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Previous Article | Next Article 
Molecular and Cellular Biology, December 2004, p. 10161-10168, Vol. 24, No. 23
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.23.10161-10168.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Skirball Institute of Biomolecular Medicine,1 Departments of Cell Biology, Medicine, and Pharmacology, New York University School of Medicine, New York, New York,5 Department of Biochemistry,2 Howard Hughes Medical Institute, University of Michigan School of Medicine, Ann Arbor, Michigan,3 Department of Biochemistry, McGill University, Montreal, Quebec, Canada4
Received 11 May 2004/ Returned for modification 6 July 2004/ Accepted 31 August 2004
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ABSTRACT |
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), thus attenuating mRNA translation and activating a gene expression program known as the integrated stress response. It has been noted that conditions associated with eIF2 phosphorylation, notably accumulation of unfolded proteins in the endoplasmic reticulum (ER), or ER stress, are also associated with activation of nuclear factor kappa B (NF-
B) and that eIF2 phosphorylation is required for NF-B activation by ER stress. We have used a pharmacologically activable version of pancreatic ER kinase (PERK, an ER stress-responsive eIF2 kinase) to uncouple eIF2 phosphorylation from stress and found that phosphorylation of eIF2 is both necessary and sufficient to activate both NF-B DNA binding and an NF-B reporter gene. eIF2 phosphorylation-dependent NF-B activation correlated with decreased levels of the inhibitor IB protein. Unlike canonical signaling pathways that promote IB phosphorylation and degradation, eIF2 phosphorylation did not increase phosphorylated IB levels or affect the stability of the protein. Pulse-chase labeling experiments indicate instead that repression of IB translation plays an important role in NF-B activation in cells experiencing high levels of eIF2 phosphorylation. These studies suggest a direct role for eIF2 phosphorylation-dependent translational control in activating NF-B during ER stress. |
INTRODUCTION |
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). Phosphorylated eIF2 inhibits its guanine nucleotide exchange factor, eIF2B, and thereby inhibits the exchange reaction required to generate active GTP-bound eIF2. As a consequence, regulated phosphorylation of eIF2 serves to modulate mRNA translation rates (18, 20). In addition to its negative impact on global protein synthesis, eIF2 phosphorylation also promotes gene-specific upregulation of the translation of certain mRNAs. The two known examples of this involve the yeast transcription factor GCN4 (19) and the mammalian transcription factor ATF4 (12). Regulated gene expression appears to be an important consequence of eIF2 phosphorylation, as mutations that interfere with eIF2 phosphorylation lead to an important defect in stress-induced gene expression (16, 28, 39).
Four known eIF2 kinases couple seemingly unrelated stressful conditions to the aforementioned common translational regulatory event. PKR responds to double-stranded RNA in virally infected cells (23), GCN2 is activated by uncharged tRNAs in amino acid-starved cells (20), HRI is activated by heme depletion in erythroid precursor cells (3), and PERK is activated by unfolded proteins in the endoplasmic reticulum (ER), or ER stress (37). Mutations in each of these four kinases have been produced, and their phenotypes reveal the importance of eIF2 phosphorylation in stressed cells (6).
Nuclear factor kappa B (NF-B) encompasses a family of stress-induced transcription factors. Like the more ancient eIF2 phosphorylation-dependent signaling, NF-B signaling is also triggered by diverse stressful conditions, and activated NF-B has broad effects on gene expression (38). Several studies have suggested cross talk between the eIF2 phosphorylation pathway and NF-B activation. The double-stranded-RNA-activated eIF2 kinase PKR was noted to phosphorylate the NF-B inhibitor, IB (26), and genetic and pharmacological interventions that interfere with PKR activity attenuated NF-B activation by cytokines (4, 27, 47) or viruses (9, 43). There is some uncertainty regarding the role of eIF2 phosphorylation in NF-B activation by PKR, as the latter contributes to NF-B activation by both kinase-dependent (9) and kinase-independent (8) mechanisms.
Conditions that promote accumulation of unfolded proteins in the endoplasmic reticulum lead to high levels of eIF2 phosphorylation (34, 35), which is mediated by the ER-localized kinase PERK (14, 15). These same conditions activate NF-B (32). A recent study has found that ER stress-mediated NF-B activation was attenuated both in PERK–/– cells and, importantly, in cells bearing two mutant alleles of EIF2A in which serine 51 (the substrate of the stress-inducible kinases) had been mutated to an alanine. These mutant eIF2A/A cells were also defective in NF-B activation by amino acid starvation, as were cells lacking GCN2 (21), the kinase that phosphorylates eIF2 in amino acid-starved cells.
Together these observations point to a nonredundant role for eIF2 phosphorylation in NF-B activation under various stress conditions. But they provide little insight into the mechanisms involved. One of the best-characterized aspects of NF-B regulation is the phosphorylation-dependent, proteasome-mediated degradation of its inhibitor, IB. However, it is not clear if and how eIF2 phosphorylation ties in to IB levels. Because the stressful conditions used to promote eIF2 phosphorylation have multiple other effects (reviewed in reference 17), it is not even known whether eIF2 phosphorylation plays a permissive role or an instructive role in NF-B activation, nor is it known whether the phosphorylated form of eIF2 is affecting NF-B activation as a modified translation initiation factor or by some other means. In an effort to answer some of these questions, we have probed NF-B activation in an experimental system that uncouples eIF2 phosphorylation from stress signaling and discovered that translational repression of IB can account for activation of NF-B under conditions of eIF2 phosphorylation.
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MATERIALS AND METHODS |
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The Fv2E-PERK+ wild-type mouse embryonic fibroblasts described above were transiently transfected using Fugene lipid-based gene transfer reagent (catalog no. 1814443; Roche, Indianapolis, Ind.) with luciferase reporter plasmids containing a minimal rat angiotensinogen promoter driven by four wild-type or mutant NF-B binding sites from the rat angiotensinogen gene, as previously described (36). One day later the cells were treated for 1 h with the indicated concentration of AP20187 (gift of ARIAD Pharmaceuticals, Cambridge, Mass.), washed free of the activator (to allow translation to recover), and harvested for use in a luciferase assay 24 h later.
Cells were treated with thapsigargin (catalog no. T9033; Sigma, St. Louis, Mo.) at a final concentration of 400 nM or cycloheximide (catalog no. C7698; Sigma) at 20 µg/ml. Unless otherwise indicated, AP20187 was used at a concentration of 10 nM. Cells were treated with 20 ng of tumor necrosis factor alpha (TNF-; catalog no. T7539; Sigma)/ml with or without the proteasome inhibitor MG132 (catalog no. 474790; Calbiochem-Novobiochem, San Diego, Calif.) at 10 µM.
Immunoblotting and immunoprecipitation.
Total IB was detected with a purified rabbit immune serum (catalog no. 9242; Cell Signaling, Beverly, Mass.), and IB phosphorylated on serine 32 and 36 was detected with an epitope-specific antiserum (catalog no. 9246; Cell Signaling). GADD34 was detected with an antiserum directed to the N terminus of the mouse protein raised in our lab (30). PERK was detected with a 1:1 mixture of two rabbit antisera (NY97, which detects the unphosphorylated form of the protein, and NY201, which detects predominantly the hyperphosphorylated forms of the protein) as described previously (2). Total eIF2 was detected with a monoclonal antibody to human eIF2, a gift of the late Edward Henshaw (40), and phosphorylated eIF2 was detected with an epitope-specific antiserum (catalog no. RG0001; Research Genetics, Huntsville, Ala.).
Pulse-chase labeling experiments were carried out in the Fv2E-PERK+ wild-type mouse embryonic fibroblasts described above. Cells were switched to Dulbecco's modified Eagle's medium containing 10% of the normal content of methionine and cysteine (these levels of methionine and cysteine are sufficient to suppress activation of the eIF2 kinase GCN2 yet are compatible with high-level incorporation of labeled amino acids into newly synthesized proteins) 15 min before addition of TRANSlabel (MP Biomedical, Irvine, Calif.) 35S-labeled methionine-cysteine mixture at 200 µCi/ml for 10 min. The labeling pulse was terminated by washing the unincorporated label and flooding the cells with complete medium. Following the indicated chase period, during which cells were exposed to AP20187 and/or MG132, the cells were lysed in RIPA buffer (20 mM Tris [pH 8.5], 100 mM NaCl, 0.2% sodium deoxycholate, 0.2% NP-40, 0.2% Triton X-100, 0.1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 4 µg of aprotinin/ml, 2 µg of pepstatin/ml), and the lysate was clarified by centrifugation at 14,000 x g for 15 min, precleared on protein A-Sepharose beads (catalog no. 10-1042; Zymed, South San Francisco, Calif.), and subjected to immunoprecipitation with prebound anti-IB rabbit immunoglobulin G (catalog no. SC-371 AC; Santa Cruz Biotech, Santa Cruz, Calif.). Radiolabeled proteins found in the immunoprecipitate were resolved by reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the dried gel was exposed to autoradiography using a phosphoimaging cassette (Molecular Dynamics, Sunnyvale, Calif.).
EMSA.
NF-B DNA binding activity in nuclear extracts was detected by an electrophoretic mobility shift assay (EMSA) performed as previously described (21, 36). The indicated molar excess of unlabeled competitor probe or 1 µl of purified anti-p65 (catalog no. SC-7151; Santa Cruz Biotech) or anti-CHOP antiserum (45) was added to the binding reaction together with the radiolabeled probe.
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RESULTS |
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phosphorylation in NF-B activation (21), we performed EMSA on nuclear extracts prepared from unstressed cells and cells that had been treated with thapsigargin (Fig. 1A). Thapsigargin-mediated ER calcium depletion leads to rapid onset of ER stress, eIF2 phosphorylation (detected here by immunoblotting with an antiserum specifically reactive with the phosphorylated form), and subsequent ATF4-mediated activation of downstream gene expression, measured here by accumulation of the GADD34 target gene. A protein complex rapidly formed on the NF-B binding site in nuclear extracts of treated wild-type cells but not in extracts from cells homozygous for the EIF2AA/A mutation that substitutes the serine at position 51 of eIF2 with an alanine and thereby prevents regulatory phosphorylation. Reduced levels of the NF-B inhibitory protein IB, detected by immunoblotting, preceded the induction of NF-B EMSA activity in thapsigargin-treated cells. The recovery of IB levels at longer treatment points correlated with the induction of the GADD34 phosphatase and the dephosphorylation of eIF2 (Fig. 1B).
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phosphorylation in NF-B activation, we made use of an experimental system that uncouples eIF2 phosphorylation from stress signaling. PERK, the ER stress-inducible eIF2 kinase, is normally activated by oligomerization in the plane of the ER membrane (2). We fused PERK's eIF2 kinase domain to a protein module with two high-affinity binding sites for the otherwise inert bivalent compound AP20187. When expressed in cells, this artificial kinase, Fv2E-PERK, is subordinate to AP20187 treatment (28) and is activated independently of any stress signaling. AP20187 treatment led to high-level eIF2 phosphorylation in Fv2E-PERK+ cells but had no effect on the parental cells lacking the artificial kinase (Fig. 2A). Fv2E-PERK was readily activated in mutant EIF2AA/A cells, but this predictably failed to induce eIF2 phosphorylation. EMSA of nuclear extracts showed that AP20187 induced NF-B activity in Fv2E-PERK+ wild-type (EIF2AS/S) cells but not in the mutant EIF2AA/A cells (Fig. 2B). Homologous competition binding assays and antibody supershift experiments confirmed the identity of the NF-B protein-DNA complex detected in the assay (Fig. 2C).
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phosphorylation and activation of NF-B DNA binding activity, we measured the activity of a transfected reporter gene driven by four copies of a wild-type NF-B binding site. A brief (60-min) pulse of AP20187 induced marked activation of the wild-type reporter gene (measured 24 h later [Fig. 3]). No activation of a reporter gene driven by mutant NF-B sites was observed. In addition, endogenous NF-B target genes, such as those encoding the major histocompatibility complex heavy chains (H2-Q8, H2-2KF, H2-K2, and H2-D1) and ß2 microglobulin (Qb-1), were induced in the Fv2E-PERK+ cells by AP20187 treatments and in wild-type mouse fibroblasts by exposure to tunicamycin (National Center for Biotechnology Information Gene Expression Omnibue [GEO] data set GDS405).
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phosphorylation and NF-B activation correlated with a time-dependent decrease in IB levels that was not observed in the mutant EIF2AA/A cells (Fig. 4A). Interestingly, Fv2E-PERK activation had no measurable effect on levels of the p65 NF-B subunit, which is consistent with the known stability of that protein (24) and with the induction of NF-B binding activity that we observe. eIF2 levels were similarly stable, attesting to the effect's specificity to IB (Fig. 4B). Canonical activators of NF-B access signal transduction pathways that promote phosphorylation of the inhibitor IB on serines 32 and 36 (38). A ubiquitin ligase complex recognizes the phosphorylated form of IB, and polyubiquitinated IB is degraded by the proteasome. Fv2E-PERK activation by AP20187 did not promote a measurable increase in levels of phosphorylated IB, which remained undetectable. However, phosphorylated IB was readily detectable in lysates of cells treated with the proteasome inhibitor, MG132, which stabilizes the phosphorylated form of the protein (Fig. 4B).
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B is difficult to detect, rendering an Fv2E-PERK-mediated increase in IB phosphorylation potentially easy to miss. Therefore, to determine if the eIF2 phosphorylation-dependent decline in IB levels correlated with any increased phosphorylation on serines 32 and 36, we exposed the AP20187-treated cells to the proteasome inhibitor MG132. As expected, proteasome inhibition markedly increased the levels of phosphorylated IB in tumor necrosis factor alpha-treated cells (Fig. 5A). Interestingly, proteasome inhibition led to only modest stabilization of total IB, an observation that is consistent with the existence of proteasome-independent mechanisms for IB degradation (5, 11).
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B levels in cells that were otherwise unperturbed (Fig. 5A, compare lanes 1 and 3, and B, compare lane 1 with lanes 2, 4, 6, 8, and 10). This observation is consistent with a relatively high basal phosphorylation-dependent turnover of IB in these cells. The decline in IB levels effected by Fv2E-PERK was only slightly attenuated by proteasome inhibition (compare Fig. 4, lanes 4 to 6, with 5B, lanes 7, 9, and 11). Furthermore, proteasome inhibition promoted some eIF2 phosphorylation (Fig. 5, lanes 8 and 10), presumably mediated by proteotoxic stress. Remarkably, however, Fv2E-PERK activation and eIF2 phosphorylation not only failed to increase IB phosphorylation but also significantly attenuated the accumulation of phosphorylated IB in proteasome-inhibited cells (Fig. 5B, compare odd- and even-numbered lanes). These observations indicate that eIF2 phosphorylation does not activate NF-B by accessing one of the canonical IB phosphorylation-promoting pathways and must use a different mechanism.
The original descriptions of IB emphasized the lability of the factor, as translational inhibitors were noted to promote NF-B DNA binding activity (1, 42). Given that eIF2 phosphorylation also inhibits protein synthesis, we decided to explore this facet of NF-B activation in more detail. NF-B DNA binding activity was increased by cycloheximide treatment of wild-type cells, as previously reported (42), and this correlated with reduced levels of the inhibitor, IB (Fig. 6A). Cycloheximide treatment led to no measurable decrease in p65 or eIF2 protein levels, attesting to the stability of these proteins. The effects of cycloheximide on levels of phosphorylated IB also resembled those of Fv2E-PERK activation (Fig. 4B) in that no increase in the phosphorylated protein was observed in cells treated with cycloheximide alone. Proteasome inhibitor, by itself, led to a progressive increase in levels of phosphorylated IB, whereas the addition of cycloheximide strongly attenuated this increase (Fig. 6B).
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phosphorylation (21, 22) (Fig. 6A), an effect that might be attributed to loss of the labile eIF2 phosphatase CReP (22). To study the role of eIF2 phosphorylation in cycloheximide-mediated activation of NF-B, we treated mutant EIF2AA/A cells with the protein synthesis inhibitor and studied NF-B activation by EMSA and IB levels by immunoblotting. The EIF2AA/A genotype, which inhibits regulatory phosphorylation of eIF2, had no measurable effect on NF-B activation, IB phosphorylation, or total IB levels in cycloheximide-treated cells (Fig. 6C). These observations suggest that inhibition of new protein synthesis can adequately explain the effects of cycloheximide on NF-B activity without evoking an additional role for eIF2 phosphorylation.
Induced degradation of IB plays an important role in canonical activation of NF-B. To address the possibility that eIF2 phosphorylation might affect this aspect of IB metabolism (independently of IB phosphorylation), we performed pulse-chase labeling experiments, tracking the fate of newly synthesized IB. The basal turnover of IB in murine fibroblasts proved very high. Less than 30% of the signal measured at the end of the 10-min labeling pulse was present after a 20-min chase. Furthermore, activation of Fv2E-PERK during the chase had no measurable effect on the decay of the IB signal (Fig. 7A). Addition of proteasome inhibitor during the chase stabilized IB somewhat; however, in that context, too, activation of Fv2E-PERK during the chase did not accelerate IB degradation and may have even contributed modestly to its stability (Fig. 7B). We conclude that IB turns over rapidly in murine fibroblasts and that eIF2 phosphorylation does not exert its effects on the levels of the inhibitor by further enhancing its degradation.
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B in untreated cells with those in cells treated with AP20187, cycloheximide, the ER stress-promoting agent thapsigargin, and the canonical NF-B activator TNF-. The amount of radiolabeled IB immunoprecipitated with a specific antibody following a short labeling pulse was markedly diminished by activation of the eIF2 kinase Fv2E-PERK by AP20187, by treatment with cycloheximide, or by exposure to conditions that cause ER stress (thapsigargin) (Fig. 7C). The effect of thapsigargin on IB synthesis depended on eIF2 phosphorylation, since it was abolished in the EIF2AA/A mutant cells (Fig. 7C), and the decline in IB synthesis paralleled the global inhibition in protein synthesis in the cells exposed to conditions promoting eIF2 phosphorylation (Fig. 7D). By contrast, exposure to the canonical NF-B activator, TNF-, increased IB synthesis, suggesting a completely different mechanism of action. These observations are consistent with a role for inhibited synthesis of IB in mediating the effects of eIF2 phosphorylation on NF-B activation both in ER-stressed cells and following activation of Fv2E-PERK. |
DISCUSSION |
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is conserved among the eukaryotes and represents one of the oldest pathways for stress-induced gene expression. Furthermore, eIF2 phosphorylation is concerned mostly with autonomous cell adaptations to stress. NF-B signaling, on the other hand, is found in metazoans, and canonical activators of NF-B signaling, such as cytokines, are intercellular signaling molecules. However, over the years evidence that autonomous cell phenomena, such as ER stress, are also associated with NF-B activation has accrued, with the suggestion that ancient, autonomous cell signaling pathways might be linked to NF-B activation.
This study confirms the established role of eIF2 phosphorylation in NF-B activation by ER stress (21). Using an inducible system that uncouples eIF2 phosphorylation from other stress signals, we find that eIF2 phosphorylation can have an instructive role in NF-B activation. In other words, activation of an eIF2 kinase provides a signal sufficient for NF-B activation in cultured mouse fibroblasts. Our study also reveals significant differences between the mechanism used by canonical inducers of NF-B and the consequences of eIF2 phosphorylation. Unlike canonical inducers of NF-B, eIF2 phosphorylation promoted neither phosphorylation nor degradation of IB. Instead, our data argue that the major impact of eIF2 phosphorylation on NF-B activation is inhibition of the synthesis of the labile inhibitor IB.
The mechanism uncovered in this study suggests that the link between eIF2 phosphorylation and NF-B activation depends on the lability of the inhibitor, which, in turn, likely depends on basal levels of signaling through the canonical pathway(s) that activates NF-B. Indeed, the rapid accumulation of phosphorylated IB in mouse fibroblasts treated with proteasome inhibitor is consistent with high basal levels of IB kinase activity in these cells. It is worth noting that both eIF2 phosphorylation and cycloheximide treatment disproportionately reduced the levels of phosphorylated IB, compared with their effect on the levels of total IB. Inhibited protein synthesis may attenuate basal activity of an IB kinase and account for some of this effect. Alternatively, newly synthesized IB might constitute a preferred substrate for its kinases. The plausibility of the latter explanation is supported by evidence for the existence of multiple pools of IB in cells (25, 33, 41). The existence of more than one pool of IB might also explain the discrepancy between the short half-life of newly synthesized IB (measured by the pulse-chase method [Fig. 7A and B]) and the much longer half-life inferred from the gradual decline in total IB protein levels in the cycloheximide-treated and Fv2E-PERK-activated cells (Fig. 4, 5B, and 6A and B). However, these potential complexities of IB metabolism do not weaken our conclusion that attenuated synthesis of the inhibitor plays a major role in mediating activation of NF-B by eIF2 phosphorylation in mouse fibroblasts.
Our findings are at odds with those reported by Jiang and colleagues, who found no decrease in steady-state IB levels in thapsigargin-treated cells and instead uncovered evidence for dissociation of the IB-NF-B complex under those conditions (see Fig. 6 in reference 21). We have no explanation for these differences; however, we do note that since the submission of the present study Wu and colleagues have reported that induction of NF-B DNA binding activity in cells exposed to UV light is also associated with eIF2 phosphorylation-dependent repression of IB synthesis (46).
Our study does not address the physiological significance of the link between eIF2 phosphorylation and NF-B activation. It is worth noting that we have but an incomplete understanding of the relative significance of regulated protein synthesis versus activation of gene expression programs as readouts of eIF2 phosphorylation. In yeast it is fairly clear that mutations in the transcription factor GCN4 phenocopy mutations in the upstream kinase GCN2 or in the gene encoding its substrate SUI2 (yeast eIF2) (6, 7). In mammalian cells too, some of the phenotypes of loss of PERK gene function or the EIF2AA/A genotype are mimicked by mutations in the gene encoding the downstream transcription factor ATF4 (16, 29, 39). Furthermore, in both yeast and mammalian cells, translation activation of the transcription factors GCN4 and ATF4 occurs at levels of eIF2 phosphorylation that have only a modest impact on global protein synthesis (7, 44; Lu et al., unpublished observation). By contrast, our proposed mechanism of cross talk between eIF2 phosphorylation and NF-B signaling is proportional to the repression of IB translation. Such levels of repression are easily attained in thapsigargin-treated cells (14) or in Fv2E-PERK+ cells activated by AP20187 (28) and are clearly sufficient to activate NF-B in cultured mouse fibroblasts (Fig. 1A and 2B) (21).
The extent to which translational repression contributes to NF-B activation in more physiological contexts in which eIF2 kinases are activated is not known. However, we note that endogenous proinflammatory NF-B target genes, such as those encoding the major histocompatibility complex heavy and light chains, the interleukin 17 receptor, and a complement receptor-related protein, were all induced in the Fv2E-PERK+ cells by AP20187 treatment and in wild-type mouse fibroblasts by exposure to tunicamycin (National Center for Biotechnology Information GEO data set GDS405). The PERK-dependent induction of NF-B target genes by tunicamycin is potentially significant, as global repression of mRNA translation is relatively modest under those conditions (14), mimicking physiological stress situations. Furthermore, loss-of-function mutations in the eIF2 kinase PERK or HRI or the EIF2AA/A genotype all predispose cells to programmed cell death under physiologically stressful conditions (10, 13, 14, 39, 48); however, the role of defective activation of NF-B in this phenotype, if any, remains to be explored.
Translational repression in response to activation of eIF2 kinases tends to be transient (34, 35). Translational recovery is mediated in part by activation of GADD34, an eIF2-specific regulatory subunit of a holophosphatase complex (30, 31), which is itself a target of the eIF2 phosphorylation-dependent gene expression program, the integrated stress response (16, 29, 30). GADD34-mediated translational recovery is therefore likely to reestablish IB translation and reverse the effects of eIF2 phosphorylation on NF-B activity, since the stress response is attenuated (Fig. 1B). Furthermore, while activation of NF-B proceeds through utilization of preformed components, the response in terms of target gene expression depends on new protein synthesis. Thus, the biphasic nature of the inhibition of protein synthesis, which is inherent to stressful conditions that promote eIF2 phosphorylation, is also predicted to contribute to the expression of NF-B target genes.
In conclusion, our study indicates that the pathways promoting eIF2 phosphorylation and those that activate NF-B interact through translational repression of the inhibitor IB. Our study also suggests that the importance of this link is likely to be influenced by signaling through canonical NF-B activation pathways that define the turnover rate of IB. As such, eIF2 phosphorylation and the consequent inhibition of eIF2B might modulate NF-B signaling by parallel pathways active in stressed cells.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grants ES08681 and DK47119 (to D.R.) and DK42394 (to R.J.K.). D.R. is a Scholar of the Ellison Medical Foundation.
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FOOTNOTES |
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