![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Previous Article | Next Article 
Molecular and Cellular Biology, October 2003, p. 6973-6981, Vol. 23, No. 19
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.19.6973-6981.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital,1 Department of Medicine, McGill University, Montreal, Quebec, Canada2
Received 30 December 2002/ Returned for modification 18 February 2003/ Accepted 19 June 2003
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IRP1 and IRP2 are homologous to mitochondrial aconitase (5, 8, 23), an enzyme of the citric acid cycle, and in fact, IRP1 is its cytosolic counterpart. The active site of aconitase contains a cubane [4Fe-4S] cluster (1). In IRP1, this cluster is assembled within iron-replete cells and prevents IRE binding. However, iron starvation, nitrogen monoxide (NO), and extracellular H2O2 (5, 23) trigger the cluster's destabilization. The resulting switch to apo-IRP1 is associated with the loss of aconitase and acquisition of IRE-binding activity. The mechanism for IRP2 regulation is distinct and does not involve iron-sulfur cluster biochemistry. While IRP2 is stable in iron-starved and hypoxic cells, it undergoes proteasomal degradation in the presence of iron, oxygen (16), or the nitrosonium cation (NO+) (19).
It has been proposed that the activities of IRP1 and IRP2 may also be regulated by phosphorylation (8). The physiological significance of this finding remains elusive. Both IRP1 and IRP2 can be subjected to phosphorylation by protein kinase C (PKC) (9, 27). The rate of IRP1 phosphorylation by PKC is negatively affected by the presence of the iron-sulfur cluster (26). Experiments with synthetic oligopeptides showed that the conserved Ser-138 and Ser-711 residues are targets of phosphorylation in vitro and implied that phosphorylation may also occur in vivo. Ser-138 is located within the region involved in RNA binding and assembly of the [4Fe-4S] cluster, which spans residues 116 to 151.
More direct evidence for the involvement of Ser-138 in the control of the iron-sulfur cluster assembly was provided by experiments in an aconitase-deficient Saccharomyces cerevisiae strain. Aerobic growth of the aco1 cells depends on complementation with iron-loaded IRP1 (i.e., cytosolic aconitase). The replacement of Ser-138 in IRP1 with phosphomimetic residues (glutamic or aspartic acid) impairs aerobic growth and prevents [4Fe-4S] cluster assembly (3). The sensitivity of these mutants to oxidative destabilization of the iron-sulfur cluster in the context of yeast has been confirmed by electron paramagnetic resonance spectroscopy (4). Here, we further investigate the role of Ser-138 in the function of IRP1 by studying the properties of nonphosphorylatable IRP1S138A and phosphomimetic IRP1S138E mutants in mammalian cells.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Plasmid construction. The S138A and S138E point mutations were introduced by site-directed mutagenesis with the ExSite PCR-based protocol (Stratagene), according to the manufacturer's recommendations. The plasmid pSG5-hIRP1 (29) was utilized as the template with the following oligonucleotides: for S138A, CAA CAG AAG GGC AGA CGC CTT ACA GAA GAA TCA AG and the reverse complement CTT GAT TCT TCT GTA AGG CGT CTG CCC TTC TGT TG and for S138E, CAA CAG AAG GGC AGA CGA ATT ACA GAA GAA TCA AG and the reverse complement CTT GAT TCT TCT GTA ATT CGT CTG CCC TTC TGT TG. All mutations were confirmed by sequencing. For the generation of recombinant protein in bacteria, a 1.5-kb PstI/EcoRV fragment from pSG5-hIRP1S138A or pSG5-hIRP1S138E was subcloned into the corresponding sites of pT7-hIRP1 (13), to yield pT7-hIRP1S138A and pT7-hIRP1S138E, respectively. For the expression of His-tagged proteins in eukaryotic cells, the 2.9-kb IRP1 cassette was excised from pT7-hIRP1, pT7-hIRP1S138A, and pT7-hIRP1S138E with XbaI/HindIII and transferred, following blunt-end formation with the Klenow fragment, into the BamHI site of pUHD10-3 (29) to yield 10-3-His-hIRP1, 10-3-His-hIRP1S138A, and 10-3-His-hIRP1S138E, respectively.
Expression and purification of recombinant IRP1. pT7-hIRP1, pT7-hIRP1S138A, and pT7-hIRP1S138E were expressed in Escherichia coli BL21(DE3), and recombinant human IRP1 was purified by affinity chromatography on Ni2+-nitrilotriacetic acid beads (13), which was followed by a second purification step with anion-exchange chromatography on a Resource Q fast-performance liquid chromatography column (Pharmacia).
Cell culture and transfections. Human embryonic kidney HEK293 cells and murine B6 fibroblasts were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U of penicillin per ml, and 0.1 ng of streptomycin per ml. Transient transfections were performed with the Lipofectamine Plus reagent (Gibco BRL). HEK293 cells were cotransfected with pSG5-hIRP1S138A or pSG5-hIRP1S138E and neoCMVtBL (20) with the calcium phosphate method, and stable clones were selected in media containing 400 µg of G418/ml. B6 cells were transfected as described above with 15-1-neo (12), containing the tetracycline repressor (tTA), to generate a tTA-B6 clone by selection in the presence of 250 µg of G418/ml. The tTA-B6 cells were then cotransfected with 10-3-His-hIRP1, 10-3-His-hIRP1S138A, or 10-3-His-hIRP1S138E and puromycin-resistant pBabe (29). Stable transfectants were selected in media containing 4 µg of puromycin/ml, 250 µg of G418/ml, and 2 µg of tetracycline/ml. In preliminary transient-transfection experiments, a tight induction of IRP1 expression was observed upon the removal of tetracycline. However, all isolated clones of stable transfectants displayed leaky IRP1 expression, and the withdrawal of tetracycline had minimal effects. Therefore, the cells were maintained and cultivated for all experiments without tetracycline.
Generation of IRP1 antibodies. Polyclonal IRP1 antibodies were raised by PRF & L, Inc. (Canadensis, Pa.), in a rabbit immunized against highly purified human recombinant IRP1 (2), kindly donated by Jean-Marc Moulis (Grenoble, France).
Western blotting. Cytoplasmic lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8% gels and transferred onto nitrocellulose filters. The blots were saturated with 10% nonfat milk in phosphate-buffered saline and probed with 1:1,000-diluted FLAG or ß-actin or with 1:2,000-diluted IRP1 antibodies. Dilutions were made in phosphate-buffered saline containing 0.5% Tween 20. After being washed with this solution, the blots with (monoclonal) FLAG antibodies were incubated with peroxidase-coupled rabbit anti-mouse immunoglobulin G (1:4,000 dilution) and the blots with (polyclonal) IRP1 or ß-actin antibodies were incubated with peroxidase-coupled goat anti-rabbit immunoglobulin G (1:5,000 dilution). Detection of peroxidase-coupled antibodies was performed with the enhanced chemiluminescence (ECL) method (Amersham).
Pulse-chase and immunoprecipitation. Cells were metabolically labeled for 2 h with trans-35S label (50 µCi/ml), a 70:30 mixture of [35S]methionine-cysteine. The radioactive medium was then removed, and the cells were chased in cold media in the absence or presence of 100 µM hemin or 100 µM desferrioxamine. Endogenous IRP1 or transfected IRP1S138A and IRP1S138E were analyzed by immunoprecipitation (22) with IRP1 or FLAG antibodies, respectively. Immunoprecipitated proteins were resolved by SDS-PAGE and visualized by autoradiography. Radioactive bands were quantified by phosphorimaging.
Biochemical assays. Cytoplasmic lysates or purified IRP1 preparations were analyzed for IRE-binding activity by electrophoretic mobility shift assay (EMSA) (21). Cytosolic extracts devoid of mitochondrial aconitase were prepared as previously described (7). The aconitase enzymatic assay was performed as described in reference 13. One unit is defined as the amount that converts 1 µmol of citrate, pH 7.4, at 25°C per min to isocitrate.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
IRP1S138E expressed in mammalian cells is sensitive to iron-dependent degradation. To study the function of Ser-138 in a more physiological context, His-tagged wild-type IRP1, IRP1S138A, and IRP1S138E were stably transfected into murine B6 fibroblasts. The expression of chimeric proteins was first analyzed by Western blotting with a polyclonal antibody against IRP1, which also cross-reacts with endogenous IRP1 (detection of chimeric IRP1 with commercial antibodies against the His tag was not reliable due to the poor quality of the immunoblots). The result depicted in Fig. 2A (top panel) shows that the IRP1 signal is increased by about twofold in cells transfected with wild-type IRP1 or IRP1S138E (lanes 2 and 4, respectively) and by about fourfold in cells transfected with IRP1S138A (lane 3). No differences are observed in the levels of control ß-actin (bottom panel). In line with the above results, the expression of chimeric wild-type IRP1 and IRP1S138A contributes to significant (P < 0.01) increases in cytosolic aconitase activity of about twofold and fourfold, respectively (Fig. 2B). However, the aconitase activity in cytoplasmic extracts of cells expressing IRP1S138E is similar to the activity of parent cells (Fig. 2B). We conclude that IRP1S138E expressed in B6 cells fails to function as aconitase, which is in agreement with the data shown in Fig. 1C.
|
To better characterize the effects of hemin on IRP1S138E, we utilized the His tag for purification of the transfected proteins from the crude cytoplasmic extract by means of Ni2+ affinity chromatography (Fig. 3). Analysis of the total lysate (Fig. 3A) and of the purified chimeric proteins (Fig. 3B) by EMSA confirms that the IRE-binding activity of IRP1S138E obtained from iron-loaded cells cannot be recovered in vitro with 2-mercaptoethanol (compare lanes 1 to 8 with lanes 9 to 12 in panels A and B). All affinity-purified proteins were inactive as aconitases, possibly due to the instability of this activity upon dilution. Finally, the purified material was subjected to Western blotting with the IRP1 antibody. Analysis of the total lysate (Fig. 3C) reveals a hemin-induced reduction in the intensity of the signal from IRP1S138E-expressing cells (lanes 11 and 12), while desferrioxamine produced the opposite effect (lane 10). Notably, the signal from untreated cells (lane 9) is weaker than that from iron-starved cells (lane 10). The iron manipulations leave the expression of wild-type IRP1 or IRP1S138A largely unaffected (lanes 1 to 8). Probing the filter with an antibody against ß-actin (bottom) provides a loading control. The negative effects of hemin in IRP1S138E expression are clearly demonstrated by the immunoblot of the affinity-purified proteins (Fig. 3D), where the background of endogenous IRP1 is eliminated (compare lanes 9 to 12 in panels C and D).
|
50% (lane 3). Probing with a FLAG antibody (middle panel) demonstrates that this decrease is due to an almost complete loss of transfected IRP1S138E, since under these conditions the background of endogenous IRP1 is eliminated. No differences in the levels of control ß-actin are observed (bottom panel). Notably, in untreated cells IRP1S138E is expressed at relatively lower levels than those of IRP1S138A and wild-type chimeric IRP1 (compare lanes 1 in Fig. 4).
|
6 h (top). In the presence of hemin, the half-life of IRP1S138E further decreases to 4 h (middle). The decay of IRP1S138E can be completely inhibited by desferrioxamine (bottom). We conclude that IRP1S138E undergoes iron-dependent degradation in apparently iron-replete control cells or in iron-overloaded, hemin-treated cells but that it is stabilized in iron-deficient cells treated with desferrioxamine. As an additional control, we performed a pulse-chase experiment with untransfected parent HEK293 (Fig. 5C) and B6 (Fig. 5D) cells to analyze endogenous IRP1. This experiment confirms previous findings (22, 28) by showing that the half-life of endogenous IRP1 is >12 h. It also demonstrates that IRP1 stability remains unaffected by a treatment with hemin in both cell types, in agreement with the data obtained with IRP1S138A (Fig. 5A).
|
|
Ser-138 is highly conserved in vertebrate IRP1 molecules. Ser-138 and its flanking sequences constitute a PKC phosphorylation site, which was earlier found to be conserved in mammalian (human, rabbit, rat, and mouse) IRP1 molecules (9). In the last few years, IRP1 sequence data from additional vertebrate and invertebrate species have been obtained. Multiple sequence alignment of all IRP1 molecules currently deposited in Swiss-Prot (Fig. 7) reveals that as in mammals (Homo sapiens, Oryctolagus cuniculus, Rattus norvegicus, and Mus musculus), Ser-138 is also conserved in chicken (Gallus gallus) IRP1. However, Ser-138 is replaced with an alanine in IRP1 proteins from crayfish (Pacifastacus leniusculus) and insects (Drosophila melanogaster and Manduca sexta) and in IRP1-like proteins from plants (Arabidopsis thaliana, Citrus limon, and Curcubita maxima) and the malaria parasite (Plasmodium falciparum). Interestingly, the stretches of amino acids upstream and downstream of mammalian and chicken Ser-138 sequences shown in Fig. 7 are almost identical (with the exception of the D131H and A136S substitutions in rat and chicken Ser-138 sequences, respectively). There is also some degree of conservation in the sequences surrounding Ala-138 of invertebrate IRP1 molecules, but this is clearly less prominent than in the vertebrate counterparts and restricted to fewer positions (for example, Q129, V130, N142, and E146). Taken together, Ser-138 and flanking sequences are highly conserved only in the IRP1 molecules of vertebrates, not invertebrates.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Since the IRE/IRP system is not conserved in yeast, we investigated the function of IRP1S138E in the more physiologically relevant context of mammalian cells. In B6 transfectants, the IRE-binding activity of IRP1S138E can be induced following iron chelation with desferrioxamine (Fig. 2C and 3A and B). This finding suggests that in apparently iron-replete B6 cells, IRP1S138E may assemble a [4Fe-4S] cluster, which is expected to render the protein to cytosolic aconitase and inactivate IRE binding. To directly address this possibility, we performed aconitase assays with affinity-purified wild-type and mutant IRP1s, but we were unable to detect any enzymatic activity. Conceivably, aconitase activity is more sensitive to dilution than IRE-binding activity. Nevertheless, the results with the crude lysates (Fig. 2B) are in agreement with the notion that IRP1S138E is unable to function as an aconitase in B6 transfectants. The positive effect of desferrioxamine in the IRE-binding activity of IRP1S138E may suggest that in iron-replete but not overloaded B6 cells, this mutant behaves as a null form of IRP1, possibly due to partial assembly of the iron-sulfur cluster (15). In addition, the increase in IRP1S138E steady-state levels (lanes 10 in Fig. 3C and D) indicates that desferrioxamine may stabilize IRP1S138E (see also below).
We were intrigued by the failure of 2-mercaptoethanol to recover the IRE-binding activity in extracts of iron-loaded IRP1S138E-expressing cells (Fig. 2C and 3A and B). A similar effect was previously observed with (endogenous) IRP1 in B6 cells in response to menadione-induced oxidative stress (10). Although under these conditions the loss of activity was not associated with any significant alterations in IRP1 levels (10), we show here that iron loading leads to the accelerated turnover of IRP1S138E. The iron-dependent degradation of IRP1S138E is documented for two different cell lines of mouse (Fig. 2 and 3) and human (Fig. 4 to 6) origins and for two types of modified constructs carrying an N- or C-terminal epitope tag. It should be noted that wild-type IRP1 has a relatively long half-life (>12 h), which is not dramatically affected by iron (22, 28) (Fig. 5C and D). These findings also hold true for IRP1S138A (Fig. 5A). In contrast, our data suggest that iron promotes the degradation of IRP1S138E by the proteasome (Fig. 5B and 6C and D), in a fashion akin to the well-established iron-dependent degradation of IRP2 (14, 17). The degradation kinetics appear to correlate with the level of iron loading. In apparently iron-replete control cells, the half-life of IRP1S138E is 6 h (Fig. 5B). Further iron loading with hemin accelerates the turnover of IRP1S138E and reduces its half-life to 4 h. Importantly, the degradation of IRP1S138E can be efficiently blocked by the iron chelator desferrioxamine.
We also observed that the recovery of endogenous IRE-binding activity in extracts of hemin-treated B6 transfectants by 2-mercaptoethanol was relatively poor (for example, Fig. 2C, lanes 6, 9, and 12). However, IRP1 could be completely recovered by 2-mercaptoethanol from hemin-treated parent B6 cells (Fig. 2C, lane 3). We speculate that the weak effect of 2-mercaptoethanol on endogenous IRP1 in the transfectants may be related to nonreversible inactivation of the protein, possibly via posttranslational modifications (perhaps oxidation), but the reason for this is unclear. Notably, similar effects have previously been observed in a different setting (10). In support of this interpretation, the pulse-chase experiments with B6 and HEK293 cells in the absence or presence of hemin (Fig. 5C and D) rule out the possibility that hemin may generally promote the degradation of IRP1. Thus, our results do not support a general model for heme-mediated degradation of IRP1, which had been proposed in the past (11) well before it became clear that the mechanism for IRP1 regulation involves an iron-sulfur cluster switch and that IRP2 is regulated at the level of protein stability. Nevertheless, in light of the data presented here, it might be worth considering Ser-138 phosphorylation as a possibility underlying the heme-mediated degradation of IRP1 described previously (11).
In conclusion, IRP1S138E expressed in mammalian cells under normoxic conditions not only is unable to assemble a [4Fe-4S] cluster but also undergoes iron-mediated degradation via the proteasome. Considering that Ser-138 may be a target of phosphorylation by PKC (9), our data raise the possibility for a new mode of IRP1 regulation at the level of protein stability. Preliminary experiments with HL-60 cells aiming to assess the effect of iron on the stability of endogenous IRP1 after treatment with phorbol 12-myristate 13-acetate (PMA) to activate PKC pathways were negative. The effectiveness of this treatment was monitored by immunoblot detection of PMA-induced p21WAF (6). However, it should be noted that the evidence for Ser-138 phosphorylation within IRP1 in PMA-treated HL-60 cells remains thus far indirect and has primarily been based on in vitro phosphorylation assays with oligopeptides as substrates (9). It is conceivable that in the context of the entire protein, Ser-138 may be a target for modification by other kinases. Alternatively, our culture conditions may have favored the predominance of iron-loaded [4Fe-4S] IRP1, which is not a preferable substrate for phosphorylation by PKC (26). Generation and employment of phospho-specific antibodies are expected to help towards identifying conditions for Ser-138 phosphorylation within IRP1 in vivo and studying the responses of the phosphorylated protein to iron.
The conservation of Ser-138 and the flanking sequences in the IRP1 proteins of vertebrates is indicative of the functional significance of this residue. If this assumption proves correct, the S138A substitution in crayfish and insect IRP1s and in the IRP1-like molecules of plants and the malaria parasite (Fig. 7) suggests that Ser-138 in vertebrate IRP1s has evolved to integrate a more complex repertoire of regulatory signals.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2. Brazzolotto, X., J. Gaillard, K. Pantopoulos, M. W. Hentze, and J.-M. Moulis. 1999. Human cytoplasmic aconitase (iron regulatory protein 1) is converted into its [3Fe-4S] form by hydrogen peroxide in vitro but is not activated for iron-responsive element binding. J. Biol. Chem. 274:21625-21630.
3. Brown, N. M., S. A. Anderson, D. W. Steffen, T. B. Carpenter, M. C. Kennedy, W. E. Walden, and R. S. Eisenstein. 1998. Novel role of phosphorylation in Fe-S cluster stability revealed by phosphomimetic mutations at Ser-138 of iron regulatory protein 1. Proc. Natl. Acad. Sci. USA 95:15235-15240.
4. Brown, N. M., M. C. Kennedy, W. E. Antholine, R. S. Eisenstein, and W. E. Walden. 2002. Detection of a [3Fe-4S] cluster intermediate of cytosolic aconitase in yeast expressing iron regulatory protein 1: insights into the mechanism of Fe-S cluster cycling. J. Biol. Chem. 277:7246-7254.
5. Cairo, G., and A. Pietrangelo. 2000. Iron regulatory proteins in pathobiology. Biochem. J. 352:241-250.
6. Das, D., G. Pintucci, and A. Stern. 2000. MAPK-dependent expression of p21(WAF) and p27(kip1) in PMA-induced differentiation of HL60 cells. FEBS Lett. 472:50-52.[CrossRef][Medline]
7. Drapier, J. C., H. Hirling, J. Wietzerbin, P. Kaldy, and L. C. Kühn. 1993. Biosynthesis of nitric oxide activates iron regulatory factor in macrophages. EMBO J. 12:3643-3649.[Medline]
8. Eisenstein, R. S. 2000. Iron regulatory proteins and the molecular control of mammalian iron metabolism. Annu. Rev. Nutr. 20: 627-662.[CrossRef][Medline]
9. Eisenstein, R. S., P. T. Tuazon, K. L. Schalinske, S. A. Anderson, and J. A. Traugh. 1993. Iron-responsive element-binding protein: phosphorylation by protein kinase C. J. Biol. Chem. 268:27363-27370.
10. Gehring, N., M. W. Hentze, and K. Pantopoulos. 1999. Inactivation of both RNA binding and aconitase activities of iron regulatory protein-1 by quinone-induced oxidative stress. J. Biol. Chem. 274:6219-6225.
11. Goessling, L. S., S. Daniels-McQueen, M. Bhattacharyya-Pakrasi, J.-J. Lin, and R. E. Thach. 1992. Enhanced degradation of the ferritin repressor protein during induction of ferritin messenger RNA translation. Science 256:670-673.
12. Gossen, M., and H. Bujard. 1992. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89:5547-5551.
13. Gray, N. K., S. Quick, B. Goossen, A. Constable, H. Hirling, L. C. Kühn, and M. W. Hentze. 1993. Recombinant iron regulatory factor functions as an iron-responsive element-binding protein, a translational repressor and an aconitase: a functional assay for translational repression and direct demonstration of the iron switch. Eur. J. Biochem. 218:657-667.[Medline]
14. Guo, B., J. D. Phillips, Y. Yu, and E. A. Leibold. 1995. Iron regulates the intracellular degradation of iron regulatory protein 2 by the proteasome. J. Biol. Chem. 270:21645-21651.
15. Haile, D. J., T. A. Rouault, J. B. Harford, M. C. Kennedy, G. A. Blondin, H. Beinert, and R. D. Klausner. 1992. Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding. Proc. Natl. Acad. Sci. USA 89:11735-11739.
16. Hanson, E. S., and E. A. Leibold. 1999. Regulation of the iron regulatory proteins by reactive nitrogen and oxygen species. Gene Expr. 7:367-376.[Medline]
17. Iwai, K., R. D. Klausner, and T. A. Rouault. 1995. Requirements for iron-regulated degradation of the RNA binding protein, iron regulatory protein 2. EMBO J. 14:5350-5357.[Medline]
18. Johansson, H. E., and E. C. Theil. 2002. Iron-response element (IRE) structure and combinatorial RNA regulation, p. 237-253. In D. M. Templeton (ed.), Molecular and cellular iron transport. Marcel Dekker, Inc., New York, N.Y.
19. Kim, S., and P. Ponka. 2002. Nitrogen monoxide-mediated control of ferritin synthesis: implications for macrophage iron homeostasis. Proc. Natl. Acad. Sci. USA 99:12214-12219.
20. Kwon, H., N. Pelletier, C. DeLuca, P. Genin, S. Cisternas, R. Lin, M. A. Wainberg, and J. Hiscott. 1998. Inducible expression of I
B
repressor mutants interferes with NF-B activity and HIV-1 replication in Jurkat T cells. J. Biol. Chem. 273:7431-7440.
21. Mueller, S., and K. Pantopoulos. 2002. Activation of iron regulatory protein-1 (IRP1) by oxidative stress. Methods Enzymol. 348:324-337.[CrossRef][Medline]
22. Pantopoulos, K., N. Gray, and M. W. Hentze. 1995. Differential regulation of two related RNA-binding proteins, iron regulatory protein (IRP) and IRPB. RNA 1:155-163.[Abstract]
23. Pantopoulos, K., and M. W. Hentze. 2000. Nitric oxide, oxygen radicals and iron metabolism, p. 293-313. In L. Ignarro (ed.), Nitric oxide. Academic Press, San Diego, Calif.
24. Ponka, P., and H. M. Schulman. 1985. Acquisition of iron from transferrin regulates reticulocyte heme synthesis. J. Biol. Chem. 260:14717-14721.
25. Rouault, T., and J. B. Harford. 2000. Translational control of ferritin synthesis, p. 655-670. In N. Sonenberg, J. W. B. Hershey, and M. B. Mathews (ed.), Translational control of gene expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
26. Schalinske, K. L., S. A. Anderson, P. T. Tuazon, O. S. Chen, M. C. Kennedy, and R. S. Eisenstein. 1997. The iron-sulfur cluster of iron regulatory protein 1 modulates the accessibility of RNA binding and phosphorylation sites. Biochemistry 36:3950-3958.[CrossRef][Medline]
27. Schalinske, K. L., and R. S. Eisenstein. 1996. Phosphorylation and activation of both iron regulatory proteins 1 and 2 in HL-60 cells. J. Biol. Chem. 271:7168-7176.
28. Tang, C. K., J. Chin, J. B. Harford, R. D. Klausner, and T. A. Rouault. 1992. Iron regulates the activity of the iron-responsive element binding protein without changing its rate of synthesis or degradation. J. Biol. Chem. 267:24466-24470.
29. Wang, J., and K. Pantopoulos. 2002. Conditional derepression of ferritin synthesis in cells expressing a constitutive IRP1 mutant. Mol. Cell. Biol. 22:4638-4651.
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| J. Bacteriol. | J. Virol. | Eukaryot. Cell |
|---|
| Microbiol. Mol. Biol. Rev. | Clin. Vaccine Immunol. | All ASM Journals |
|---|
