly different in iron-treated or control cells. After 1 h of labeling, the amount of labeled IRP2 in iron-treated cells decreased slightly compared with the amount of IRP2 in control cells. The decrease in IRP2 label at 1 h is presumably due to the increase in the degradation of newly synthesized protein. We conclude that the iron-mediated reduction in IRP2 levels is due to an increased rate of degradation without changes in the rate of IRP2 synthesis.
Figure 5:
Effect
of iron on the rate of IRP2 synthesis. A, FTO2B cells were
grown in the presence (lanes 5-8) or the absence (lanes 1-4) of 50 µg/ml FAC for 2.5 h. The cells
were incubated in methionine-free medium for 15 min with or without
FAC. After 15 min, the cells were labeled with 100 µCi/ml
TranS-label and were then harvested after 10, 20, 40, and
60 min. IRP2 was immunoprecipitated using anti-IRP2 antisera and
analyzed by 8% SDS-PAGE. Molecular weight standards and IRP2 are
indicated. PI, control 60-min lysate immunoprecipitated with
preimmune serum. B, the synthesis data in A was
quantified by densitometry and the integrated density of labeled IRP2
bands was plotted. These data represent the results from two
experiments.
Figure 6:
Effect of cycloheximide on the
iron-mediated degradation of IRP2 by iron. FTO2B cells were grown in
the presence (lanes 3-5) or absence (lanes 1 and 2) of 50 µg/ml FAC (Iron), 20 µg/ml
cycloheximide (Cyx) (lanes 6-8), or FAC plus
cycloheximide (Iron + Cyx) (lanes 9-11)
for 0-4 h. A, equal amounts of protein (50 µg) were
subjected to 8% SDS-PAGE for immunoblot analysis using anti-IRP2
antisera. Molecular weight standards and the positions of IRP2 and a
nonspecific immunoreactive band (ns) are indicated. B, equal amount of protein (10 µg) from extracts in A was incubated with P-labeled IRE followed by
electrophoresis of the RNA-protein by 5% native polyacrylamide gels.
The positions of IRP1
IRE and IRP2
IRE complexes are
indicated.
We also determined if transcription is required for the degradation of IRP2 induced by iron. FTO2B cells were treated with the transcription inhibitor, actinomycin D alone, or in the presence or absence of FAC for 0, 1, 2.5, and 4 h, and IRP1 and IRP2 RNA binding activities and IRP2 levels were measured (Fig. 7, A and B). Actinomycin D alone had no effect on IRP1 or IRP2 RNA binding activities (Fig. 7B) or IRP2 protein levels (Fig. 7A). When cells were treated with FAC and actinomycin D, IRP1 and IRP2 RNA binding activities and IRP2 levels decreased, but not to the levels observed with iron alone. These data indicated that the iron-mediated degradation of IRP2 requires protein synthesis, but to a lesser extent transcription, suggesting that the synthesis of a labile protein is required for IRP2 degradation.
Figure 7: Effect of actinomycin D on the iron-mediated degradation of IRP2. FTO2B cells were grown in the presence (lanes 5-7) or absence (lanes 1-4) of 50 µg/ml FAC (Iron), 10 µM actinomycin D (lanes 8-10), or FAC plus actinomycin D (Act D + Iron) (lanes 11-13) for 0-4 h. Immunoblot analysis (A) and RNA band shift assays (B) were carried out as described in the legend to Fig. 6.
Figure 8:
Effect of a proteasome inhibitor on IRP2
iron-mediated degradation. A, FTO2B cells were pretreated with
100 µM MG-132 in 1.0% dimethyl sulfoxide
(MeSO) for 1 h prior to the addition of 50 µg/ml FAC
for 1-4 h (lanes 9-11). Cells were also treated
with MG-132 for 1-5 h (lanes 2-5) or FAC in
Me
SO for 1-4 h (lanes 6-8). CO control, untreated cells harvested at 0 h. The top panel is an immunoblot using anti-IRP2 antisera, and the bottom
panel is an RNA band shift assay. The positions of IRP1 and IRP2
are indicated.
Because MG-132 also inhibits calpains and lysosomal cysteine
proteases, such as cathepsin B, ()we tested whether calpain
II (N-acetyl-leucinyl-leucinyl-methional-H), a cysteine
protease inhibitor, and the lysosomal inhibitors, ammonium chloride and
chloroquine, prevented iron-mediated IRP2 degradation. Previous studies
demonstrated that calpain II inhibitors have little effect on
proteasome function(33) . Fig. 9A shows that
the treatment of cells with calpain II inhibitor in the presence of FAC
has no effect on IRP2 iron-mediated degradation. Ammonium chloride also
did not inhibit IRP2 degradation by iron (Fig. 9B). We
conclude from these studies that the proteasomes, and not the
lysosomes, are required for iron-mediated degradation of IRP2.
Figure 9: Effect of calpain II and lysosomal inhibitors on IRP2 iron-mediated degradation. A, cells were pretreated with 100 µM calpain II inhibitor in 0.4% dimethylformamide for 1 h prior to the addition of 50 µg/ml FAC (lanes 6-8) or in FAC in DMF (lanes 2-4) for 1-4 h. CO control, untreated cells harvested at 0 h. B, cells were pretreated with 20 mM ammonium chloride (lanes 5-7) or 0.15 mM chloroquine (lanes 8-10) for 1 h prior to the addition of 50 µg/ml FAC for 1-4 h. Cells were also treated with FAC (lanes 2-4) for 1-4 h. The top panels of A and B are immunoblots using anti-IRP2 antisera, and the bottom panels are RNA band shift assays. The positions of IRP1 and IRP2 are indicated.
In this paper we report the differential regulation of IRP1 and IRP2 by iron in mammalian cells. IRP1 exhibits two functions in cells dependent on iron levels: IRP1 with an 4Fe-4S cluster functions as an cytosolic aconitase converting citrate into isocitrate when iron is abundant and as an RNA binding apoprotein regulating the translation and stabilization of IRE-containing mRNAs when iron is scarce(18, 34, 35, 36) . The switch between the 4Fe-4S form and the apoprotein forms occurs without changes in IRP1 levels(12, 26) . By contrast, IRP2 lacks aconitase activity and functions solely as an RNA binding protein(20) . Our results indicate that IRP2 is regulated by specific proteolysis induced by iron in a variety of cells types and that the proteasome is responsible for IRP2 degradation.
Our data
suggest a mechanism for the iron-mediated degradation of IRP2. When
intracellular iron is scarce, IRP2 binds IREs with high affinity. An
increase in intracellular iron results in the induction of a labile
protein that is required for IRP2 degradation. Although we do not know
the identity and function of this protein, it is possible that it is a
targeting protein that binds IRP2 via the 73-amino acid domain, marking
it for degradation. Iron could also cause the assembly of an 4Fe-4S
cluster in IRP2, similar to the cluster in IRP1. Rat IRP2 contains the
3 conserved cysteines that coordinate the 4Fe-4S cluster in
IRP1(27, 29) . In addition, the presence of 4
cysteines and 1 histidine in the 73-amino acid insertion of IRP2
suggests that this region might also participate in iron
binding(29) . Preliminary data suggests that in vitro reconstitution of IRP2 with iron results in loss in RNA binding
activity. ()Thus, according to our model, cluster assembly
woud lead to a conformational change in IRP2 and subsequent loss in RNA
binding activity. IRP2 would then be recognized by the targeting
protein and rapidly degraded by the proteasome. Finally, our data
indicate that the decrease IRP2 RNA binding activity mediated by iron
is also prevented when IRP2 proteolysis is blocked either by MG-132 or
by cycloheximide. One possibility to explain these data is that the
putative Fe-S cluster is unstable in IRP2 and is disassembled during
extract purification, leading to the generation of an apoprotein
containing RNA binding activity.
The 26 S proteasome contains subunits which are important in the degradation of ubiquitin-conjugated proteins(30, 31) . We have not detected higher molecular weight IRP2 complexes by gel electrophoresis, which might be suggestive of ubiquitination of IRP2. However, since ubiquitin-conjugated proteins are very labile, they are generally difficult to detect. The 26 S proteasome also degrades non-ubiquitinated proteins(31, 37) . The signals required for targeting non-ubiquitinated proteins to the proteasome are poorly understood; however, it is possible that the putative targeting protein discussed above could mark IRP2 for degradation by the proteasome.
Although we cannot eliminate the possibility that MG-132
may affect other unknown proteases and enzymatic activities in cells,
the utilization of these inhibitors both in vitro and in
vivo have demonstrated the specificity and effectiveness of these
compounds against the proteasome(33) . Our data suggested that
MG-132 may increase cellular iron levels, perhaps by blocking the
degradation of iron transporter proteins. Peptide-aldehyde inhibitors
have been used to demonstrate the role of the proteasome in the
generation of peptides presented on the major histocompatibility class
I molecules (33) and in the proteolytic processing of the
transcription factor NF-B1(32) .
The structural determinants required for IRP2 iron-mediated degradation are unknown. IRP2 does not contain PEST regions (sequences rich in proline, glutamine, serine, and threonine) which are commonly found in proteins that are rapidly degraded(38) . However, IRP2, the 73-amino acid insertion, contains a site that is susceptible to proteolysis during purification and results in the production of an 83,000-Da proteolytic polypeptide(20) . The cleavage site has the sequence SQ- IENTP and is not a known protease cleavage sequence. Whether proteolysis at this site represents a physiological mechanism for iron-mediated degradation or whether the 73-amino acid insertion is a determinant required for degradation remains to be determined.
The biological relevance of two IRPs in cells is unclear. Both IRP1 and IRP2 bind IREs with high affinity (20, 24, 27) and function as translational repressors of IRE-containing RNAs in vitro(20) . First, it is possible that IRP2 binds to a subset of IRE-containing mRNAs containing slightly different sequences. A recent study using in vitro synthesized IREs demonstrated that mouse IRP2 has a preference for specific IRE sequences, suggesting that IRP2 may bind to specific IRE-containing mRNAs in vivo(39) . Second, since IRP2 is present in the highest amounts in skeletal muscle and heart, this suggests that IRP2 may regulate muscle-specific mRNAs(29) . Third, IRP2 RNA binding activity is decreased in the livers of rats treated with chemicals to induce oxidative stress (22) and increased in regenerating rat livers(21) , suggesting that IRP2 is regulated under a variety of physiological states. It is unclear whether these effects are due to alterations in intracellular iron levels or to stimuli other than iron.
A recent study suggested that iron-mediated regulation of IRP2 degradation may be cell-specific(27) . A c-myc-tagged recombinant IRP2 expressed in HeLa cells treated with iron or hemin for 16 h resulted in a decrease in RNA binding activity, but no change in the amount of protein. Our experiments analyzing the iron-mediated regulation of endogenous IRP2 in HeLa cells treated with iron for up to 24 h showed a steady decrease in RNA binding activity and protein levels up to 6 h, after which RNA binding activity and protein levels gradually increased. The half-life of recombinant IRP2 expressed in RD-4 cells was greater than 24 h in desferrioxamine-treated cells and 6 h in iron-treated cells(27) . By contrast, our data indicated that the half-life of endogenous IRP2 in untreated FTO2B cells was 6 h and 1.5 h in iron-treated cells. The discrepancies between these studies may reflect differences in experimental design due to use of overexpressed protein or to different cell growth conditions.
The regulation of gene expression by specific proteolysis provides a way by which cells can change the concentration of specific proteins depending on the metabolic state of the cell. The iron-dependent regulation of IRP2 turnover may be similar to the mechanisms regulating the mammalian enzyme ornithine decarboxylase. Ornithine decarboxylase is the first enzyme in the polyamine biosynthesis pathway and is degraded when intracellular polyamine levels increase(40) . Polyamines induce antizyme, a protein which binds with high affinity to ornithine decarboxylase (41, 42) and targets ornithine decarboxylase for degradation by the proteasome(37) . Thus, it is possible that IRP2, like ornithine decarboxylase, may utilize other proteins that specify its degradation during changes in intracellular iron levels. The characterization of the other components responsible for IRP2 iron-mediated degradation will provide a clearer understanding of the mechanism by which IRP2 is targeted and degraded by the proteasome.