JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M607365200 on November 17, 2006

J. Biol. Chem., Vol. 282, Issue 3, 1757-1768, January 19, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/3/1757    most recent
M607365200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kaur, S.
Right arrow Articles by Platanias, L. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kaur, S.
Right arrow Articles by Platanias, L. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Regulatory Effects of Mammalian Target of Rapamycin-activated Pathways in Type I and II Interferon Signaling*

Surinder Kaur{ddagger}, Lakhvir Lal{ddagger}, Antonella Sassano{ddagger}, Beata Majchrzak-Kita§, Maya Srikanth{ddagger}, Darren P. Baker, Emmanuel Petroulakis||, Nissim Hay**, Nahum Sonenberg||, Eleanor N. Fish§, and Leonidas C. Platanias{ddagger}1

From the {ddagger}Robert H. Lurie Comprehensive Cancer Center and Division of Hematology-Oncology, Northwestern University Medical School and Lakeside Veterans Affairs Medical Center, Chicago, Illinois 60611, the §Division of Cell and Molecular Biology, Toronto Research Institute, University Health Network and Department of Immunology, University of Toronto, Toronto, Ontario M5G2M1, Canada, Biogen Idec Inc., Cambridge, Massachusetts 02142, the ||Department of Biochemistry, McGill University, Montreal, Quebec H3G1Y6, Canada, and the **Department of Molecular Genetics, University of Illinois, Chicago, Illinois 60607

Received for publication, August 3, 2006 , and in revised form, November 17, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms regulating initiation of mRNA translation for the generation of protein products that mediate interferon (IFN) responses are largely unknown. We have previously shown that both Type I and II IFNs engage the mammalian target of rapamycin (mTOR), resulting in downstream phosphorylation and deactivation of the translational repressor 4E-BP1 (eIF4E-binding protein 1). In the current study, we provide direct evidence that such regulation of 4E-BP1 by IFN{alpha} or IFN{gamma} results in sequential dissociation of 4E-BP1 from eukaryotic initiation factor-4E and subsequent formation of a functional complex between eukaryotic initiation factor-4E and eukaryotic initiation factor-4G, to allow initiation of mRNA translation. We also demonstrate that the induction of key IFN{alpha}- or IFN{gamma}-inducible proteins (ISG15 (interferon-stimulated gene 15) and CXCL10) that mediate IFN responses are enhanced in 4E-BP1 (4E-BP1-/-) knockout MEFs, as compared with wild-type 4E-BP1+/+ MEFs. On the other hand, IFN-dependent transcriptional regulation of the Isg15 and Cxcl10 genes is intact in the absence of 4E-BP1, as determined by real time reverse transcriptase-PCR assays and promoter assays for ISRE and GAS, establishing that 4E-BP1 plays a selective negative regulatory role in IFN-induced mRNA translation. Interestingly, the induction of expression of ISG15 and CXCL10 proteins by IFNs was also strongly enhanced in cells lacking expression of the tuberin (TSC2-/-) or hamartin (TSC1-/-) genes, consistent with the known negative regulatory effect of the TSC1-TSC2 complex on mTOR activation. In other work, we demonstrate that the induction of an IFN-dependent antiviral response is strongly enhanced in cells lacking expression of 4E-BP1 and TSC2, demonstrating that these elements of the IFN-activated mTOR pathway exhibit important regulatory effects in the generation of IFN responses. Taken altogether, our data suggest an important role for mTOR-dependent pathways in IFN signaling and identify 4E-BP1 and TSC1-TSC2 as key components in the generation of IFN-dependent biological responses.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Type I ({alpha}, beta, {delta}, {epsilon}, {kappa}, and {omega}) and II ({gamma}) interferons (IFNs)2 are pleiotropic cytokines that exhibit important antiviral, immunomodulatory, and growth inhibitory properties via engagement of widely expressed cell surface-specific receptors (1–5). The IFNs constitute the first line of the immune antiviral defense and are key components of the immune surveillance against tumors (1–5). In addition, because of their important biological properties, different recombinant IFNs ({alpha}, beta, and {gamma}) have been used extensively as therapeutic agents in a variety of clinical syndromes in humans, and certain IFN-subtypes are approved for the treatment of human malignancies, viral syndromes, and neurologic diseases (6–10).

Over the years, the mechanisms of IFN signal transduction have been the focus of attention by many research groups and have come under extensive investigation. Since the demonstration of the existence of STAT pathways that regulate transcription of interferon-inducible genes (2, 3), there has been gradual accumulation of evidence pointing toward a complicated network of multiple signaling pathways, whose coordinated function is necessary for the generation of IFN biological responses (4, 5). For instance, it has now become apparent that, in addition to different combinations of IFN-activated JAKs and STATs, the coordinated function of the phosphatidylinositol (PI) 3-kinase and the p38 mitogen-activated protein kinase pathways are essential for optimal IFN-dependent transcriptional regulation (5). The PI 3'-kinase pathway appears to be required for IFN-inducible transcriptional activation by regulating phosphorylation of STAT1 on serine 727 (11, 12), probably via intermediate engagement and activation of protein kinase C-{delta} (13–16). Activation of the p38 mitogen-activated protein kinase appears to be also necessary for Type I, but not II, IFN-dependent gene transcription, via a STAT-independent mechanism (17–20). Although the precise mechanisms by which this signaling cascade facilitates gene transcription remain unknown, there is strong evidence implicating this pathway in the generation of the growth-inhibitory properties of IFN{alpha} on normal and leukemic hematopoietic cells as well as in the induction of antiviral responses (19–22).

Despite the important conceptual advances in our understanding of the mechanisms of IFN transcriptional regulation over the last few years, much less is known about the means and pathways that regulate mRNA translation and ultimately protein expression of IFN-sensitive genes. It has been previously shown that Type I (23) and II (24) IFNs induce phosphorylation/activation of mTOR, in a PI 3'-kinase-dependent manner (23, 24). These original observations have suggested that the PI 3-kinase-mTOR pathway may be of importance in IFN signaling, since it results in downstream activation of the p70 S6 kinase and phosphorylation/deactivation of the translational repressor 4E-BP1 (eIF4E-binding protein 1) (23, 24). To directly address the functional relevance of this pathway in IFN signaling, we used cells from mice with targeted disruption of the 4e-bp1 and Tsc2 genes. Our data demonstrate that up-regulation of expression of key IFN-regulated proteins is enhanced in 4e-bp1 and Tsc2 knock-out cells, whereas there are no changes in IFN-dependent gene transcription, suggesting selective effects on the initiation of cap-dependent translation. Importantly, the generation of IFN-inducible antiviral responses is augmented in cells with targeted disruption of these genes, suggesting important functional roles for these proteins in IFN signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Reagents—Human recombinant IFN{alpha} was provided by Hoffman-La Roche. Recombinant mouse IFNbeta was provided by Biogen Inc. Recombinant mouse IFN{gamma} was from PBL Biomedical Laboratories (Piscataway, NJ), whereas human IFN{gamma} was obtained from InterMune, Inc. A rabbit polyclonal antibody against mouse ISG15 (25) was kindly provided by Dr. Dong-Er Zhang (Scripps Research Institute, La Jolla, CA). Anti-4E-BP1 and anti-eIF4E antibodies were from Cell Signaling (Beverly, MA). Anti-IP10 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) or Abcam (Cambridge, MA). Anti-eIF4G antibodies were purchased from Santa Cruz Biotechnology or Cell Signaling. An antibody against tubulin was from Abcam. The FRAP/mTOR inhibitor, rapamycin, and the PI 3'-kinase inhibitor LY294002 were obtained from Calbiochem. The cDNA cloned into the pcDNA3–4e-bp1 construct has been previously described (26). KT1 and NB4 cells were grown in RPMI supplemented with 10% fetal calf serum and antibiotics. U2OS cells were grown in McCoy medium, supplemented with 10% fetal calf serum and antibiotics. Immortalized mouse embryonic fibroblasts (MEFs) from 4E-BP1 knock-out mice (27) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics. Immortalized TSC2+/- and TSC2-/- MEFs (28, 29) and TSC1+/+ and TSC1-/- MEFs (30) were from Dr. Kwiatkowski and were grown in Dulbecco's modified Eagle's medium, 10% fetal calf serum, and antibiotics.

Cell Lysis Immunoprecipitations and Immunoblotting— Cells were treated with the indicated interferons for the indicated times and lysed in phosphorylation lysis buffer (PLB) supplemented with phenylmethylsulfonyl fluoride, aprotinin, and orthovanadate, as previously described (31). In the experiments to determine the effects of pharmacological inhibition of mTOR or PI 3'-kinase, rapamycin was used at final concentrations of 10–20 nM, and LY294002 was used at final concentrations of 10–50 µM. Immunoprecipitations and immunoblotting using an ECL method were performed as previously described (23, 24). For the co-immunoprecipitation experiments, an equal amount of protein from total cell lysates was incubated with a monoclonal eIF4E antibody (SC 9976), and the immune complexes were precipitated using BioMag beads. Alternately, the lysates were incubated with eIF4E antibody directly conjugated to agarose. The beads were washed three times with PLB, and protein bound to beads was extracted by boiling the beads in Laemmli sample buffer. Equal volumes of supernatants from the same experiment were loaded on 12.5 and/or 7% gels for analysis of 4E-BP1 and/or eIF4G, respectively.

Quantitative RT-PCR (TaqMan)—Cells were treated with 5000 IU/ml IFN{alpha} or 2500 IU/ml IFN{gamma} for 6 h, and RNA was isolated using the RNeasy kit (Qiagen). 1 µg of total cellular mRNA was reverse transcribed into cDNA using the Omniscript RT kit and oligo(dT) primer (Qiagen). Real time reverse transcriptase PCR for the Isg15 and Ip10 genes was carried out by an ABI7900 sequence detection system (Applied Biosystems) using commercially available FAM-labeled probes and primers (Applied Biosystems). Relative quantitation of mRNA levels was plotted as -fold increase as compared with untreated samples. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization (32). {Delta}Ct values (target gene Ct minus GAPDH Ct) for each triplicate sample were averaged, and {Delta}{Delta}Ct was calculated as previously described. mRNA amplification was determined by the formula 2-{Delta}{Delta}Ct (32).

Luciferase Reporter Assays—Luciferase reporter assays were performed as previously described (13, 17). Briefly, cells were transfected with a beta-galactosidase expression vector and either an ISRE luciferase construct or a luciferase reporter gene containing eight GAS elements linked to a minimal prolactin promoter (8x GAS), using Superfect transfection reagent as per the manufacturer's recommended procedure (Qiagen). The ISRE luciferase construct containing the wild type ISG15 ISRE (17) was from Dr. Richard Pine (Public Health Research Institute, New York). The 8x GAS construct was from Dr. Christopher Glass (University of California, San Diego) (33). 48 h after transfection, triplicate cultures were left untreated or treated with 5 x 103 units/ml of IFN{alpha} or 2500 units/ml of IFN{gamma}, and luciferase activity was measured in cell lysates.

Antiviral Assays—The antiviral effects of mouse IFN{alpha} on mouse embryonic fibroblasts were determined by assaying its activity against encephalomyocarditis virus (EMCV) infection as in our previous studies (19).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
It is well established that the translational repressor 4E-BP1 is inactivated in response to phosphorylation by a variety of stimuli (35–37). We have previously shown that different Type I IFNs induce phosphorylation of 4E-BP1 on threonines 37/46 and threonine 70 (23) and that such phosphorylation may result in its dissociation from eIF4E, as shown indirectly in m7GDP-agarose binding experiments (23). To directly examine the effects of IFN{alpha} on the association of eIF4E with eIF4G and 4E-BP1, we performed studies in the IFN-sensitive KT-1 cell line. KT-1 cells were left untreated or treated with human IFN{alpha}, and, after cell lysis, lysates were immunoprecipitated with an anti-eIF4E antibody. After IFN{alpha} treatment of the cells, there was an increase in the amounts of eIF4G associated with eIF4E, whereas such enhanced association was blocked by pretreatment of the cells with the mTOR inhibitor rapamycin or the PI 3'-kinase inhibitor LY294002 (Fig. 1A). On the other hand, the amount of 4E-BP1 bound to eIF4E decreased dramatically after treatment of cells with IFN{alpha} (Fig. 1B). Similar immunoprecipitation experiments were also carried out using IFN{gamma} instead of IFN{alpha}. After IFN{gamma} treatment of U2OS cells, there was an increase in the amount of eIF4G associated with eIF4E (Fig. 1D) and a corresponding decrease in the amounts of eIF4E-bound 4E-BP1 protein (Fig. 1E). Such a decrease in the eIF4E-bound 4E-BP1 was reversible by pretreatment of the cells with rapamycin or LY294002 (Fig. 1E), indicating that such an event is also PI 3-kinase-mTOR-dependent. It should be pointed out that we have previously shown that IFN{gamma} induces phosphorylation of 4E-BP1 in all sites required for its dissociation from 4E-BP1 (Thr37/46, Ser65, and Thr70) (24). The induction of IFN-dependent dissociation of 4E-BP1 from eIF4E and association of eIF4G with eIF4E was prolonged and could be detected in long time course experiments (Fig. 1, G–L). Altogether, these studies indicated that dissociation of 4E-BP1 from eIF4E during IFN{alpha} or IFN{gamma} treatment of sensitive cells results in an enhanced interaction of eIF4E with eIF4G in an mTOR-dependent manner and the formation of the complex that is known to regulate initiation of cap-dependent mRNA translation.


Figure 1
View larger version (46K):
[in this window]
[in a new window]

  		FIGURE 1.
IFN-dependent assembly of cap-dependent translation initiation complex. A–F, KT1 (A–C) or U2OS (D–F) cells were preincubated with rapamycin or LY294002 for 60–90 min, and they were subsequently treated with IFN{alpha} (A–C) or IFN{gamma} (D–F) for 30 min, as indicated. The cells were lysed in PLB, and equal amounts of cell lysates were immunoprecipitated (IP) with an anti-eIF4E monoclonal antibody. Immune complexes were resolved either in a 7% SDS-PAGE for analysis of eIF4G (A and D) or in a 12.5% SDS-PAGE for analysis of 4E-BP1 (B and E) and eIF4E (C and F). Immunoblotting with the indicated antibodies was performed for the detection of eIF4E, 4E-BP1-eIF4E, and eIF4G-eIF4E complexes. G and H, KT1 cells were preincubated with rapamycin or LY294002 for 90 min, and they were subsequently treated with IFN{alpha} for 24 h, as indicated, in the continuous presence or absence of the inhibitors. Equal amounts of cell lysates were immunoprecipitated with an anti-eIF4E monoclonal antibody. Immune complexes were resolved separately for analysis of eIF4G (G) or eIF4E (H). I and J, NB4 cells were preincubated with rapamycin or LY294002 for 90 min, and they were subsequently treated with IFN{gamma} for 24 h in the continuous absence or presence of the inhibitors, as indicated. The cells were then lysed in PLB, and equal amounts of cell lysates were immunoprecipitated with an anti-eIF4E monoclonal antibody. Immune complexes were resolved separately for analysis of eIF4G (I) or eIF4E (J). K and L, NB4 cells were preincubated with rapamycin or LY294002 for 90 min, and they were subsequently treated with IFN{gamma} for 24 h in the continuous absence or presence of the inhibitors, as indicated. The cells were then lysed in PLB, and equal amounts of cell lysates were immunoprecipitated with an anti-eIF4E monoclonal antibody. Immune complexes were analyzed on 12.5% gel for analysis of eIF4E-bound 4E-BP1 (K). The blot shown in K was stripped and probed with eIF4E antibody (L).

 


Figure 2
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 2.
Enhanced IFN{alpha}-inducible expression of ISG15 protein in the absence of 4E-BP1. A and B, 4E-BP1+/+ or 4E-BP1-/- MEFs were treated with mouse IFN{alpha} for the indicated times. Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-ISG15 antibody (A). The same blot was reprobed with an anti-tubulin antibody (B) to control for protein loading. C, the signals for ISG15 and tubulin from three independent experiments (including the one shown in A and B) were quantitated by densitometry, and the intensity of ISG15 expression relative to tubulin expression was calculated. Data are expressed as the mean of ratios of ISG15 to tubulin levels ± S.E. for each experimental condition. D and E, 4E-BP1+/+ or 4E-BP1-/- MEFs were treated with mouse IFNbeta for the indicated times. Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-ISG15 antibody (D), and the same blot shown in D was reprobed with an anti-tubulin antibody (E) to control for protein loading. F, the signals for ISG15 and tubulin from three independent experiments (including the one shown in D and E) were quantitated by densitometry, and the intensity of ISG15 expression relative to tubulin expression was calculated. Data are expressed as the mean of ratios of ISG15 to tubulin levels ± S.E. for each experimental condition. G, 4E-BP1-/- cells were nucleofected with a pCDNA3-4ebp1 construct or the pCDNA3 vector alone using the MEF2 nucleofector kit (Amaxa). 24 h after nucleofection, the cells were either left untreated or treated with mouse IFN{alpha} as indicated. Equal amounts of protein from total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-ISG15 antibody. H, the same blot from the experiment shown in G was reprobed with an anti-tubulin antibody, to control for protein loading. I, equal amounts of protein from the same experiment were analyzed separately by SDS-PAGE and immunoblotted with an anti-4E-BP1 antibody to document expression of 4E-BP1 in the nucleofected MEFs. J, the signals for ISG15 and tubulin or GAPDH from three independent experiments (including the experiment shown in G–I) were quantitated by densitometry, and the intensity of ISG15 expression relative to tubulin or GAPDH expression was calculated. The IFN{alpha} treatment times in the three independent experiments included were at 30 or 48 h. Data are expressed as the mean of ratios of ISG15 to tubulin or GAPDH levels ± S.E. for each experimental condition. K and L, 4E-BP1+/+ MEFs or 4E-BP1-/- MEFs nucleofected with a pCDNA3-4ebp1 construct or the pCDNA3 vector alone were either left untreated (UT) or treated with IFN{alpha} or IFN{gamma} for 30 h as indicated. Equal amounts of protein from total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-4E-BP1 antibody to examine the relative expression of 4E-BP1 (K). The same blot was reprobed with an anti-tubulin antibody (L) to control for protein loading. M and N, 4E-BP1-/- MEFs were nucleofected with a pCDNA3-4ebp1 construct or the pCDNA3 vector alone and were subsequently incubated in the presence or absence of IFN{alpha} for 90 min, as indicated. The cells were lysed in PLB, and equal amounts of cell lysates were immunoprecipitated (IP) with an anti-eIF4E monoclonal antibody. Immune complexes were resolved by SDS-PAGE and immunoblotted with an anti-4E-BP1 antibody (M). The blot shown in L was stripped and reprobed with an anti-eIF4E antibody (N). WT, wild type.

 
We subsequently sought to examine whether engagement of 4E-BP1 in IFN signaling regulates ultimate expression of protein products that participate in the generation of IFN responses. We determined the role of 4E-BP1 in the expression of ISG15, a Type I IFN-induced protein product that mediates IFN responses by regulating ISGylation (38, 39). In addition, we examined the role of 4E-BP1 in the expression of CXCL10, a Type II IFN-induced protein product/chemokine that participates in the generation of IFN{gamma}-dependent apoptosis (40). For this purpose, we used MEFs from mice with targeted disruption of the 4E-BP1 gene (27). 4E-BP1+/+ and 4E-BP1-/- MEFs were incubated in the presence or absence of mouse IFN{alpha}, and cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-ISG15 antibody. There was induction of expression of the ISG15 protein in both wild-type and 4E-BP1 knock-out cells, but the induction of expression of ISG15 was stronger in the 4E-BP1-/- MEFs as compared with the +/+ MEFs (Fig. 2, A–C). Similar enhancement of ISG15 expression in 4E-BP1-/- MEFs, as compared with the parental MEFs, was observed when another mouse Type I IFN, IFNbeta, was used in similar experiments (Fig. 2, D–F). On the other hand, in experiments in which 4E-BP1 was ectopically reexpressed using a pCDNA3-4e-bp1 construct, we found that such ectopic expression results in partial reversal of the IFN{alpha}-inducible expression of ISG15 (Fig. 2, G–J), further demonstrating that 4E-BP1 plays a negative regulatory role on the expression of ISG15 protein by IFN{alpha}. It should be pointed out that ectopically expressed 4E-BP1 protein in the knock-out MEFs associated with eIE4E (Fig. 2, K–N), indicating that the reversal of enhanced mRNA translation/protein expression seen in cells lacking 4E-BP1 results from direct inhibitory effects of the ectopically expressed protein on the formation of complexes that regulate cap-dependent translation.

Similar to what we observed in the case of ISG15 expression by IFN{alpha}, there was stronger IFN{gamma}-inducible expression of the CXCL10 protein in cells with targeted disruption of mouse 4E-BP1, as compared with parental MEFs (Fig. 3, A–C). On the other hand, ectopic reexpression of 4E-BP1 in 4E-BP1-/- MEFs resulted in a decrease in the expression of CXCL10 protein as compared with cells transfected with empty vector alone (Fig. 3, D–G), indicating a negative regulatory effect of 4E-BP1 on CXCL10 expression.

The known ability of 4E-BP1 to repress the function of eIF4E, taken together with our findings demonstrating enhanced ISG15 and CXCL10 expression in cells lacking 4E-BP1, strongly suggested that such enhanced expression reflects potentiation of mRNA translation for selected IFN-regulated genes. To exclude the possibility that such enhanced protein expression reflects indirect effects on IFN-dependent gene transcription, luciferase reporter assays were carried out in 4E-BP1+/+ and 4E-BP1-/- MEFs. As shown in Fig. 4, there was no enhancement of IFN{alpha}-inducible transcription via ISRE elements (Fig. 4A), nor was there significantly enhanced IFN{gamma}-transcription via GAS elements (Fig. 4B) in 4E-BP1 knock-out cells. Moreover, when the induction of transcription of the Isg15 and Cxcl10 genes was compared in 4E-BP1+/+ and 4E-BP1-/- MEFs using real time RT-PCR, similar levels of induction were observed (Fig. 4, C and D). Thus, although induction of ISG15 and CXCL10 protein expression by Type I and II IFNs is enhanced in the absence of 4E-BP1, targeted disruption of the 4e-bp1 gene does not alter IFN-inducible transcription, consistent with a selective effect on the initiation of IFN-dependent mRNA translation.

We subsequently sought to evaluate the functional relevance of 4E-BP1 in the generation of IFN responses. A universal property of all different Type I IFNs is their ability to generate antiviral responses in IFN-sensitive cells. We examined the antiviral properties of mouse IFN{alpha}4 against EMCV infection in 4E-BP1 knock-out MEFs as compared with parental wild-type MEFs. Interestingly, 4E-BP1-/- cells were more resistant to infection with EMCV, and much higher titers were required to achieve viral infectivity as compared with 4E-BP1+/+ cells (Fig. 5). Most importantly, despite the fact that much higher viral titers were used in such experiments in the 4E-BP1-/- cells (1:50 dilution) compared with wild-type MEFs (1:1000 dilution), the sensitivity of these cells to the antiviral effects of mouse IFN{alpha}4 was dramatically enhanced as compared with the 4E-BP1+/+ MEFs (Fig. 5). Thus, cells with targeted disruption of the 4e-bp1 gene exhibit enhanced sensitivity to the antiviral effects of IFN{alpha}, indicating that 4E-BP1 plays an important negative regulatory role in the induction of the biological effects of IFNs.


Figure 3
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 3.
Enhanced expression of the CXCL10 by IFN{gamma} in cells with targeted disruption of the 4e-bp1 gene. A and B, 4E-BP1+/+ or 4E-BP1-/- MEFs were either left untreated or treated with IFN{gamma} as indicated. The cells were lysed, and equal amounts of protein were resolved by SDS-PAGE and immunoblotted with an anti-CXCL10 (anti-IP10) antibody (A), and the same blot shown in A was reprobed with anti-tubulin antibody (B) to control for protein loading. C, the signals for CXCL10 and tubulin from three independent experiments (including the one shown in A and B) were quantitated by densitometry, and the intensity of IP10 expression relative to tubulin expression was calculated. The IFN{gamma} treatment times in the three independent experiments included were at 30 or 48 h. Data are expressed as the mean of ratios of IP10 to tubulin levels ± S.E. for each experimental condition. D, 4E-BP1-/- cells were nucleofected with the pCDNA3-4ebp1 construct or control pCDNA3 vector alone. 24 h after nucleofection, the cells were either left untreated or treated with IFN{gamma} as indicated. Equal amounts of protein from total cell lysates was resolved by SDS-PAGE and immunoblotted with an anti-CXCL10 (IP10) antibody. E, the same blot from the experiment shown in D was reprobed with an anti-tubulin antibody to control for protein loading. F, equal amounts of protein from the same experiment were analyzed separately by SDS-PAGE and immunoblotted with an anti-4E-BP1 antibody to document expression of 4E-BP1 in the nucleofected MEFs. G, the signals for IP10 and tubulin from two independent experiments (including the one shown in D and E) were quantitated by densitometry, and the intensity of IP10 expression relative to tubulin expression was calculated. Data are expressed as the mean of ratios of CXCL10 to tubulin levels ± S.E. for each experimental condition.

 
The Tsc2 gene product (tuberin) is a part of a protein complex that also includes the Tsc1 gene product (hamartin) and negatively regulates activation of mTOR (35, 41). Since our data indicated that knock-out of the downstream negative mTOR effector, 4E-BP1, results in enhanced IFN responses, we sought to determine whether TSC2, an upstream negative mTOR regulator, also controls the generation of IFN responses. We initially examined the induction of ISG15 expression in cells with targeted deletion of the Tsc2 gene (28), in response to mouse IFN{alpha} or IFNbeta. Treatment of TSC2+/- cells with mouse IFN{alpha} resulted in an increase in ISG15 protein expression (Fig. 6, A and B), but such an IFN{alpha}-inducible protein expression was further enhanced in TSC2-/- cells (Fig. 6, A–C). Similarly, IFNbeta treatment also resulted in enhanced expression of ISG15 in TSC2-/- cells as compared with TSC2+/- cells (Fig. 6, D–F). In other studies, we compared the induction of CXCL10 protein expression in response to IFN{gamma} in TSC2+/- and TSC2-/- MEFs. As shown in Fig. 6, G–I, the IFN{gamma}-inducible protein expression of CXCL10 was very intense in the TSC2-/- MEFs but very weak in the TSC2+/- MEFs. Similar results were obtained when TSC1-deficient MEFs were used (Fig. 7, A–F), consistent with the known negative regulatory effects of the TSC1-TSC2 complex on mTOR activation and downstream regulation of mRNA translation.

In subsequent studies, we used TSC2-/- MEFs in which the Tsc2 gene was stably reexpressed, using retroviral transduction (42). As shown in Fig. 8, ectopic reexpression of TSC2 in TSC2-/- MEFs reversed the enhanced ISG15 (Fig. 8, A–C) and CXCL10 (Fig. 8, D–F) expression in response to IFN{alpha} or IFN{gamma}, respectively. Thus, as in the case of the translational repressor 4E-BP1, the negative regulators of mTOR activation, TSC1 and TSC2, negatively control induction of protein expression in response to Type I and II IFNs.

To exclude the possibility that such differential regulation of protein expression does not reflect indirect effects on transcriptional regulation, the IFN-dependent gene transcription of the Isg15 or Cxcl10 genes was assessed in TSC2+/- and TSC2-/- MEFs. The absence of TSC2 expression did not have consistent and/or significant effects on the transcriptional activation of the Isg15 or Cxcl10 genes, determined by real time RT-PCR (Fig. 9, A and B), suggesting that the negative regulation of the expression of these proteins by TSC2 does not reflect suppressive effects on the transcription of their respective genes.

To examine the functional relevance of TSC2 in IFN signaling, we determined the induction of antiviral responses in TSC2-/- MEFs, as compared with TSC2+/- MEFs. Remarkably, as in the case of the 4E-BP1 knock-out MEFs, much higher titers of EMCV were necessary to elicit viral cytopathic effects in the TSC2 knock-out cells as compared with TSC2 heterozygous cells (Fig. 10). Moreover, as in the case of 4E-BP1-/- cells, TSC2-/- cells were more sensitive to the antiviral effects of IFN{alpha} (Fig. 10), consistent with an important negative regulatory role of TSC2 in the generation of IFN antiviral responses.


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 4.
IFN-inducible gene transcription is intact in the absence of 4E-BP1. A and B, 4E-BP1+/+ or 4E-BP1-/- MEFs were transfected with a beta-galactosidase expression vector and either ISRE (A) or GAS (B) luciferase plasmids. 48 h after transfection, triplicate cultures were either left untreated or treated with IFN{alpha} (A) or IFN{gamma} (B) for 6 h, and luciferase reporter assays were carried out. The data are expressed as relative luciferase units for each condition, normalized for beta-galactosidase activity. Data represent means ± S.E. values of three experiments for A and six experiments for B. C, 4E-BP1+/+ or 4E-BP1-/- MEFs were incubated for 6 h at 37°C in the absence or presence of mouse IFN{alpha}. Expression of mRNA for the Isg15 gene was evaluated by quantitative RT-PCR (TaqMan). GAPDH was used for normalization. Data are expressed as -fold increase over IFN{alpha}-untreated samples and represent mean ± S.E. of three experiments. D, 4E-BP1+/+ or 4E-BP1-/- MEFs were incubated for 6 h at 37°C in the absence or presence of mouse IFN{gamma}. Expression of mRNA for the Cxcl10 (Ip10) gene was evaluated by quantitative RT-PCR (TaqMan). GAPDH was used for normalization. Data are expressed as -fold increase over IFN{gamma}-untreated samples and represent mean ± S.E. of three experiments.

 


Figure 5
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 5.
Enhanced IFN{alpha}-dependent antiviral responses in cells with targeted disruption of the 4e-bp1 gene. 4E-BP1+/+ and 4E-BP1-/- MEFs were incubated, in triplicates, with the indicated doses of mouse IFN{alpha}. The cells were subsequently challenged with EMCV, and the direct cytopathic effect was quantified 24 h later. Data are expressed as the percentage of protection from the cytopathic effects of EMCV.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A major mechanism by which IFNs exert their effects is the up-regulation of expression of genes, whose products act as regulators and mediators of IFN responses (1–6). It is now clear that the key regulatory mechanisms for initiation of transcriptional regulation by IFNs involve engagement of the classic JAK-STAT pathways (2–5). In addition, there is evidence that pathways downstream of the p38 mitogen-activated protein kinase are also required for optimal gene transcription (5, 43). Although the initial events that mediate transcription of interferon-targeted genes have been clarified, the subsequent events that account for mRNA translation and protein expression of specific gene products induced by IFNs are not well defined. The ability of IFNs to block cell growth and inhibit viral replication is associated with a global negative effect on mRNA translation. In fact, it has been previously demonstrated in several studies that IFNs are capable of suppressing viral RNA translation of a variety of viruses (44–47) and that such effects contribute to the generation of antiviral responses. On the other hand, IFNs also induce mRNA translation and protein synthesis of selective interferon-sensitive genes that act as direct or indirect mediators of their pleiotropic biological effects, but the knowledge of the mechanisms that regulate such events has been very limited so far.


Figure 6
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 6.
TSC2 negatively regulates the induction of ISG15 and CXCL10 protein expression by interferons. A, TSC2+/- and TSC2-/- MEFs were treated with mouse IFN{alpha} for the indicated times. Cell lysates were resolved by SDS-PAGE and immunoblotted with antibody against ISG15. B, the same blot shown in A was reprobed with an anti-tubulin antibody to control for protein loading. C, the signals for ISG15 and tubulin or GAPDH from three independent experiments (including the one shown in A and B) were quantitated by densitometry, and the intensity of ISG15 expression relative to tubulin or GAPDH expression was calculated. Data are expressed as the mean of ratios of ISG15 to tubulin or GAPDH levels ± S.E. for each experimental condition. D, TSC2+/- and TSC2-/- MEFs were treated with mouse IFNbeta for the indicated times. Cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-ISG15 antibody. E, the same blot shown in D was reprobed with an anti-tubulin antibody to control for protein loading. F, the signals for ISG15 and tubulin from two independent experiments (including the one shown in D and E) were quantitated by densitometry, and the intensity of ISG15 expression relative to tubulin expression was calculated. The IFNbeta treatment times in the two independent experiments included were 24 and 48 h. Data are expressed as the mean of ratios of ISG15 to tubulin levels ± S.E. for each experimental condition. G, TSC2+/- and TSC2-/- MEFs were treated with mouse IFN{gamma} as indicated. Cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-CXLC10 (IP10) antibody. H, the same blot shown in G was reprobed with an anti-tubulin antibody to control for protein loading. I, the signals for IP10 and tubulin from three independent experiments were quantitated by densitometry, and the intensity of IP10 expression relative to tubulin expression was calculated. The IFN{gamma} treatment times in the three independent experiments included were 24–30 h. Data are expressed as the mean of ratios of IP10 to tubulin levels ± S.E. for each experimental condition.

 


Figure 7
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 7.
TSC1 negatively regulates the induction of ISG15 and IP10 protein expression by interferons. A, TSC1+/+ and TSC1-/- MEFs were treated with mouse IFN{alpha} for the indicated times. Cell lysates were resolved by SDS-PAGE and immunoblotted with antibody against ISG15. B, the same blot shown in A was reprobed with an anti-tubulin antibody to control for protein loading. C, the signals for ISG15 and tubulin from two independent experiments (including the one shown in A and B) were quantitated by densitometry, and the intensity of ISG15 expression relative to tubulin expression was calculated. Data are expressed as the mean of ratios of ISG15 to tubulin levels ± S.E. for each experimental condition. D, TSC1+/+ and TSC1-/- MEFs were treated with mouse IFN{gamma} for the indicated times. Cell lysates were resolved by SDS-PAGE and immunoblotted with antibody against IP10. E, the same blot shown in D was reprobed with an anti-tubulin antibody to control for protein loading. F, the signals for IP10 and tubulin from two independent experiments (including the one shown in D and E) were quantitated by densitometry, and the intensity of IP10 expression relative to tubulin expression was calculated. Data are expressed as means of ratios of IP10 to tubulin levels ± S.E. for each experimental condition.

 


Figure 8
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 8.
Reconstitution of TSC2 in TSC2-/- MEFs reverses the enhanced induction of ISG15 and IP10 protein expression by IFNs. A, TSC2+/- MEFs, TSC2-/- MEFs, or TSC2-/- MEFs stably transfected with Tsc2 or control empty vector were treated with mouse IFN{alpha} for the indicated times. Cell lysates were resolved by SDS-PAGE and immunoblotted with antibody against ISG15. B, the blot shown in A was reprobed with an anti-tubulin antibody to control for protein loading. C, the signals for ISG15 and tubulin from two independent experiments (including the one shown in A and B) were quantitated by densitometry, and the intensity of ISG15 expression relative to tubulin expression was calculated. Data are expressed as the mean of ratios of ISG15 to tubulin levels ± S.E. for each experimental condition. D, TSC2+/- MEFs, TSC2-/- MEFs, or TSC2-/- MEFs stably transfected with Tsc2 or control empty vector were treated with mouse IFN{gamma} for the indicated times. Cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-IP10 antibody. E, the blot shown in D was reprobed with an anti-tubulin antibody to control for protein loading. F, the signals for IP10 and tubulin from two independent experiments (including the one shown in D and E) were quantitated by densitometry, and the intensity of IP10 expression relative to tubulin expression was calculated. Data are expressed as the mean of ratios of IP10 to tubulin levels ± S.E. for each experimental condition.

 
In previous work, we demonstrated that Type I (23, 48) and II (24) IFNs are capable of engaging the mTOR/PI 3-kinase cascade in different cell types and have identified p70 S6K/rpS6 and 4E-BP1 as putative downstream effectors of this signaling pathway (23, 24). These findings have suggested a potential mechanism by which IFNs regulate translational control of IFN-specific genes, but the precise functional relevance of mTOR and downstream pathways in IFN signaling had not been defined. A recent study has suggested that mTOR signals may play positive roles in the generation of IFN biological responses, as evidenced by the inhibition of IFN{alpha}-induced apoptosis in cells in which a kinase-deficient mTOR mutant was expressed or by treatment of cells with the mTOR inhibitor rapamycin (49). However, the functional relevance of specific effectors downstream of mTOR that may be involved in the generation of IFN responses has remained unknown to date.


Figure 9
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 9.
Deletion of the Tsc2 gene does not affect IFN-dependent gene transcription. A, TSC2+/- or TSC2-/- MEFs were incubated for 6 h at 37 °C in the absence or presence of mouse IFN{alpha}. Expression of mRNA for the Isg15 gene was evaluated by quantitative RT-PCR (TaqMan). GAPDH was used for normalization. Data are expressed as -fold increase over IFN{alpha}-untreated samples and represent mean ± S.E. of three experiments. B, TSC2+/- or TSC2-/- cells were incubated for 6 h at 37°C in the absence or presence of mouse IFN{gamma}. Expression of mRNA for the Cxcl10 (Ip10) gene was evaluated by quantitative RT-PCR (TaqMan). GAPDH was used for normalization. Data are expressed as -fold increase over IFN{gamma}-untreated samples and represent mean ± S.E. of five experiments.

 
In the present study, we used cells from mice with targeted disruption of the 4e-bp1 gene to evaluate generation of IFN responses in that context (27). We found that the Type I or II IFN-inducible expression of two ISG products, ISG15 and CXCL10, known to participate in the generation of the biological effects of IFNs, is enhanced in the absence of 4E-BP1. On the other hand, there was no enhancement of Type I or II IFN-dependent gene transcription via ISRE or GAS elements in cells with targeted disruption of the 4e-bp1 gene, indicating that the enhanced protein expression seen in such cells reflects selective effects on post-transcriptional/translational control of these ISGs. Such control apparently results from enhanced capdependent translation, as shown by our finding that IFN-dependent phosphorylation/inactivation of 4E-BP1 and dissociation of 4E-BP1 from eIF4E result in the formation of the eIF4E-eIF4G translation initiation complex. Importantly, the induction of IFN-dependent antiviral effects was strongly enhanced in 4E-BP1 knock-out MEFs, demonstrating that the 4E-BP1 protein plays an important regulatory role in the generation of IFN responses. Similar studies were performed to examine the role of TSC2 (tuberin), a negative regulator of mTOR activation (19, 40), in IFN-dependent translational regulation of ISG-protein products. Similar to what we observed in 4E-BP1-/- MEFs, we found enhanced IFN-inducible expression of the ISG15 and CXCL10 proteins and antiviral responses in TSC2 knock-out cells. Thus, 4E-BP1-/- and TSC2-/- cells appear to have the same phenotype in terms of induction of IFN responses, a finding consistent with their negative regulatory properties upon cap-dependent translation upstream (TSC2) or downstream (4E-BP1) of mTOR.


Figure 10
View larger version (15K):
[in this window]
[in a new window]

  		FIGURE 10.
Enhanced IFN{alpha}-dependent antiviral responses in the absence of the Tsc2 gene. TSC2+/- and TSC2-/- MEFs were incubated, in triplicates, with the indicated doses of mouse IFN{alpha}. The cells were subsequently challenged with EMCV, and the direct cytopathic effect was quantified 24 h later. Data are expressed as the percentage of protection from the cytopathic effects of EMCV.

 
Altogether, our data provide direct evidence for a link between IFN-activated JAK-STAT pathways and the IFN-inducible mTOR pathway. Although these pathways are activated independently of each other and mTOR inhibition does not affect the formation of STAT complexes and gene transcription (23, 24), it appears that they cooperate in the control of common biological responses via regulation of expression of common protein mediators. For instance, it is well established that the promoter of the Isg15 gene contains three ISRE elements and that the STAT pathway regulates its transcriptional activation (50). In fact, the original definition of ISREs was based on their identification and description of their presence in the Isg15 gene promoter (50, 51). Our findings now provide direct evidence that translational control of the Isg15 gene product is regulated by cap-dependent translation, as evidenced by the enhanced up-regulation of IFN{alpha}-dependent ISG15 protein expression in cells with targeted disruption of the 4e-bp1 gene. Consistent with this observation, similar up-regulation is also observed in TSC2 knock-out cells, firmly establishing that negative effectors upstream or downstream of mTOR abrogate IFN-dependent induction of ISG15, whose transcriptional activation is regulated by the classical JAK-STAT pathway. A similar regulatory mechanism appears to exist in the Type II (IFN{gamma}) system, as evidenced by the coordinated function of the mTOR and STAT pathways in the regulation of expression of the Cxcl10 gene, which contains GAS elements in its promoter.

Altogether, our findings address outstanding important issues regarding the generation of IFN-induced protein products via the engagement of the JAK-STAT pathway, by definitively defining the requirement of mTOR-generated signals in the process. At the same time, they raise new questions relating to the overall cellular function of mTOR and its downstream effectors. It is generally established and accepted that activation of the mTOR pathway mediates progrowth and survival signals (35, 36, 41). Moreover, mTOR has been implicated in the pathophysiology of several malignancies, and its pharmacological targeting is currently under extensive investigation for the treatment of various cancers (34, 41, 52–56). The fact that the same pathway can selectively mediate signals for the biological effects of IFNs, cytokines, with antitumor and antiviral properties suggests the existence of an IFN-regulated specific mechanism that transiently diverts mTOR and its effectors from the transmission of mitogenic signals and redirects cap-dependent translation to proteins that exhibit opposing biological effects. Future efforts to identify such mechanisms and define IFN-specific regulators of the mTOR pathway should lead to a better understanding of the mechanisms of IFN-cellular signaling and, possibly, to the identification of novel new pharmacological targets for the treatment of viral syndromes and malignancies.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants CA77816, CA100579, and CA94079 (to L. C. P.), by a grant from the Department of Veterans Affairs (to L. C. P.), and Canadian Institutes of Health Research Grant MOP15094 (to E. N. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Robert H. Lurie Comprehensive Cancer Center, 303 E. Superior St., Lurie 3-107, Chicago, IL 60611. Tel.: 312-503-4267; Fax: 312-908-1372; E-mail: l-platanias{at}northwestern.edu.

2 The abbreviations used are: IFN, interferon; STAT, signal transducer and activator of transcription; ISRE, interferon-stimulated response element; GAS, IFN{gamma}-activated site; PI, phosphatidylinositol; mTOR, mammalian target of rapamycin; eIF4E, eukaryotic initiation factor-4E; eIF4G, eukaryotic initiation factor-4G; JAK, Janus kinase; MEF, mouse embryonic fibroblast; PLB, phosphorylation lysis buffer; EMCV, encephalomyocarditis virus; RT, reverse transcriptase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pestka, S., Langer, J. A., Zoon, K. C., and Samuel, C. E. (1987) Annu. Rev. Biochem. 56, 727-777[CrossRef][Medline] [Order article via Infotrieve]
  2. Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H., and Schreiber, R. D. (1998) Annu. Rev. Biochem. 67, 227-264[CrossRef][Medline] [Order article via Infotrieve]
  3. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415-1420[Abstract/Free Full Text]
  4. Platanias, L. C., and Fish, E. N. (1999) Exp. Hematol. 27, 1583-1592[CrossRef][Medline] [Order article via Infotrieve]
  5. Platanias, L. C. (2005) Nat. Rev. Immunol. 5, 375-386[CrossRef][Medline] [Order article via Infotrieve]
  6. Parmar, S., and Platanias, L. C. (2003) Curr. Opin. Oncol. 15, 431-439[CrossRef][Medline] [Order article via Infotrieve]
  7. Asselah, T., Ripault, M. P., Castelnau, C., Giuily, N., Boyer, N., and Marcellin, P. (2005) J. Clin. Virol. 34, S115-S124
  8. Derwenskus, J., and Lublin, F. D. (2006) Adv. Neurol. 98, 257-271[Medline] [Order article via Infotrieve]
  9. Javed, A., and Reder, A. T. (2006) Pharmacol Ther. 110, 35-56[CrossRef][Medline] [Order article via Infotrieve]
  10. Butterfield, J. H. (2005) Acta Haematol. 114, 26-40[CrossRef][Medline] [Order article via Infotrieve]
  11. Nguyen, H., Ramana, C. V., Bayes, J., and Stark, G. R. (2001) J. Biol. Chem. 276, 33361-33368[Abstract/Free Full Text]
  12. Kaur, S., Uddin, S., and Platanias, L. C. (2005) J. Interferon Cytokine Res. 25, 780-787[CrossRef][Medline] [Order article via Infotrieve]
  13. Uddin, S., Sassano, A., Deb, D. K., Verma, A., Majchrzak, B., Rahman, A., Malik, A. B., Fish, E. N., and Platanias, L. C. (2002) J. Biol. Chem. 277, 14408-14416[Abstract/Free Full Text]
  14. Deb, D. K., Sassano, A., Lekmine, F., Majchrzak, B., Verma, A., Kambhampati, S., Uddin, S., Rahman, A., Fish, E. N., and Platanias, L. C. (2003) J. Immunol. 171, 267-273[Abstract/Free Full Text]
  15. DeVries, T. A., Rachelle, L., Kalkofen, A., Matassa, A., and Reyland, M. E. (2004) J. Biol. Chem. 279, 45603-45612[Abstract/Free Full Text]
  16. Zhao, K. W., Li, D., Zhao, Q., Huang, Y., Silverman, R. H., Sims, P. J., and Chen, G. Q. (2005) J. Biol. Chem. 280, 42707-42714[Abstract/Free Full Text]
  17. Uddin, S., Majchrzak, B., Woodson, J., Arunkumar, P., Alsayed, Y., Pine, R., Young, P. R., Fish, E. N., and Platanias, L. C. (1999) J. Biol. Chem. 274, 30127-30131[Abstract/Free Full Text]
  18. Uddin, S., Lekmine, F., Sharma, N., Majchrzak, B., Mayer, I., Young, P. R., Bokoch, G. M., Fish, E. N., and Platanias, L. C. (2000) J. Biol. Chem. 275, 27634-27640[Abstract/Free Full Text]
  19. Li, Y., Sassano, A., Majchrzak, B., Deb, D. K., Levy, D. E., Gaestel, M., Nebreda, A. R., Fish, E. N., and Platanias, L. C. (2004) J. Biol. Chem. 279, 970-979[Abstract/Free Full Text]
  20. Katsoulidis, E., Li, Y., Mears, H., and Platanias, L. C. (2005) J. Interferon Cytokine Res. 25, 749-756[CrossRef][Medline] [Order article via Infotrieve]
  21. Verma, A., Deb, D. K., Sassano, A., Uddin, S., Varga, J., Wickrema, A., and Platanias, L. C. (2002) J. Biol. Chem. 277, 7726-7735[Abstract/Free Full Text]
  22. Mayer, I. A., Verma, A., Grumbach, I. M., Uddin, S., Lekmine, F., Ravandi, F., Majchrzak, B., Fujita, S., Fish, E. N., and Platanias, L. C. (2001) J. Biol. Chem. 276, 28570-28577[Abstract/Free Full Text]
  23. Lekmine, F., Uddin, S., Sassano, A., Parmar, S., Brachmann, S. M., Majchrzak, B., Sonenberg, N., Hay, N., Fish, E. N., and Platanias, L. C. (2003) J. Biol. Chem. 278, 27772-27780[Abstract/Free Full Text]
  24. Lekmine, F., Sassano, A., Uddin, S., Smith, J., Majchrzak, B., Brachmann, S. M., Hay, N., Fish, E. N., and Platanias, L. C. (2004) Exp. Cell Res. 295, 173-182[CrossRef][Medline] [Order article via Infotrieve]
  25. Malakhova, O. A., Yan, M., Malakhov, M. P., Yuan, Y., Ritchie, K. J., Kim, K. I., Peterson, L. F., Shuai, K., and Zhang, D. E. (2003) Genes Dev. 17, 455-460[Abstract/Free Full Text]
  26. Andersen, J. B., Aaboe, M., Borden, E. C., Goloubeva, O. G., Hassel, B. A., and Orntoft, T. F., (2006) Br. J. Cancer 94, 1465-1471[CrossRef][Medline] [Order article via Infotrieve]
  27. Tsukiyama-Kohara, K., Poulin, F., Kohara, M., DeMaria, C. T., Cheng, A., Wu, Z., Gingras, A. C., Katsume, A., Elchebly, M., Spiegelman, B. M., Harper, M. E., Tremblay, M. L., and Sonenberg, N. (2001) Nat. Med. 7, 1128-1132[CrossRef][Medline] [Order article via Infotrieve]
  28. Zhang, H., Cicchetti, G., Onda, H., Koon, H. B., Asrican, K., Bajraszewski, N., Vazquez, F., Carpenter, C. L., and Kwiatkowski, D. J. (2003) J. Clin. Invest. 112, 1223-1233[CrossRef][Medline] [Order article via Infotrieve]
  29. Hahn-Windgassen, A., Nogueira, V., Chen, C. C., Skeen, J. E., Sonenberg, N., and Hay, N. (2005) J. Biol. Chem. 280, 32081-32089[Abstract/Free Full Text]
  30. Kwiatkowski, D. J., Zhang, H., Bandura, J. L., Heiberger, K. M., Glogauer, M., el-Hashemite, N., and Onda, H. (2002) Hum. Mol. Genet. 11, 525-534[Abstract/Free Full Text]
  31. Kaur, S., Parmar, S., Smith, J., Katsoulidis, E., Li, Y., Sassano, A., Majchrzak, B., Uddin, S., Tallman, M. S., Fish, E. N., and Platanias, L. C. (2005) Exp. Hematol. 33, 550-557[CrossRef][Medline] [Order article via Infotrieve]
  32. Livak, K. J., and Schmittgen, T. D. (2001) Methods 25, 402-408[CrossRef][Medline] [Order article via Infotrieve]
  33. Horvai, A. E., Xu, L., Korzus, E., Brard, G., Kalafus, D., Mullen, T.-M., Rose, D. W., Rosenfeld, M. G., and Glass, C. K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1074-1079[Abstract/Free Full Text]
  34. Sawyers, C. L. (2003) Cancer Cell 4, 343-348[CrossRef][Medline] [Order article via Infotrieve]
  35. Hay, N., and Sonenberg, N. (2004) Genes Dev. 18, 1926-1945[Abstract/Free Full Text]
  36. Holcik, M., and Sonenberg, N. (2005) Nat. Rev. Mol. Cell. Biol. 6, 318-327[CrossRef][Medline] [Order article via Infotrieve]
  37. Richter, J. D., and Sonenberg, N. (2005) Nature 433, 477-480[CrossRef][Medline] [Order article via Infotrieve]
  38. Ritchie, K. J., Hahn, C. S., Kim, K. I., Yan, M., Rosario, D., Li, L., De La Torre, J. C., and Zhang, D. E. (2004) Nat. Med. 10, 1374-1378[CrossRef][Medline] [Order article via Infotrieve]
  39. Ritchie, K. J., and Zhang, D. E. (2004) Semin. Cell Dev. Biol. 15, 237-246[CrossRef][Medline] [Order article via Infotrieve]
  40. Zhang, H. M., Yuan, J., Cheung, P., Chau, D., Wong, B. W., McManus, B. M., and Yang, D. (2005) Mol. Cell. Biol. 25, 6247-6258[Abstract/Free Full Text]
  41. Hay, N. (2005) Cancer Cell 8, 179-183[CrossRef][Medline] [Order article via Infotrieve]
  42. Skeen, J. E., Bhaskar, P. T., Chen, C. C., Chen, W. S., Peng, X. D., Nogueira, V., Hahn-Windgassen, A., Kiyokawa, H., and Hay, N. (2006) Cancer Cell 10, 269-280[CrossRef][Medline] [Order article via Infotrieve]
  43. Platanias, L. C. (2003) Pharmacol. Ther. 98, 129-142[CrossRef][Medline] [Order article via Infotrieve]
  44. Guo, J., Hui, D. J., Merrick, W. C., and Sen, G. C. (2000) EMBO J. 19, 6891-6899[CrossRef][Medline] [Order article via Infotrieve]
  45. Friedman, R. M., Metz, D. H., Esteban, R. M., Tovell, D. R., Ball, L. A., and Kerr, I. M. (1972) J. Virol. 10, 1184-1198[Abstract/Free Full Text]
  46. Samuel, C. E. (1991) Virology 183, 1-11[CrossRef][Medline] [Order article via Infotrieve]
  47. Wang, C., Pflugheber, J., Sumpter, R., Jr., Sodora, D. L., Hui, D., Sen, G. C., and Gale, M., Jr. (2003) J. Virol. 77, 3898-3912[Abstract/Free Full Text]
  48. Parmar, S., Smith, J., Sassano, A., Uddin, S., Katsoulidis, E., Majchrzak, B., Kambhampati, S., Eklund, E. A., Tallman, M. S., Fish, E. N., and Platanias, L. C. (2005) Blood 106, 2436-2443[Abstract/Free Full Text]
  49. Thyrell, L., Hjortsberg, L., Arulampalam, V., Panaretakis, T., Uhles, S., Dagnell, M., Zhivotovsky, B., Leibiger, I., Grander, D., and Pokrovskaja, K. (2004) J. Biol. Chem. 279, 24152-24162[Abstract/Free Full Text]
  50. Martensen, P. M., and Justesen, J. (2004) J. Interferon Cytokine Res. 24, 1-19[CrossRef][Medline] [Order article via Infotrieve]
  51. Reich, N., Evans, B., Levy, D., Fahey, D., Knight, E., Jr., and Darnell, J. E., Jr. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6394-6398[Abstract/Free Full Text]
  52. Inoki, K., Corradetti, M. N., and Guan, K. L. (2005) Nat. Genet. 37, 19-24[CrossRef][Medline] [Order article via Infotrieve]
  53. Astrinidis, A., and Henske, E. P. (2005) Oncogene 24, 7475-7481[CrossRef][Medline] [Order article via Infotrieve]