Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro
 and in vivo

Abstract Abstract 2908 Introduction: The eukaryotic initiation factor 4E (eIF4E) is a critical regulator in protein synthesis. It has been shown that overexpression and/or activation of eIF4E is critical for oncogenic protein synthesis. The precise role of protein translation in multiple myeloma (MM) is less clear. Recently it has been shown that eIF4E protein levels are higher in primary CD138+ MM cells than in normal plasma cells (Li, Jin et al. 2011) and that mutations in genes related to mRNA translation are involved in the pathogenesis of multiple (Chapman, Lawrence et al. 2011). Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. In this study, we analyzed the effects of introduction of ectopic eIF4E in MM cell lines compared with their parent cells in vitro and in vivo. Results: To examine the effect of overexpressed eIF4E in MM, we transduced MM cell lines with lentiviral particles encoding human eIF4E with GFP as selection marker. Introduction of ectopic eIF4E significantly increased critical factors for myeloma cell growth such as myc, cyclin D1, C/EBP beta and IRF4 as detected by western blotting. Overexpression of eIF4E resulted in a significant (p<0.001) increase of DNA synthesis compared to empty vector control (EV) cells. Cell cycle analysis revealed a decreasing number of cells in G0/G1 phase (62% vs 49%) and cells arresting in the G2/M phase (14% vs 23%), not affecting cell apoptosis. Overexpression of eIF4E further led to the significant increase (p=0.004) of clonogenic tumor growth with expansion of clonogenic colony numbers (22.3 ± 2.5 vs 40.3 ± 2.1) and size. To determine whether overexpressed eIF4E also affects MM tumor growth in vivo, we generated subcutaneous MM xenografts in severe combined immunodeficient x beige (SCID/bg) mice using the EV and eIF4E-OE-U266 cells. In contrast to EV-U266 tumors, animals bearing eIF4E-OE-U266 xenografts showed a significant increase (p<0.001) of tumor growth by 180% after 13 days. Conclusion: Here we show that eIF4E, a key player in translational machinery, promotes multiple myeloma cell growth both in vitro and in vivo. When eIF4E is overexpressed, it enhances protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation. Disclosures: Lentzsch: Celgene: Consultancy, Research Funding.

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Evidence that the dephosphorylation of Ser535 in the ∊-subunit of eukaryotic initiation factor (eIF) 2B is insufficient for the activation of eIF2B by insulin

Biochemical Journal ◽

10.1042/bj20020677 ◽

2002 ◽

Vol 367 (2) ◽

pp. 475-481 ◽

Cited By ~ 34

Author(s):

Xuemin WANG ◽

Maarten JANMAAT ◽

Anne BEUGNET ◽

Fiona E.M. PAULIN ◽

Christopher G. PROUD

Keyword(s):

Protein Synthesis ◽

Glycogen Synthase ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Nucleotide Exchange ◽

Ovary Cells

Eukaryotic initiation factor (eIF) 2B is a guanine-nucleotide exchange factor that plays a key role in the regulation of protein synthesis. It is activated by insulin, serum and other agents that stimulate general protein synthesis. The largest (∊) subunit of eIF2B is a substrate for glycogen synthase kinase (GSK)-3 in vitro, and phosphorylation by GSK3 inhibits the activity of eIF2B. The site of phosphorylation has previously been identified as Ser535. GSK3 is inactivated by phosphorylation in response to insulin or serum. In Chinese-hamster ovary cells, insulin and serum bring about the dephosphorylation of Ser535in vivo, concomitantly with the phosphorylation of GSK3, and these effects are mediated through signalling via phosphoinositide 3-kinase. We have made use of inhibitors of GSK3 to determine whether GSK3 is responsible for phosphorylation of Ser535in vivo and to explore the role of phosphorylation of Ser535 in the regulation of eIF2B. Treatment of cells with LiCl or with either of two recently developed GSK3 inhibitors, SB-415286 and SB-216763, brought about the dephosphorylation of Ser535, which strongly indicates that this site is indeed a target for GSK3 in vivo. However, these compounds did not elicit significant activation of eIF2B, indicating, consistent with conclusions from one of our previous studies, that additional inputs are required for the activation of eIF2B. Our results also show that each of the inhibitors used affects overall protein synthesis and have additional effects on translation factors or signalling pathways apparently unrelated to their effects on GSK3, indicating that caution must be exercised when interpreting data obtained using these compounds.

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Adenovirus-mediated eukaryotic initiation factor 4E binding protein-1 in combination with rapamycin inhibits tumor growth of pancreatic ductal adenocarcinoma in vivo

International Journal of Oncology ◽

10.3892/ijo_00000251 ◽

2009 ◽

Author(s):

Mishra

Keyword(s):

Tumor Growth ◽

Pancreatic Ductal Adenocarcinoma ◽

Binding Protein ◽

Initiation Factor ◽

Ductal Adenocarcinoma ◽

Eukaryotic Initiation Factor ◽

Eukaryotic Initiation Factor 4E

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Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4546-4553.1994 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4546-4553

Author(s):

K V Ramaiah ◽

M V Davies ◽

J J Chen ◽

R J Kaufman

Keyword(s):

Protein Synthesis ◽

Initiation Factor ◽

Guanine Nucleotide ◽

Eukaryotic Initiation Factor ◽

Nucleotide Exchange ◽

Wild Type ◽

Guanine Nucleotide Exchange ◽

Initiation Factor 2 ◽

Exchange Activity

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2 alpha phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2 alpha 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2 alpha impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2 alpha and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2 alpha inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.

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Regulation of eukaryotic initiation factor-2B activity in muscle of diabetic rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1993.264.1.e101 ◽

1993 ◽

Vol 264 (1) ◽

pp. E101-E108 ◽

Cited By ~ 20

Author(s):

A. M. Karinch ◽

S. R. Kimball ◽

T. C. Vary ◽

L. S. Jefferson

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Cardiac Muscle ◽

Casein Kinase Ii ◽

Diabetic Rats ◽

Casein Kinase ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Fast Twitch

Peptide-chain initiation is inhibited in fast-twitch skeletal muscle, but not heart, of diabetic rats. We have investigated mechanisms that might maintain eukaryotic initiation factor (eIF)-2B activity, preventing loss of efficiency of protein synthesis in heart of diabetic rats but not in fast-twitch skeletal muscle. There was no change in the amount or phosphorylation state of eIF-2 in skeletal or cardiac muscle during diabetes. In contrast, eIF-2B activity was decreased in fast-twitch but not slow-twitch muscle from diabetic animals. NADP+ inhibited partially purified eIF-2B in vitro, but addition of equimolar NADPH reversed the inhibition. The NADPH-to-NADP+ ratio was unchanged in fast-twitch muscle after induction of diabetes but was increased in heart of diabetic rats, suggesting that NADPH also prevents inhibition of eIF-2B in vivo. The activity of casein kinase II, which can phosphorylate and activate eIF-2B in vitro, was significantly lower in extracts of fast-twitch, but not cardiac muscle, of diabetic rats compared with controls. The results presented here demonstrate that changes in eIF-2 alpha phosphorylation are not responsible for the effect of diabetes on eIF-2B activity in fast-twitch skeletal muscle. Modulation of casein kinase II activity may be a factor in the regulation of protein synthesis in muscle during acute diabetes. The activity of eIF-2B in heart might be maintained by the increased NADPH/NADP+.

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Cap-binding protein (eukaryotic initiation factor 4E) and 4E-inactivating protein BP-1 independently regulate cap-dependent translation.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5450 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5450-5457 ◽

Cited By ~ 47

Author(s):

D Feigenblum ◽

R J Schneider

Keyword(s):

Heat Shock ◽

Translation Initiation ◽

Binding Protein ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Metaphase Arrest ◽

Animal Cells ◽

Eukaryotic Initiation Factor 4E ◽

Dependent Protein

Cap-dependent protein synthesis in animal cells is inhibited by heat shock, serum deprivation, metaphase arrest, and infection with certain viruses such as adenovirus (Ad). At a mechanistic level, translation of capped mRNAs is inhibited by dephosphorylation of eukaryotic initiation factor 4E (eIF-4E) (cap-binding protein) and its physical sequestration with the translation repressor protein BP-1 (PHAS-I). Dephosphorylation of BP-I blocks cap-dependent translation by promoting sequestration of eIF-4E. Here we show that heat shock inhibits translation of capped mRNAs by simultaneously inducing dephosphorylation of eIF-4E and BP-1, suggesting that cells might coordinately regulate translation of capped mRNAs by impairing both the activity and the availability of eIF-4E. Like heat shock, late Ad infection is shown to induce dephosphorylation of eIF-4E. However, in contrast to heat shock, Ad also induces phosphorylation of BP-1 and release of eIF-4E. BP-1 and eIF-4E can therefore act on cap-dependent translation in either a mutually antagonistic or cooperative manner. Three sets of experiments further underscore this point: (i) rapamycin is shown to block phosphorylation of BP-1 without inhibiting dephosphorylation of eIF-4E induced by heat shock or Ad infection, (ii) eIF-4E is efficiently dephosphorylated during heat shock or Ad infection regardless of whether it is in a complex with BP-1, and (iii) BP-1 is associated with eIF-4E in vivo regardless of the state of eIF-4E phosphorylation. These and other studies establish that inhibition of cap-dependent translation does not obligatorily involve sequestration of eIF-4E by BP-1. Rather, translation is independently regulated by the phosphorylation states of eIF-4E and the 4E-binding protein, BP-1. In addition, these results demonstrate that BP-1 and eIF-4E can act either in concert or in opposition to independently regulate cap-dependent translation. We suggest that independent regulation of eIF-4E and BP-1 might finely regulate the efficiency of translation initiation or possibly control cap-dependent translation for fundamentally different purposes.

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ANG II activates effectors of mTOR via PI3-K signaling in human coronary smooth muscle cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00040.2004 ◽

2004 ◽

Vol 287 (3) ◽

pp. H1232-H1238 ◽

Cited By ~ 33

Author(s):

Sassan Hafizi ◽

Xuemin Wang ◽

Adrian H. Chester ◽

Magdi H. Yacoub ◽

Christopher G. Proud

Keyword(s):

Protein Synthesis ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Signaling Pathway ◽

Initiation Factor ◽

Ang Ii ◽

Eukaryotic Initiation Factor ◽

Mtor Signaling Pathway ◽

Phosphatidylinositol 3 Kinase ◽

Eukaryotic Initiation Factor 4E

We have previously shown that the vasoconstrictive peptide angiotensin II (ANG II) is a hypertrophic agent for human coronary artery smooth muscle cells (cSMCs), which suggests that it plays a role in vascular wall thickening. The present study investigated the intracellular signal transduction pathways involved in the growth response of cSMCs to ANG II. The stimulation of protein synthesis by ANG II in cSMCs was blocked by the immunosuppressant rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway that includes the 70-kDa S6 kinase (p70S6k) and plays a key role in cell growth. The inhibitory effect of rapamycin was reversed by a molar excess of FK506; this indicates that both agents act through the common 12-kDa immunophilin FK506-binding protein. ANG II caused a rapid and sustained activation of p70S6k activity that paralleled its phosphorylation, and both processes were blocked by rapamycin. In addition, both of the phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002 abolished the ANG II-induced increase in protein synthesis, and wortmannin also blocked p70S6k phosphorylation. Furthermore, ANG II triggered dissociation of the translation initiation factor, eukaryotic initiation factor-4E, from its regulatory binding protein 4E-BP1, which was also inhibited by rapamycin and wortmannin. In conclusion, we have shown that ANG II activates components of the rapamycin-sensitive mTOR signaling pathway in human cSMCs and involves activation of phosphatidylinositol 3-kinase, p70S6k, and eukaryotic initiation factor-4E, which leads to activation of protein synthesis. These signaling mechanisms may mediate the growth-promoting effect of ANG II in human cSMCs.

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Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells

Biochemical Journal ◽

10.1042/bj3600087 ◽

2001 ◽

Vol 360 (1) ◽

pp. 87-95 ◽

Cited By ~ 14

Author(s):

Duraisamy SENTHIL ◽

Jennifer L. FAULKNER ◽

Goutam GHOSH CHOUDHURY ◽

Hanna E. ABBOUD ◽

Balakuntalam S. KASINATH

Keyword(s):

Protein Synthesis ◽

Angiotensin Ii ◽

Insulin Signalling ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Tubular Epithelial Cells ◽

Eukaryotic Initiation Factor 4E ◽

Proximal Tubular Epithelial Cells ◽

Proximal Tubular ◽

Stimulation Of

Interaction between angiotensin II, which binds a G-protein-coupled receptor, and insulin, a ligand for receptor tyrosine kinase, was examined in renal proximal tubular epithelial cells. Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) which follows the release of the factor from a heterodimeric complex by phosphorylation of its binding protein, 4E-BP1. Angiotensin II (1nM) or insulin (1nM) individually stimulated 4E-BP1 phosphorylation. However, pre-incubation with angiotensin II abrogated insulin-induced phosphorylation of 4E-BP1, resulting in persistent binding to eIF4E. Although angiotensin II and insulin individually activated phosphoinositide 3-kinase and extracellular signal-regulated kinase (ERK)-1/−2-type mitogen-activated protein (MAP) kinase, pre-incubation with angiotensin II abolished insulin-induced stimulation of these kinases, suggesting more proximal events in insulin signalling may be intercepted. Pretreatment with angiotensin II markedly inhibited insulin-stimulated tyrosine phosphorylation of insulin-receptor β-chain and insulin-receptor substrate 1. Losartan prevented angiotensin II inhibition of insulin-induced ERK-1/−2-type MAP kinase activation and 4E-BP1 phosphorylation, suggesting mediation of the effect of angiotensin II by its type 1 receptor. Insulin-stimulated de novo protein synthesis was also abolished by pre-incubation with angiotensin II. These data show that angiotensin II inhibits 4E-BP1 phosphorylation and stimulation of protein synthesis induced by insulin by interfering with proximal events in insulin signalling. Our data provide a mechanistic basis for insulin insensitivity induced by angiotensin II.

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Phosphorylation of Eukaryotic Initiation Factor 2 by Heme-Regulated Inhibitor Kinase-Related Protein Kinases in Schizosaccharomyces pombe Is Important for Resistance to Environmental Stresses

Molecular and Cellular Biology ◽

10.1128/mcb.22.20.7134-7146.2002 ◽

2002 ◽

Vol 22 (20) ◽

pp. 7134-7146 ◽

Cited By ~ 57

Author(s):

Ke Zhan ◽

Krishna M. Vattem ◽

Bettina N. Bauer ◽

Thomas E. Dever ◽

Jane-Jane Chen ◽

...

Keyword(s):

Protein Synthesis ◽

Schizosaccharomyces Pombe ◽

Environmental Stresses ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Α Subunit ◽

Eukaryotic Initiation Factor 2 ◽

Initiation Factor 2 ◽

Eif2α Kinase

ABSTRACT Protein synthesis is regulated by the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) in response to different environmental stresses. One member of the eIF2α kinase family, heme-regulated inhibitor kinase (HRI), is activated under heme-deficient conditions and blocks protein synthesis, principally globin, in mammalian erythroid cells. We identified two HRI-related kinases from Schizosaccharomyces pombe which have full-length homology with mammalian HRI. The two HRI-related kinases, named Hri1p and Hri2p, exhibit autokinase and kinase activity specific for Ser-51 of eIF2α, and both activities were inhibited in vitro by hemin, as previously described for mammalian HRI. Overexpression of Hri1p, Hri2p, or the human eIF2α kinase, double-stranded-RNA-dependent protein kinase (PKR), impeded growth of S. pombe due to elevated phosphorylation of eIF2α. Cells from strains with deletions of the hri1+ and hri2+ genes, individually or in combination, exhibited a reduced growth rate when exposed to heat shock or to arsenic compounds. Measurements of in vivo phosphorylation of eIF2α suggest that Hri1p and Hri2p differentially phosphorylate eIF2α in response to these stress conditions. These results demonstrate that HRI-related enzymes are not unique to vertebrates and suggest that these eIF2α kinases are important participants in diverse stress response pathways in some lower eukaryotes.

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A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5328 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5328-5334 ◽

Cited By ~ 118

Author(s):

N Méthot ◽

M S Song ◽

N Sonenberg

Keyword(s):

Aspartic Acid ◽

Ribosomal Subunit ◽

Direct Interaction ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Ribosome Binding ◽

Initiation Factors ◽

Self Association

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3.

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