Initiation Factor 4F Complex Research Articles

AICAR treatment for 14 days normalizes obesity-induced dysregulation of TORC1 signaling and translational capacity in fasted skeletal muscle

The aim of this study was to determine the effect of 14 days of 5-aminoimidazole-4-carboxamide-1β-4-ribofuranoside (AICAR) treatment on mammalian target of rapamycin (mTOR) signaling and mTOR-regulated processes (i.e., translation initiation) in obese mouse skeletal muscle. Our hypothesis was that daily treatment (14 days) with AICAR would normalize obesity-induced alterations in skeletal muscle mTOR signaling and mTOR-regulated processes to lean levels and positively affect muscle mass. Fourteen-week-old male, lean (L; 31.3 g body wt) wild-type and ob/ob (O; 59.6 g body wt) mice were injected with the AMP-activated kinase (AMPK) activator AICAR (A) at 0.5 mg·g body wt(-1)·day(-1) or saline control (C) for 14 days. At 24 h after the last injection (including a 12-h fast), all mice were killed, and the plantar flexor complex muscle (gastrocnemius, soleus, and plantaris) was excised for analysis. Muscle mass was lower in OC (159 ± 12 mg) than LC, LA, and OA (176 ± 10, 178 ± 9, and 166 ± 16 mg, respectively) mice, independent of a body weight change. A decrease in obese muscle mass corresponded with higher muscle cross section staining intensity for lipid and glycogen, higher blood glucose and insulin levels, and lower nuclear-enriched fractions for peroxisome proliferator-activated receptor-γ coactivator-1α protein expression in OC skeletal muscle, which was normalized with AICAR treatment. AMPK and acetyl-cocarboxylase phosphorylation was reduced in OC mice and augmented by AICAR treatment in OA mice. Conversely, OC mice displayed higher activation of downstream targets (S6 kinase-1 and ribosomal protein S6) of mTOR and lower raptor-associated mTOR than LC mice, which were reciprocally altered after 14 days of AICAR treatment. Dysregulation of translational capacity was improved in OA mice, as assessed by sucrose density gradient fractionation of ribosomes, total and ribosome-associated RNA content, eukaryotic initiation factor 4F complex formation, and eukaryotic initiation factor 4G phosphorylation. These data show that short-term (14 days) AMPK agonist treatment augments regulatory processes in atrophic obese mouse skeletal muscle through the normalization of mTOR signaling and mRNA translation closer to lean levels.

HDAC2 Promotes eIF4E Sumoylation and Activates mRNA Translation Gene Specifically

Histone deacetylases (HDACs) are best known as transcription corepressors through deacetylating histone tails. Here we show that HDAC2 is also involved in cap-dependent mRNA translation by promoting sumoylation of eukaryotic translation initiation factor 4E (eIF4E), which is independent of its deacetylase activity. By stimulating eIF4E sumoylation, HDAC2 induces the formation of the active eukaryotic initiation factor 4F (eIF4F) complex and induces the protein synthesis of a subset of eIF4E-responsive genes that are essential for cell proliferation and preventing apoptosis. These data demonstrate that HDAC2 has an unexpected sumoylation-promoting activity and regulates cap-dependent mRNA translation.

The Antidepressant Sertraline Inhibits Translation Initiation by Curtailing Mammalian Target of Rapamycin Signaling

Sertraline, a selective serotonin reuptake inhibitor, is a widely used antidepressant agent. Here, we show that sertraline also exhibits antiproliferative activity. Exposure to sertraline leads to a concentration-dependent decrease in protein synthesis. Moreover, polysome profile analysis of sertraline-treated cells shows a reduction in polysome content and a concomitant increase in 80S ribosomes. The inhibition in translation caused by sertraline is associated with decreased levels of the eukaryotic initiation factor (eIF) 4F complex, altered localization of eIF4E, and increased eIF2alpha phosphorylation. The latter event leads to increased REDD1 expression, which in turn impinges on the mammalian target of rapamycin (mTOR) pathway by affecting TSC1/2 signaling. Sertraline also independently targets the mTOR signaling pathway downstream of Rheb. In the Emu-myc murine lymphoma model where carcinogenesis is driven by phosphatase and tensin homologue (PTEN) inactivation, sertraline is able to enhance chemosensitivity to doxorubicin. Our results indicate that sertraline exerts antiproliferative activity by targeting the mTOR signaling pathway in a REDD1-dependent manner.

JNK inhibition arrests cotranslational degradation

Adhesion to fibronectin stimulates protein synthesis (translation) of fibroblasts. Protein synthesis stimulation is dependent from the activation of β1-integrin. β1-Integrin elicits a PI3K cascade that modulates eIF4F (eukaryotic initiation factor 4F) complex formation. In the attempt to further dissect elements of the PI3K cascade that might be responsible for fibronectin-stimulated translation, we used pharmacological inhibitors of known kinases. We found that JNK inhibition, by SP600125 treatment, increased 35S-methionine incorporation. Paradoxically, the increase in methionine incorporation was associated to a reduction of initiation of translation. These data imply that, during the adhesion of fibroblasts to fibronectin, conspicuous protein degradation occurs. Indeed, we found that inhibition of the proteasome by MG132 also increased methionine incorporation. Cotranslational degradation depended on PI3K activation. In spite of this, serum promoted translation, but not cotranslational degradation. The crosstalk between translation and degradation was further analyzed by studying the phosphorylation of initiation factors. Briefly, inhibition of JNK leads to eIF2α phosphorylation, which may account for the decrease in initiation of translation. In conclusion, β1-integrin-activated translation causes the synthesis of short-lived proteins, whose degradation is controlled by the JNK pathway. We hypothesize that JNK is a general regulator of cotranslational degradation.

C-Myc and eIF4F Constitute a Feedforward Loop That Regulates Cell Growth: Implications for Anticancer Therapy

The Myc/Max/Mad family of transcription factors and the eukaryotic initiation factor 4F (4F) complex play fundamental roles in regulating cell growth, proliferation, differentiation, and oncogenic transformation. Recent findings indicate that the role of Myc during cell growth and proliferation is linked to an increase in eIF4F activity in a feedforward relationship, providing a possible molecular mechanism of cell transformation by Myc. Developing therapeutics to inhibit eIF4F and/or Myc could be a potential treatment for a wide range of human cancers.

Silibinin inhibits translation initiation: implications for anticancer therapy

Silibinin is a nontoxic flavonoid reported to have anticancer properties. In this study, we show that silibinin exhibits antiproliferative activity on MCF-7 breast cancer cells. Exposure to silibinin leads to a concentration-dependent decrease in global protein synthesis associated with reduced levels of eukaryotic initiation factor 4F complex. Moreover, polysome profile analysis of silibinin-treated cells shows a decrease in polysome content and translation of cyclin D1 mRNA. Silibinin exerts its effects on translation initiation by inhibiting the mammalian target of rapamycin signaling pathway by acting upstream of TSC2. Our results show that silibinin blocks mammalian target of rapamycin signaling with a concomitant reduction in translation initiation, thus providing a possible molecular mechanism of how silibinin can inhibit growth of transformed cells.

Antitumor Activity and Mechanism of Action of the Cyclopenta[b]benzofuran, Silvestrol

BackgroundFlavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model.Methodology/Principal FindingsAmong a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs.Conclusions/SignificanceOur results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.

Expression of poly(A)‐binding protein is upregulated during recovery from heat shock in HeLa cells

Induction of heat shock proteins (HSPs) helps cells to survive severe hyperthermal stress and removes toxic unfolded proteins. At the same time, the cap-dependent translation of global cellular mRNA is inhibited, due to the loss of function of eukaryotic initiation factor (eIF)4F complex. It has been previously reported that, following heat shock, HSP27 binds to the insoluble granules of eIF4G and impedes its association with cytoplasmic poly(A)-binding protein (PABP) 1 and eIF4E. In the studies reported here, in addition to heat shock, we have included results of our investigation on the association between eIF4G, PABP1 and HSP27 during recovery from heat shock, when cap-dependent mRNA translation resumes. We showed here that in the heat-shocked cells, the PABP1-eIF4G complex dissociated, and both polypeptides translocated with the HSP27 to the nucleus. During recovery after heat shock, PABP1 and eIF4G were redistributed into the cytoplasm and colocalized with each other. In addition, PABP1 expression was upregulated and its translation efficiency was increased during the recovery period, possibly to meet additional demands on the translation machinery. HSP27 remained associated with the eIF4G-PABP1 complex during recovery from heat shock. Therefore, our results raise the possibility that the association of HSP27 with eIF4G may not be sufficient to suppress cap-dependent translation during heat shock. In addition, we provide evidence that the terminal oligopyrimidine cis-element of PABP1 mRNA is responsible for the preferential increase of PABP1 mRNA translation in cells undergoing recovery from heat shock.

Blocking eukaryotic initiation factor 4F complex formation does not inhibit the mTORC1-dependent activation of protein synthesis in cardiomyocytes

Activation of the mammalian target of rapamycin complex 1 (mTORC1) causes the dissociation of eukaryotic initiation factor 4E complex (eIF4E)-binding protein 1 (4E-BP1) from eIF4E, leading to increased eIF4F complex formation. mTORC1 positively regulates protein synthesis and is implicated in several diseases including cardiac hypertrophy, a potentially fatal disorder involving increased cardiomyocyte size. The importance of 4E-BP1 in mTORC1-regulated protein synthesis was investigated by overexpressing 4E-BP1, which blocks eIF4F formation in isolated primary cardiomyocytes without affecting other targets for mTORC1 signaling. Interestingly, blocking eIF4F formation did not impair the degree of activation of overall protein synthesis by the hypertrophic agent phenylephrine (PE), which, furthermore, remained dependent on mTORC1. Overexpressing 4E-BP1 also only had a small effect on PE-induced cardiomyocyte growth. Overexpressing 4E-BP1 did diminish the PE-stimulated synthesis of luciferase encoded by structured mRNAs, confirming that such mRNAs do require eIF4F for their translation in cardiomyocytes. These data imply that the substantial inhibition of cardiomyocyte protein synthesis and growth caused by inhibiting mTORC1 cannot be attributed to the activation of 4E-BP1 or loss of eIF4F complexes. Our data indicate that increased eIF4F formation plays, at most, only a minor role in the mTORC1-dependent activation of overall protein synthesis in these primary cells but is required for the translation of structured mRNAs. Therefore, other mTORC1 targets are more important in the inhibition by rapamycin of the rapid activation of protein synthesis and of cell growth.

AMPing Down Leucine Action in Skeletal Muscle

In recent years, several investigative teams have tested leucine as a therapeutic agent to support muscle growth. Although some studies have shown that BCAA or leucine support muscle protein synthesis (MPS) (1), others have found that leucine is ineffective at stimulating muscle protein anabolism under certain conditions, e.g. sepsis, alcohol intoxication, and glucocorticoid excess in aged rats. During these catabolic states, MPS is either precluded or blunted in response to leucine, leading the authors of the current highlighted paper, Pruznak et al. (2), which was published in the October, 2008 issue of The Journal of Nutrition, to describe the muscle as being in a state of leucine resistance. Understanding the molecular basis for why muscle is unresponsive to supplemental leucine can be used to identify novel therapeutic targets and/or optimize current nutritional support strategies. The BCAA are known for their unique ability to stimulate MPS independently of other amino acids. When administered orally, leucine alone stimulates MPS in exercised or fooddeprived rats (3). The mechanism for the anabolic effect involves stimulation of mRNA translation initiation by increasing eukaryotic initiation factor (EIF) 4F complex assembly and signaling via mammalian target of rapamycin (mTOR) (3). The mTOR kinase facilitates cell growth and proliferation by nutrients and insulin through its association with the regulatory proteins Raptor (regulatory associated protein of mTOR), mLST8/GbL, and PRAS40 (proline-rich Akt substrate of 40 kDa) in a complex referred to as mTOR complex 1 (mTORC1) (4,5). Over the past decade, many laboratories have sought to define how leucine stimulates mTORC1 activity. Although it is clear that leucine does not activate the insulin/insulin-like growth factor/phosphatidylinositol 3-kinase/protein kinase B signal transduction pathway, identification of the relevant molecules upstream of mTORC1 remains elusive. To understand why leucine is ineffective at inducing MPS under certain stress conditions in vivo, Pruznak et al. (2) examined the relationship between leucine and AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that serves as a major energy sensor in the cell. Activated when ATP becomes limiting, AMPK stimulates catabolic pathways and inhibits anabolic pathways in an effort to supply ATP for cell survival. As such, AMPK is a negative upstream regulator of mTORC1, reducing MPS under conditions of energetic stress such as hypoxia, intense exercise, or glucose starvation (6). AMPK inhibits mTORC1 signaling in 2 ways: via phosphorylation of Raptor, which leads to decreased mTOR kinase activity, and via phosphorylation of tuberin (also known as tuberous sclerosis complex 2) at Ser1345 (7), converting the mTOR activator, Ras homolog enhanced in brain, to its inactive form. Activationof AMPK canbemimickedusing5-aminoimidazole4-carboxamide-1–4-ribofuranoside (AICAR), a cell-permeable compound whose phosphorylated metabolite activates AMPK without perturbing the cellular concentrations of ATP, ADP, or AMP in skeletal muscle (8). In the October issue, Pruznak et al. (2) utilizes AICAR treatment in rats to very effectively block the stimulation of skeletal MPS, EIF4F complex assembly, and mTORC1 signaling by oral administration of leucine (Fig. 1). Importantly, the overall block in leucine action is more complete than that achieved by other inhibitors tested previously in rats (e.g. rapamycin), demonstrating that cellular energy state plays a dominant role in the regulation of MPS by amino acids in vivo. These findings are supported by recently published work showing that leucine stimulation of mTOR and MPS is inhibited by AICAR treatment of C2C12 cells (9). Pruznak et al. (2) further advance our understanding of how leucine transmits mTORC1 signaling in vivo by identifying PRAS40 as a mediator of leucine’s effects. PRAS40 is a Raptorbinding protein that is phosphorylated directly by mTORC1 at several sites, including Ser183, and by protein kinase B at Thr246. Whereas studies using cells in culture show that phosphorylation of PRAS40 at Ser183 is modulated by amino acid-activated mTORC1 (4), the study of Pruznak et al. (2) finds that leucine enhances PRAS40 phosphorylation at the insulin-activated Thr246 site. Interpretation of this result should be viewed cautiously, because it is possible that the transient rise in insulin following leucine administration may have contributed to 1 Author disclosures: T. Anthony and J. Anthony, no conflicts of interest.

Protein Synthesis Escapes mTORC1 Control and Constitutes a Promising Therapeutic Target in Acute Myeloid Leukemia.

The deregulation of translation, leading to a preferential translation of oncogene-related mRNAs, markedly contributes to the malignant phenotype in human cancers. The assembly of the eukaryotic translation Initiation Factor 4F (eIF4F) complex is limiting for malignancy-related translation events and is hence an attractive anticancer therapeutic target. The Mammalian Target Of Rapamycin Complex 1 (mTORC1) is thought to positively regulate eIF4F assembly and subsequent oncogenic protein synthesis through eIF4E Binding Protein 1 (4E-BP1) phosphorylation. The mTORC1 inhibitor rapamycin has been developed as an anti-cancer agent but is of limited efficacy in the treatment of Acute Myeloid Leukemia (AML). We show in our study that in primary AML cells, 4E-BP1 phosphorylation is mTORC1-independent and is in fact controlled by the Pim- 2 serine/threonine kinase: neither the rapamycin derivate RAD001 nor Raptor siRNA inhibited 4E-BP1 phosphorylation contrary to siRNA-mediated Pim-2 knock-down. We utilized the 4E-BP1 mimetic 4EGI-1, as a direct inhibitor of translation in AML. In contrast to RAD001, 4EGI-1totally inhibited eIF4F assembly;markedly decreased the proportion of polysome-bound c-Myc mRNA; anddramatically decreased the accumulation of oncogenic proteins that are regulated at the translational level.Moreover, 4EGI-1 demonstrated potent anti-leukemic activity ex vivo in primary AML blast cells through a selective suppression of proteins implicated in proliferation and in anti-apoptotic mechanisms. Significantly, these effects occurred with a minimal degree of toxicity against normal hematopoietic processes. Our present results therefore strongly implicate an mTORC1-independent mechanism of deregulated synthesis of oncogenic proteins in human myeloid leukemogenesis. The direct inhibition of the eIF4F translation initiating complex, by 4EGI-1, by the recently developed eIF4E antisense oligonucleotides or by specific Pim-2 inhibitors when available, thus represents an attractive option for the development of new therapies for AML.

C-Myc and eIF4F Are Components of a Feedforward Loop that Links Transcription and Translation

The Myc/Max/Mad family of transcription factors and the eukaryotic initiation factor 4F (eIF4F) complex play fundamental roles in regulating cell growth, proliferation, differentiation, and oncogenic transformation. eIF4F is involved in the recruitment of ribosomes to mRNAs and is thought to generally be the rate-limiting phase of translation. Here, we show that c-Myc directly activates transcription of the three subunits of eIF4F (eIF4E, eIF4AI, and eIF4GI). These transcriptional effects are mediated through canonical E-boxes (5'CACGTG3') present in the promoters of these genes. In addition, the c-Myc antagonist Mad1 down-regulates the expression of eIF4F subunits. We also show that MycER activation stimulates protein synthesis at the level of translation initiation. Increased eIF4F levels result in stimulation of c-Myc mRNA translation specifically, as assessed by quantitative reverse transcription-PCR. We use a murine model of lymphomagenesis to show the expression of eIF4F subunits is also up-regulated by c-Myc in vivo. Our results suggest the presence of a feedforward loop involving c-Myc and eIF4F that serves to link transcription and translation and that could contribute to the effects of c-Myc on cell proliferation and neoplastic growth.

Cap-dependent eukaryotic initiation factor-mRNA interactions probed by cross-linking

Cap-dependent ribosome recruitment to eukaryotic mRNAs during translation initiation is stimulated by the eukaryotic initiation factor (eIF) 4F complex and eIF4B. eIF4F is a heterotrimeric complex composed of three subunits: eIF4E, a 7-methyl guanosine cap binding protein; eIF4A, a DEAD-box RNA helicase; and eIF4G. The interactions of eIF4E, eIF4A, and eIF4B with mRNA have previously been monitored by chemical- and UV-based cross-linking approaches aimed at characterizing the initial protein/mRNA interactions that lead to ribosome recruitment. These studies have led to a model whereby eIF4E interacts with the 7-methyl guanosine cap structure in an ATP-independent manner, followed by an ATP-dependent interaction of eIF4A and eIF4B. Herein, we apply a splint-ligation-mediated approach to generate 4-thiouridine-containing mRNA adjacent to a radiolabel group that we utilize to monitor cap-dependent cross-linking of proteins adjacent to, and downstream from, the cap structure. Using this approach, we demonstrate interactions between eIF4G, eIF4H, and eIF3 subunits with the mRNA during the cap recognition process.

A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

Insulin-like growth factor 1 receptor (IGF-1R) is important in cancer cell growth and survival and has been implicated in cancer pathophysiology and treatment. Here we report a novel function for IGF-1R in p53-dependent apoptotic response. We show that inhibition or loss of IGF-1R activity reduces translational synthesis of p53 and Mdm2 protein. Notably, IGF-1R inhibition increases p53 protein stability by reducing p53 ubiquitination and maintains p53 at low levels by decreasing p53 synthesis, thus rendering p53 insensitive to stabilization after DNA damage. The accumulation and apoptosis of DNA-damage–induced p53 is therefore reduced in Igf-1r−/− mouse embryonic fibroblasts or tumor cells treated with the IGF-1R inhibitor. Furthermore, we find that inhibition of IGF-1R reduces p53 and Mdm2 translation through a gene-specific mechanism mediated by the respective 5′ untranslated region of p53 and mdm2 messenger RNA. The eukaryotic translation initiation factor 4F complex is also involved in this translational inhibition. These results demonstrate an unexpected role for translational control by IGF-1R in p53-mediated apoptosis.

Rapamycin Limits Formation of Active Eukaryotic Initiation Factor 4F Complex Following Meal Feeding in Rat Hearts ,

Feeding promotes protein synthesis in cardiac muscle through a stimulation of the messenger RNA translation initiation phase of protein synthesis by enhancing assembly of active eukaryotic initiation factor (eIF)4F complex. The experiments reported herein examined the potential role for a rapamycin-sensitive signaling pathway in increasing formation of active eIF4G-eIF4E complex during meal feeding. Hearts from male Sprague-Dawley rats fed a meal consisting of rat nonpurified diet were sampled prior to and 3 h following the meal in the presence or absence of treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) complex 1. Rapamycin prevented the meal feeding-induced stimulation of myocardial protein synthesis. Inhibition of mTOR with rapamycin decreased the association of rapamycin-associated TOR protein with mTOR and prevented the feeding-induced assembly of eIF4G-eIF4E complex. In contrast, the abundance of eIF4E binding protein-1 (4E-BP1)-eIF4E complex was unaffected by either meal feeding or rapamycin. Pretreatment with rapamycin completely prevented the feeding-induced phosphorylation of eIF4G(Ser1108), whereas the inhibitor only partially attenuated meal feeding-induced 70-kDa ribosomal protein S6 kinase1(Thr389) phosphorylation and extent of 4E-BP1 in the γ-form. Meal feeding-induced phosphorylation of protein kinase B on either Ser473 or Thr308 was unaffected by rapamycin. These findings suggest the extent of phosphorylation of eIF4G following meal feeding occurs by a rapamycin-sensitive mechanism in cardiac muscle. Furthermore, the rapamycin-sensitive reductions in phosphorylation of eIF4G may also lead to decreased formation of active eIF4G-eIF4E complex.

Compartmentalisation and localisation of the translation initiation factor (eIF) 4F complex in normally growing fibroblasts

Previous observations of association of mRNAs and ribosomes with subcellular structures highlight the importance of localised translation. However, little is known regarding associations between eukaryotic translation initiation factors and cellular structures within the cytoplasm of normally growing cells. We have used detergent-based cellular fractionation coupled with immunofluorescence microscopy to investigate the subcellular localisation in NIH3T3 fibroblasts of the initiation factors involved in recruitment of mRNA for translation, focussing on eIF4E, the mRNA cap-binding protein, the scaffold protein eIF4GI and poly(A) binding protein (PABP). We find that these proteins exist mainly in a soluble cytosolic pool, with only a subfraction tightly associated with cellular structures. However, this “associated” fraction was enriched in active “eIF4F” complexes (eIF4E.eIF4G.eIF4A.PABP). Immunofluorescence analysis reveals both a diffuse and a perinuclear distribution of eIF4G, with the perinuclear staining pattern similar to that of the endoplasmic reticulum. eIF4E also shows both a diffuse staining pattern and a tighter perinuclear stain, partly coincident with vimentin intermediate filaments. All three proteins localise to the lamellipodia of migrating cells in close proximity to ribosomes, microtubules, microfilaments and focal adhesions, with eIF4G and eIF4E at the periphery showing a similar staining pattern to the focal adhesion protein vinculin.

Insulin and IGF-I stimulate the formation of the eukaryotic initiation factor 4F complex and protein synthesis in C2C12 myotubes independent of availability of external amino acids

The objective of this study was to investigate the effect of insulin and IGF-I on protein synthesis and translation initiation in C2C12 myotubes in nutrient-deprived Dulbecco's phosphate buffered saline (DPBS). The results showed that insulin and IGF-I increased protein synthesis by 62% and 35% respectively in DPBS, and the effect was not affected by rapamycin, but was blocked by LY294002. Insulin and IGF-I stimulated eukaryotic initiation factor 4E (eIF4E) binding protein (4EBP1) phosphorylation in a dose-dependent manner, and the stimulation was independent of availability of external amino acids. Both LY294002 and rapamycin blocked the insulin and IGF-I-induced increases in 4EBP1 phosphorylation. The results also showed that insulin and IGF-I were able to stimulate PKB/Akt phosphorylation, glycogen synthase kinase (GSK) 3beta phosphorylation and mTOR phosphorylation in DPBS. Insulin and IGF-I increased the amount of eIF4G associated with eIF4E in nutrient-deprived C2C12 myotubes. The amount of 4EBP1 associated with eIF4E was decreased after insulin or IGF-I stimulation. We conclude that in C2C12 myotubes, insulin and IGF-I may regulate protein synthesis and translation initiation independent of external amino acid supply via the phosphatidylinositol-3 kinase-PKB/Akt-mTOR pathway.

Meal Feeding Alters Translational Control of Gene Expression in Rat Liver

Meal feeding after a period of food deprivation results in a subsequent increase in the protein and RNA content of the liver. To gain insight into the mechanisms involved in the response to food intake, changes in the association of selected mRNAs with polysomes were examined. On the day of the study, rat livers were collected at 0, 15, 60, and 180 min after the start of feeding and analyzed for biomarkers of the translational control of protein synthesis. Protein synthesis was increased within 60 min and was sustained for 180 min. Assembly of the active eukaryotic initiation factor (eIF) 4F complex was elevated within 15 min, as indicated by the relative association of eIF4E . eIF4G, but returned to the basal value within 180 min. Phosphorylation of the ribosomal protein (rp) S6 kinase S6K1 and its substrate rpS6 was increased within 15 min and was sustained for at least 180 min. Both eIF4F assembly and activation of S6K1 have been linked to upregulated translation of a subset of mRNAs. To identify translationally regulated mRNAs, polysomal (i.e., actively translated) and nonpolysomal (nontranslated) fractions were isolated and subjected to microarray analysis. The mRNAs encoding 78 proteins, including 42 proteins involved in protein synthesis, exhibited increased abundance in polysomes in response to feeding. Overall, the results demonstrate that protein synthesis as well as ribosomal protein mRNA translation undergo rapid and sustained stimulation in the liver after meal feeding and thus contribute to the previously observed increases in protein and RNA content.

Suppression of human Mnk1 by small interfering RNA increases the eukaryotic initiation factor 4F activity in HEK293T cells

Short-interfering RNAs (siRNAs) have proved to be a useful tool in studying gene function in plants, invertebrates and mammalian systems. Herein, we report the use of siRNAs for targeting the human MAP kinase-interacting kinase Mnk1 gene. This study demonstrates the efficacy of the designed siRNA in silencing Mnk1 in the human cell line HEK293T and shows that Mnk1 suppression decreases eukaryotic initiation factor 4E phosphorylation without causing any change in global protein synthesis rate and cell proliferation. Interestingly, suppression of Mnk1 results in a significant increase in eukaryotic initiation factor 4F complex formation after 72 h of transfection.

Cell cycle-dependent phosphorylation of the translational repressor eIF-4E binding protein-1 (4E-BP1)

A fundamental control point in the regulation of the initiation of protein synthesis is the formation of the eukaryotic initiation factor 4F (eIF-4F) complex. The formation of this complex depends upon the availability of the mRNA cap binding protein, eIF-4E, which is sequestered away from the translational machinery by the tight association of eIF-4E binding proteins (4E-BPs). Phosphorylation of 4E-BP1 is critical in causing its dissociation from eIF-4E, leaving 4E available to form translationally active eIF-4F complexes, switching on mRNA translation. In this report, we provide the first evidence that the phosphorylation of 4E-BP1 increases during mitosis and identify Ser-65 and Thr-70 as phosphorylated sites. Phosphorylation of Thr-70 has been implicated in the regulation of 4E-BP1 function, but the kinase phosphorylating this site was unknown. We show that the cyclin-dependent kinase, cdc2, phosphorylates 4E-BP1 at Thr-70 and that phosphorylation of this site is permissive for Ser-65 phosphorylation. Crucially, the increased phosphorylation of 4E-BP1 during mitosis results in its complete dissociation from eIF-4E.