(Received for publication, October 30, 1995; and in revised form, January 4, 1996)
From the
Iron regulatory proteins (IRPs) are RNA-binding proteins that
post-transcriptionally regulate synthesis of iron uptake (transferrin
receptor) and storage (ferritin) proteins. Our previous work
demonstrating that IRP1 is phosphorylated by protein kinase C supported
the hypothesis that factors in addition to iron modulate IRP function.
We have investigated changes in activity and expression of both IRP1
and IRP2 during phorbol 12-myristate 13-acetate (PMA)-induced
differentiation of HL-60 cells. In contrast to IRP1, IRP2 was highly
phosphorylated in untreated cells. PMA stimulated phosphorylation of
IRP1 and IRP2 by at least 2-3-fold without affecting
incorporation of [S]methionine into the
proteins. IRP1 and IRP2 isolated from PMA-treated cells displayed
different phosphopeptides. Phosphorylation of IRPs was associated with
a 2-fold increase in high affinity RNA binding activity without
altering K
, and this was accompanied by a
50% increase in transferrin receptor mRNA abundance. PMA acted on a
latent pool of binding activity that is present in a nonaconitase
oxidized form and is largely composed of a stable but inactive species
of IRP2. Desferal and hemin modulated iron-responsive element binding
activity in HL-60 cells without affecting the phosphorylation state of
IRP1. Hemin appeared to reduce the abundance of phosphorylated IRP2.
Thus, multiple factors affect the function of both IRPs and indicate
that extracellular agents may program changes in cellular iron
metabolism by altering the phosphorylation state of these regulatory
RNA-binding proteins.
Iron is an essential nutrient for the growth and optimal health
of virtually all
organisms(1, 2, 3, 4) . However,
excessive levels of iron can be toxic due to the increased potential
for generation of reactive oxygen metabolites. Consequently, regulatory
mechanisms have evolved that allow cells to maintain iron homeostasis
in response to variations in the availability of and/or requirement for
iron. Two proteins that contribute to the maintenance of iron
homeostasis are ferritin, an intracellular iron storage protein, and
transferrin receptor (TfR), ()a membrane protein that
transports iron into the cell. In nearly all cell types of higher
eucaryotes, ferritin and TfR synthesis is coordinated in a divergent
manner in response to the level of intracellular
iron(1, 2, 3, 4) . In the presence
of excess intracellular iron, ferritin synthesis is elevated to
accommodate the need for additional iron storage capacity, whereas TfR
synthesis is diminished to minimize the potentially toxic effects of
intracellular iron overload. The opposite response is exhibited in
iron-depleted cells; TfR synthesis is increased, whereas ferritin
synthesis is reduced.
Iron-dependent changes in ferritin and TfR synthesis are regulated primarily at the post-transcriptional level through specific regulated protein-RNA interactions(1, 2, 3, 4) . The translation and stability of ferritin and TfR mRNAs, respectively, is modulated by cytosolic proteins (iron regulatory proteins (IRPs)). IRPs influence mRNA utilization by binding to specific stem-loop motifs (iron-responsive elements (IRE)) present in the 5`- or 3`-untranslated regions of ferritin and TfR mRNAs, respectively(5, 6, 7, 8) . In addition, the mRNA encoding erythroid 5-aminolevulinate synthase, the rate-limiting enzyme in formation of heme, contains an IRE in its 5`-untranslated region and appears to be translationally regulated by iron(9, 10, 11) . Thus, IRPs are sensory and regulatory components of a homeostatic system that coordinates the synthesis of proteins involved in storage, uptake, and metabolic utilization of iron.
A fundamental question regarding the regulation of ferritin and TfR synthesis that has received much attention concerns how changes in cytoplasmic iron levels are transmitted into alterations in IRE RNA binding activity. For IRP1 much evidence has accumulated in support of the ``aconitase model'' as one mechanism by which iron regulates RNA binding activity of IRP1 without variation in abundance of the protein (12, 13, 14, 15) . However, iron-dependent changes in degradation of IRP1 appears to represent another means for affecting function of this binding protein at least in some cell types(16) . For IRP2, iron appears to largely act by influencing the steady state level of the protein since in some, but not all, iron-loaded cells the rate of degradation of IRP2 is enhanced as compared with the rate observed in iron-depleted cells(17, 18, 19) . In some cell types iron appears to modulate IRP2 RNA binding activity without affecting the steady state level of the protein(18) . However, the biochemical nature of this stably inactive pool of IRP2 has not been addressed.
Because IRPs are central regulators of iron utilization, they serve as likely focal points at which extracellular agents could act to influence the rate of uptake or metabolic fate of iron. Several physiological and pathological situations are associated with alterations in the uptake, storage, or utilization of iron, and it is apparent that multiple effectors can influence IRP function(20, 21, 22, 23) . These include iron-independent increases in TfR expression and iron uptake that occur during cellular proliferation such as after lymphocyte activation(24, 25, 26) . In addition, certain cell types exhibit specialized requirements for iron during or after they differentiate. For example, when monocytes differentiate into macrophages or during erythropoiesis, stimulation of iron uptake and alteration in iron storage occur and agents other than iron appear to be the effectors (26, 27) . Under such circumstances alterations of IRP function through the action of extracellular agents could provide a means for inducing changes in cellular iron utilization.
We have recently begun to describe a novel mechanism by which a variety of extracellular agents may influence cellular iron metabolism as a result of phosphorylation of IRP1(23) . In HL-60 cells, PMA-induced phosphorylation of IRP1 was associated with a rapid stimulation of IRE binding activity; however, the potential role of IRP2 was not examined nor were the biochemical characteristics of the latent pool of binding activity on which PMA acted. In the present paper we describe experiments that define the forms of IRPs present in untreated HL-60 cells and the effects that PMA and changes in iron status have on these forms of the binding proteins. We show that unstimulated HL-60 cells contain a significant level of latent IRE binding activity, which is largely IRP2, that is almost exclusively in a nonaconitase oxidized form. Furthermore, we show that PMA or the iron chelator desferal act on the latent forms(s) of IRP to convert them from an inactive or perhaps low affinity state to a high affinity state with respect to RNA binding with concurrent increases in the abundance of TfR mRNA. Our results further demonstrate a direct relationship between increases in phosphorylation of both IRPs and activation of their RNA binding function by compounds that activate protein kinase C and other kinases. Our results support the hypothesis that phosphoregulation and iron regulation represent different mechanisms for affecting IRP function.
Figure 1:
PMA rapidly stimulates IRE binding
activity in HL-60 cells. A, HL-60 cell cultures were incubated
for various amounts of time in the presence of 0.2 µM PMA
() or 0.02% Me
SO (
), harvested, and lysed. Gel
retardation assays were performed on lysates (total protein, 5 µg)
as indicated under ``Materials and Methods.'' Bound and free
RNA bands were visualized by autoradiography, excised, and quantified
by liquid scintillation counting. The data are the means ± S.E., n = 5 independent culture flasks/time point. B, supershift gel retardation assays were performed at 4
°C as indicated under ``Materials and Methods'' with
lysates from C58 cells in the absence (lane 1) or the presence
of either 4 (lane 2) or 20 µg (lane 3) of
anti-IRP2 IgG. For C58 lysates, IRP 1 (band a) and IRP2 (band b, lane 1) are readily separated; only IRP2 is
supershifted by the antibody (region d, lanes 2 and 3). C, extracts from both control (lanes 4 and 6) and PMA-treated (lanes 5 and 7)
HL-60 cells were subjected to supershift analysis with 10 µg of
anti-IRP2 IgG (lanes 6 and 7). For HL-60 cell
extracts, IRP1 and IRP2 co-migrate (band c); IRP2 was
supershifted with anti-IRP2 IgG (region d, lanes 6 and 7), leaving IRP1 (band c, lanes 6 and 7). The autoradiograms shown in B and C are representative of several independent
experiments.
PMA-dependent stimulation of IRE RNA binding activity could
represent changes in the affinity of interaction of the binding
proteins with RNA (K) and/or a change in the
amount of protein in the high affinity state (B
). Lysates from HL-60 cells treated with PMA
for 30 min exhibited nearly a 2-fold increase in B
for total IRE binding activity compared with uninduced cells (Fig. 2) (Table 1). In contrast, K
was unaffected by PMA treatment (Table 1). Although a
specific IRE binding activity of lower affinity (K
) was detected by RNA saturation analysis with
low specific activity RNA, it was not affected by PMA treatment of the
cells. (
)Our results support the hypothesis that PMA
treatment induces conversion of the IRPs, particularly IRP2, from an
inactive form to an active form and/or that PMA treatment stabilizes
the binding protein(s) in an active form for RNA binding.
Figure 2:
PMA increases B but
not K
in HL60 cells. Gel retardation
assays were performed using graded levels of
[
P]IRE RNA and extracts (5 µg) from control
cells (
) and cells treated with 0.2 µM PMA for 30 min
(
). The data were curve-fitted from a number of independent RNA
saturation assays using nonlinear regression (GraphPAD Software) to
obtain estimates of B
and K
. Mean B
and K
values obtained are shown in Table 1.
Figure 3:
2-Mercaptoethanol-inducible IRE binding
activity in extracts from untreated and PMA-treated HL-60 cells. Using
the same control () and PMA (
) extracts described in Fig. 1, gel retardation assays were performed in the presence of
2-mercaptoethanol. Extracts (5 µg) were incubated at room
temperature with 2% 2-ME for 10 min prior to the addition of
[
P]IRE RNA. The data are the means ±
S.E., n = 5 independent cell cultures at each time
point.
Maximal activation of IRE RNA
binding activity occurred with 10-50 mM 2-ME (Fig. 4A), a level of 2-ME that would not be sufficient
to significantly activate RNA binding by holo-IRP1. ()Furthermore, previous addition of citrate reduced the
action of 2-ME slightly, which also indicated that holo-IRP1 was not a
significant component of the latent pool present in these extracts (Fig. 4A). Although the small effect of citrate on the
action of 2-ME was not statistically significant, it was consistently
observed, suggesting that approximately 10-20% of the
2-ME-inducible pool of RNA binding activity was due to cytosolic
aconitase.
Figure 4:
Characteristics of the latent pool of IRE
binding activity in HL-60 cells. All gel retardation assays were
performed as indicated under ``Materials and Methods'' on
extracts (5 µg) from control HL-60 cells and cells incubated in the
presence of 0.2 µM PMA for 240 min. A, IRE
binding activity was determined by gel retardation analysis in extracts
from control HL-60 cells as a function of 2-mercaptoethanol
concentration in the absence () and presence (
) of 1 mM citrate. The data are the means ± S.E., n =
5 independent assays. B, extracts from control (open
bars) and PMA-treated (closed bars) HL-60 cells were
incubated in the presence and the absence of 1 mMN-ethylmaleimide for 30 min on ice, followed by titration
with various amounts of 2-mercaptoethanol for 10 min at room
temperature prior to the addition of [
P]IRE. The
autoradiogram corresponding to the quantitative data in B is
shown at the bottom of the figure. These results are
representative of at least five independent assays. Similar results
were obtained using both 0.1 and 10 mMN-ethylmaleimide.
Because the latent pool of IRE binding activity was redox
regulated, we tested the effect of NEM on IRE binding activity in
untreated and PMA-treated cells. The addition of 1 mM NEM
completely inactivated RNA binding in untreated and PMA-treated cells
when measured without any added 2-ME (Fig. 4B, lane
3). To test whether the form(s) of IRP present in the latent pool
contained disulfide or other forms of Cys that would be nonreactive
with NEM, we added gradually increasing levels of 2-ME to extracts
previously treated with NEM to determine if any IRE binding activity
could be recovered. When we added back a molar excess of 2-ME (over
NEM) to the NEM-treated extracts, we found that untreated extracts now
contained more 2-ME-inducible IRE binding activity than was present in
extracts from PMA-treated cells. Thus, untreated HL-60 cells contain a
latent pool of IRE binding activity, mostly IRP2, in which oxidation of
one or more cysteines is associated with inactivation of RNA binding.
We have found that the ``redox'' state of IRP2 varies in a
cell type-specific manner.
Figure 5:
PMA treatment fails to alter the relative
rate of synthesis of IRP1 or IRP2. HL-60 cells were incubated in the
absence and the presence of 0.2 µM PMA for various amounts
of time. 30 min before each time point, cells were collected by
centrifugation, resuspended in methionine-free media, and labeled for
30 min with [S]methionine at 75 µCi/ml.
Cells were harvested, lysed, immunoprecipitated with the appropriate
antibody, and separated by SDS-polyacrylamide gel electrophoresis as
indicated under ``Materials and Methods.'' A,
labeled lysates (30
10
cpm) from untreated cells
(30 min, lane 1; 240 min, lane 6) and cells treated
with PMA for 30 (lane 2), 60 (lane 3), 120 (lane
4), or 240 min (lane 5) were immunoprecipitated with 30
µg of anti-IRP1 IgG. B, labeled lysates from untreated (lane 7) and 4-h PMA-treated (lane 8) cells
containing 30
10
trichloroacetic acid-precipitable
counts were immunoprecipitated with 20 µg of anti-IRP2 IgG. The
results are representative of several independent labeling experiments.
The total area of each band was quantitated by computerized
densitometry as described under ``Materials and
Methods.''
Figure 6:
Acute treatment with PMA stimulates
phosphorylation of IRP1 and IRP2 in HL-60 cells. For P
labeling, flasks containing an equal number of cells were incubated in
phosphate-free medium plus [
P]orthophosphate (1
mCi/ml) for 4 h prior to the addition of 0.2 µM PMA or
0.02% Me
SO. At the appropriate time point, cells were
harvested, lysed, and immunoprecipitated with either 30 µg of
anti-IRP1 IgG (A) or 20 µg of anti-IRP2 IgG (B). Lanes 1 and 6 are control cells incubated for 30 and
240 min with Me
SO, respectively. Cells were treated with
PMA for 30 (lane 2), 60 (lane 3), 120 (lane
4), or 240 min (lane 5). The total area of each band was
quantitated by computerized densitometry as described under
``Materials and Methods.''
To determine the number of sites phosphorylated in IRP1 and IRP2 and whether or not individual sites displayed different kinetics of phosphorylation after PMA treatment, we performed phosphopeptide mapping of the proteins isolated from untreated and/or PMA treated HL-60 cells. IRP1 isolated from PMA-treated cells had one major site of phosphorylation (Fig. 7A, spot a) that comigrated with one of the sites identified when the purified protein was phosphorylated by protein kinase C (results not shown) (23) . In contrast, IRP 2 displayed one site in untreated cells (results not shown) that co-migrated with spot b in PMA-treated cells (Fig. 7, B and C). PhosphorImager analysis revealed a second site in PMA-treated cells (Fig. 7C, spot c). Both of these sites present in IRP2 differed in migration from the site observed for IRP1, indicating that they are likely to represent phosphorylation of different residues in the two related proteins. The major phosphopeptide observed in IRP2 increased in intensity by about 2-fold in PMA-treated cells (Fig. 7, B and C) as compared with untreated cells (results not shown). Given the low intensity of the second phosphorylation site observed for IRP2 in PMA-treated cells, it remains to be determined if, as compared with untreated cells, it represents a new phosphorylation site or increased phosphorylation of a site that was partially phosphorylated before the addition of PMA. Taken together, both IRP1 and IRP2 are phosphorylated in response to PMA treatment of HL60 cells but that IRP2 has a much higher basal level of phosphorylation in untreated cells.
Figure 7: Tryptic phosphopeptide mapping of IRP1 and IRP2 isolated from PMA-treated HL-60 cells. HL-60 cells were treated with PMA for 60-120 min to induce a high level of phosphorylation of IRP1 and IRP2. IRP1 and IRP2 were separately immunoprecipitated and fractionated by SDS-polyacrylamide gel electrophoresis as indicated under ``Materials and Methods.'' Bands corresponding to IRP1 and IRP2 were excised from the dried gel, eluted, subjected to tryptic digestions overnight, and then separated by chromatography and electrophoresis as described. A is an autoradiogram of an IRP1 phosphopeptide map, and B is an autoradiogram of a IRP2 phosphopeptide map; both are from PMA-treated cells. C is a PhosphorImager analysis of a IRP2 phosphopeptide map that reveals the presence of a second, less radioactive spot for IRP2 in PMA-treated cells. The origin is indicated by the dot. One spot was seen for IRP1 (A, band a), and two spots were seen for IRP2 (B and C, spots b and c). For IRP2 only spot b was observed in untreated cells (results not shown) but at a lower intensity than seen in PMA-treated cells.
Figure 8:
PMA
increases TfR mRNA level in HL-60 cells. HL-60 cells were treated with
either 0.02% MeSO (control, lane 1), 0.2
µM PMA (lane 2), 20 µM hemin (lane 3), or 100 µM desferal (lane 4)
for 5 h. Total RNA was extracted and isolated from the cells for
analysis of TfR mRNA levels using a ribonuclease protection assay
according to the manufacturer's instructions. Antisense TfR and
glyceraldehyde phosphate dehydrogenase (GAPDH) probes (500,000
cpm each) were hybridized at 45 °C for 16 h with 30 µg of total
RNA from each treated cell lysate digested with a mixture of RNase A/T1
and separated on a 5% acrylamide/8 M urea gel. TfR and
glyceraldehyde dehydrogenase probes resulted in protected fragments of
374 and 316 nucleotides, respectively. Undigested TfR and
glyceraldehyde phosphate dehydrogenase probes migrated at 429 and 403
nucleotides, respectively (lane 5). The total area of each
band was quantitated by computerized densitometry as described under
``Materials and Methods.''
Figure 9:
Contrasting effects of PMA and desferal versus hemin on IRE binding activity in HL-60 cells. HL-60
cells were incubated with 0.02% MeSO (control, lanes 1 and 5), 0.2 µM PMA (lanes 2 and 6), 20 µM hemin (lanes 3 and 7), or 100 µM desferal (lanes 4 and 8) for 4 h, harvested, and lysed. Gel retardation analysis was
performed on cell lysates (5 µg) in the absence (B) and
the presence (C) of 2% 2-mercaptoethanol as indicated under
``Materials and Methods.'' Bound and free RNA bands were
excised and quantified by liquid scintillation counting, and the data
are presented in A. The results are representative of several
independent cell culture experiments.
Differences were noted concerning the regulation of the function of IRPs by hemin and desferal as compared with PMA treatment. First, neither hemin or desferal affected total phosphorylation state of IRP1 (Fig. 10A) or the relative synthesis rates of the protein (Fig. 10B). For IRP2, hemin reduced the amount of phosphorylated protein by 30-40% (Fig. 10C) without changing synthesis of this binding protein (Fig. 10D). It remains to be seen if iron status differentially affects phosphorylation of specific sites but not the total level of phosphate in IRPs. Second, hemin reduced IRE binding activity as measured in both the absence or the presence of 2-ME (Fig. 9C), suggesting that hemin reduced the level of IRE-binding protein, presumably IRP2, present in HL-60 cells. The reduction in the level of binding activity was similar to the extent to which hemin reduced the level of phosphorylated IRP2. In contrast, the total level of 2-ME-inducible IRE binding activity was essentially identical in extracts from untreated, PMA-treated, and desferal-treated cells. Taken together, these results support the hypothesis that phosphorylation of IRP1 and IRP2 represents a means for influencing cellular iron homeostasis by factors other than iron.
Figure 10:
Effects of PMA versus hemin and
desferal on IRP1 and IRP2 phosphorylation state and relative synthesis
rate. HL-60 cells were treated with either 0.02% MeSO
(control, lanes 1, 5, 9, and 13),
0.2 µM PMA (lanes 2, 6, 10, and 14), 20 µM hemin (lanes 3, 7, 11, and 15), or 100 µM desferal (lanes 4, 8, 12, and 16) for 4 h.
For [
S]methionine labeling, cells were collected
by centrifugation after 3.5 h of treatment and resuspended in
methionine-free medium containing [
S]methionine
(75 µCi/ml) for 30 min prior to harvesting and lysing the cells.
For
P labeling, cells were incubated in phosphate-free
medium containing [
P]orthophosphate for 4 h
prior to the addition of PMA, hemin, or desferal for an additional 4 h.
An equal number of trichloroacetic acid-precipitable counts (15
10
cpm) from each
[
S]methionine-labeled lysate was
immunoprecipitated with either 30 µg of anti-IRP1 IgG (B)
or 20 µg of anti-IRP2 IgG (D) and separated by
SDS-polyacrylamide gel electrophoresis. Lysate aliquots representing an
equal number of
P labeled cells were immunoprecipitated
with 30 µg of anti-IRP1 IgG (A) or 20 µg of anti-IRP2
IgG (C). The total area of bands for IRP1 and IRP2 were
quantitated using a computerized densitometer as described under
``Materials and Methods.''
For IRPs to represent true central regulators of iron homeostasis, it seems likely that they should respond to multiple effectors. In this regard both intracellular and extracellular effectors of IRP1 have been identified. Iron is clearly an intracellular effector of IRP1 function and could be considered an allosteric regulator of the binding protein. A number of other effectors of IRP1 function have been identified including nitric oxide, oxidative stress, and, in our laboratory, phosphorylation(20, 21, 22, 23) . Modulation of IRP1 by these agents provide mechanisms to affect the uptake and metabolic fate of iron by extracellular agents. IRP2 appears to be modulated by iron as well as by changes in proliferation status of some cells. In the latter case specific agents that act on IRP2 have not been identified. Our current work extends our observations concerning the mechanisms by which phosphorylation affects the functions of IRP1 and also demonstrate that IRP2 is phosphoregulated in response to extracellular stimuli. Our results support the hypothesis that IRP1 and IRP2 are iron-based and phosphorylation-based regulators of TfR mRNA accumulation and presumably ferritin and erythroid 5-aminolevulinate synthase synthesis.
Our observations provide further insight into the mechanisms by which factors other than iron affect IRP function. We showed that extracts of HL-60 cells possess an inactive form of IRP(s) that is largely if not exclusively in a nonaconitase form and is mostly composed of IRP2. PMA appears to act on an inactive form of IRP1 and particularly IRP2 and induces their conversion to high affinity RNA-binding proteins. This action of PMA was directly associated with increased phosphorylation of the proteins. Our conclusion is based on the amount of 2-ME (10-50 mM) required to maximally activate the latent pool of binding activity in extracts from untreated HL-60 cells and that inclusion of aconitase substrates does not substantially block the 2-ME effect on RNA binding. Several pieces of evidence indicate that this latent form of IRP is functionally important. First, we showed that PMA treatment of HL-60 cells rapidly stimulated IRE binding activity, and this was found to be associated with a significant increase in TfR mRNA level. Second, we have found that the size of the latent pool of IRP is affected by cell growth state and the level of serum in which HL-60 cells are cultured (results not shown) and TfR expression is affected by serum in these cells(38) . Third, supershift analysis demonstrated that the inactive pool of binding protein in HL-60 cells is largely IRP2, and we (results not shown) and others (31, 32, 33) have observed inactive IRP2 in rodent cell lines, finding that it is not restricted to the HL60 cell system. These observations indicate that the latent form of IRP2 can be recruited into the functional high affinity pool of binding protein. To more clearly determine the mechanism(s) by which PMA affected IRP function, particularly IRP2, we examined the extent to which post-translational events affected RNA binding activity.
Our results
indicated that RNA binding by IRP2 can be modulated without changes in
the amount of binding protein. We obtained several lines of evidence
indicating that PMA treatment influenced the amount of IRP2 in the high
affinity state by altering the proportion of the protein that was
stably reduced. First, PMA stimulates IRE binding activity in HL-60
cells, as measured in the absence of dithiothreitol or 2-ME, without
affecting the total amount of binding protein, as evaluated by the 2-ME
induction assay. Further support for this conclusion came from the
observation that the ability of PMA to stimulate IRE binding activity
occurred without changes in the synthesis rates of IRPs. Thus,
oxidation or reduction of cysteine(s) appears to represent a mechanism
by which IRP2 RNA binding activity can be regulated in these cells.
Second, similar results were obtained when 1 mM dithiothreitol
was included in the cell lysis buffer, although the magnitude of the
difference between untreated and PMA-treated cells was lessened
(results not shown). Thus, phosphorylation may increase the proportion
of IRP2, which binds RNA with high affinity by inducing a structural
change in the binding protein, which causes the reduced form of the
protein to be favored, and/or by stabilizing the protein in the reduced
state during cell lysis. As noted above, the redox state of IRP2 varies
in a cell type-specific manner. In HL-60 cells ( Fig. 1and 3)
and C58 cells, only a portion of IRP2 is stably reduced. ()However, FTO-2B rat hepatoma cells contain IRP2 in a fully
reduced state, even when extracts are prepared without dithiothreitol
or 2-ME ( (17) and results not shown). Thus, RNA binding by
IRP2 can be regulated in a cell type-specific manner without large
changes in the levels of the protein through an oxidation or reduction
mechanism.
Is there a role of phosphorylation in affecting the redox state of IRP2 as there appears to be for another nucleic acid-binding protein(39) ? We note that within the 73-amino acid region found in human and rat IRP2, but not IRP1, there are two Cys residues with an adjacent Arg residue. Based on studies with model peptides(40) , such Cys residues are predicted to be especially redox active and may well contribute to the redox and/or iron regulation of IRP2. Furthermore, some of the Ser and Thr residues in the 73-amino acid region fit the consensus sequences for protein kinase C or mitogen-activated protein kinases, both of which can be activated directly or indirectly, respectively, by PMA. Thus, phosphorylation within this region might affect the redox properties of nearby Cys residues. Our results support the hypothesis that oxidation or reduction of one or more Cys in IRP2 represents a physiological mechanism for regulation of its RNA binding function.
To evaluate the role of cysteine oxidation state, we made use of our observation that NEM completely inactivated spontaneous RNA binding in extracts of untreated and PMA-treated cells and used it as a means to assess the percentage of binding activity that was in a reduced state. We found, as others have recently demonstrated(32) , that NEM can inactivate spontaneous binding activity for IRP1 and IRP2. Our results on the effect of NEM on IRP2, like those of Kuhn and Henderson(32) , differ from those of Kim et al.(41) and further suggest that the cellular form of IRP2 differs from the recombinant form of the binding protein. We also demonstrated that after inactivation of stably reduced IRPs with NEM we could still activate the latent pool with low levels of 2-ME, further indicating that oxidation or reduction of one or more critical Cys in the binding protein(s) contributed to the regulation of its RNA binding function. NEM treatment followed by the addition of 2-ME permitted us to demonstrate that PMA treatment of HL-60 cells reduced the size of the latent pool of IRP though a change in cysteine oxidation state. Also, we have found that IRP2 is reversibly inactivated by diamide (results not shown) and recently a similar phenomenon was observed in other cell types(32) . Thus, it is apparent that post-translational regulation of IRP2 can occur as a result of changes in the cysteine oxidation state without alterations in the abundance of the binding protein.
To what extent do phosphorylation-dependent and iron-dependent mechanisms overlap to regulate IRP2 function? Roles for oxidation or reduction, involving the intra- and intermolecular formation or destruction of disulfide bonds, in influencing protein function have been demonstrated in several systems (42, 43, 44, 45, 46, 47, 48) . Oxidized species of proteins such as HMG-CoA reductase(42) , the hemin-controlled protein kinase(43) , and other nucleic acid binding proteins(42, 43, 44, 45, 46, 47, 48) exist in cells, even in the presence of the cellular glutathione redox system. Furthermore, IRP1, FNR, and soxR provide striking examples of how changes in ligation state of Cys residues, as a result of formation of Fe-S clusters, greatly influence protein function(13, 49, 50) . However, in the case of IRP2 our results presented here as well as those of other investigators(17, 18, 19) indicate that irrespective of whether an Fe-S cluster forms in this binding protein, iron decreases the abundance of IRP2. Along these lines, it appears that iron reduces the abundance of the phosphorylated form of IRP2, suggesting that this form of the binding protein may be preferentially degraded when iron is in excess. However, it remains to be shown whether the phosphorylated form of IRP2 is the preferred substrate for degradation. In contrast to the effect of iron status in affecting the steady state level of IRP2, PMA affects the relative distribution of the binding protein between active (reduced) and inactive (oxidized) pools without altering the total level of the protein. Because desferal treatment affects RNA binding in HL-60 cells without apparent changes in the total level of binding protein, it appears that iron depletion may affect the abundance of the latent form of IRP2 in cells, but this remains to be investigated further. Overall, our results demonstrate that IRP2 can stably accumulate in an inactive form in cells and that multiple pathways exist for modulating the RNA binding activity of the protein.
We demonstrated that phosphorylation of IRP1 and IRP2 was
associated with activation of their RNA binding activity. Because IRP2
apparently cannot act as an aconitase even if it formed an Fe-S
cluster(17) , it is likely that phosphorylation affects other
functions. This could include an effect on the relative abundance of
the oxidized and reduced forms of IRP2 as indicated above or on the
action of iron in affecting RNA binding function perhaps through
mechanisms involving altered degradation of the protein. Given the
observations that RNA binding by IRP2 is stimulated during liver
regeneration and lymphocyte activation, it will then be of interest to
define the extent to which phosphorylation affects the function of this
binding protein during cell
proliferation(24, 25, 26, 51) . With
regard to IRP1, we also found that increased phosphorylation was
associated with a stimulation of its binding activity. These results
support the concept that within cells the Fe-S cluster in IRP1 may be
labile and that phosphorylation may affect assembly (or reassembly) of
cluster in intact cells(1, 23) . A direct effect of
phosphorylation on cluster assembly/disassembly remains to be more
directly demonstrated. However, given our observations ()that the iron-free form of IRP1 is the preferred substrate
for phosphorylation, phosphoregulation may indirectly activate RNA
binding by predominantly acting as a determinant of whether or not IRP1
functions as an aconitase or RNA-binding protein. Taken together, our
results provide support for the hypothesis that alterations in
phosphorylation state of IRP1 or IRP2 provide a means by which
extracellular agents affect iron homeostasis.