eIF4F Complex Formation Research Articles

Analysis of mRNA associated factors during bovine oocyte maturation and early embryonic development

Regulation of gene expression at the translational level is particularly essential during developmental periods, when transcription is impaired. According to the closed-loop model of translational initiation, we have analyzed components of the 5 -mRNA cap-binding complex eIF4F (eIF4E, eIF4G, eIF4A), the eIF4E repressor 4E-BP1, and 3 -mRNA poly-(A) tail-associated proteins (PABP1 and 3, PAIP1 and 2, CPEB1, Maskin) during in vitro maturation of bovine oocytes and early embryonic development up to the 16-cell stage. Furthermore, we have elucidated the activity of distinct kinases which are potentially involved in their phosphorylation. Major phosphorylation of specific target sequences of PKA, PKB, PKC, CDKs, ATM/ATR, and MAPK were observed in M II stage oocytes. Furthermore, main changes in the abundance and/or phosphorylation of distinct mRNA-binding factors occur at the transition from M II stage oocytes to 2-cell embryos. In conclusion, the results indicate that, at the transition from oocyte to embryonic development, translational initiation is regulated by striking differences in the abundance and/or phosphorylation of 5 -end and 3 -end mRNA associated factors, mainly the poly-(A) bindings proteins PABP1 and 3, their repressor PAIP2 and a Maskin-like protein with distinct eIF4E-binding properties which prevents eIF4E/cap binding and eIF4F formation in vitro. Nevertheless, from the M II stage to 16-cell embryos a substantial amount of eIF4E and, to a lesser extent, of eIF4G was precipitated by (7)m-GTP-Separose indicating eIF4F complex formation. Therefore, it is likely that in general the reduction in PABP1 and 3 abundance represses overall translation during early embryonic development.

PI3K Signaling Regulates Rapamycin-Insensitive Translation Initiation Complex Formation in Vaccinia Virus-Infected Cells

How vaccinia virus (VV) regulates assembly of the host translation initiation complex eIF4F remains unclear. Here, we show that VV activated host PI3K to stimulate downstream mammalian target of rapamycin (mTOR), a kinase that inactivates the translational repressor 4E-BP1. However, although the mTOR inhibitor rapamycin suppressed VV-induced inactivation of 4E-BP1, it failed to inhibit eIF4F assembly. In contrast, PI3K inhibition in VV-infected cells increased the abundance of hypophosphorylated 4E-BP1 and disrupted eIF4F complex formation. PI3K signaling, therefore, plays a critical role in regulating protein production during VV infection, at least in part by controlling the abundance and activity of 4E-BP1.

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.

Selection of cyclic peptide aptamers to HCV IRES RNA using mRNA display

The hepatitis C virus (HCV) is a positive strand RNA flavivirus that is a major causative agent of serious liver disease, making new treatment modalities an urgent priority. Because HCV translation initiation occurs by a mechanism that is fundamentally distinct from that of host mRNAs, it is an attractive target for drug discovery. The translation of HCV mRNA is initiated from an internal ribosomal entry site (IRES), independent of cap and poly(A) recognition and bypassing eIF4F complex formation. We used mRNA display selection technology combined with a simple and robust cyclization procedure to screen a peptide library of >10(13) different sequences and isolate cyclic peptides that bind with high affinity and specificity to HCV IRES RNA. The best peptide binds the IRES with subnanomolar affinity, and a specificity of at least 100-fold relative to binding to several other RNAs of similar length. The peptide specifically inhibits HCV IRES-initiated translation in vitro with no detectable effect on normal cap-dependent translation initiation. An 8-aa cyclic peptide retains most of the activity of the full-length 27-aa bicyclic peptide. These peptides may be useful tools for the study of HCV translation and may have potential for further development as an anti-HCV drug.

Heterogeneous Nuclear Ribonucleoprotein A1 Regulates Cyclin D1 and c-myc Internal Ribosome Entry Site Function through Akt Signaling

The translation of the cyclin D1 and c-myc mRNAs occurs via internal ribosome entry site (IRES)-mediated initiation under conditions of reduced eIF-4F complex formation and Akt activity. Here we identify hnRNP A1 as an IRES trans-acting factor that regulates cyclin D1 and c-myc IRES activity, depending on the Akt status of the cell. hnRNP A1 binds both IRESs in vitro and in intact cells and enhances in vitro IRES-dependent reporter expression. Akt regulates this IRES activity by inducing phosphorylation of hnRNP A1 on serine 199. Serine 199-phosphorylated hnRNP A1 binds to the IRESs normally but is unable to support IRES activity in vitro. Reducing expression levels of hnRNP A1 or overexpressing a dominant negative version of the protein markedly inhibits rapamycin-stimulated IRES activity in cells and correlated with redistribution of cyclin D1 and c-myc transcripts from heavy polysomes to monosomes. Importantly, knockdown of hnRNP A1 also renders quiescent Akt-containing cells sensitive to rapamycin-induced G(1) arrest. These results support a role for hnRNP A1 in mediating rapamycin-induced alterations of cyclin D1 and c-myc IRES activity in an Akt-dependent manner and provide the first direct link between Akt and the regulation of IRES activity.

Regulation of cap‐dependent translation initiation in the early stage porcine parthenotes

The binding of mRNAs to ribosomes is mediated by the protein complex eIF4F in conjunction with eIF4B (eukaryotic initiation factor 4F and 4B). EIF4F is a three subunit complex consisting of eIF4A (RNA helicase), eIF4E (mRNA cap binding protein), and eIF4G (bridging protein). The crucial role is played by eIF4E, which directly binds the 5'-cap structure of the mRNA and facilitates the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. EIF4E binding to mRNA and to other initiation factors is regulated on several levels, including its phosphorylation on Ser-209, and association with its regulatory protein 4E-binding protein (4E-BP1). In this study we document that both the translation initiation factor eIF4E and its regulator 4E-BP1 become dephosphorylated in the early stage porcine zygotes already 8 hr post-activation. Similarly, the activities of ERK1/2 MAP and Mnk1 kinases, which are both involved in eIF4E phosphorylation, gradually decrease during this period with the timing similar to that of eIF4E dephosphorylation. The formation of an active eIF4F complex is also diminished after 9-15 hr post-activation, although substantial amounts of this complex have been detected also 24 hr post-activation (2-cell stage). The overall protein synthesis in the parthenotes decreases gradually from 12 hr post-activation reaching a minimum after 48 hr (4-cell stage). Although the translation is gradually decreasing during early preimplantation development, the eIF4F complex, which is temporarily formed, might be a premise for the translation of a small subset of mRNAs at this period of development.

Selection of cyclic peptide aptamers to HCV IRES RNA using mRNA display

The hepatitis C virus (HCV) is a positive strand RNA flavivirus that is a major causative agent of serious liver disease, making new treatment modalities an urgent priority. Because HCV translation initiation occurs by a mechanism that is fundamentally distinct from that of host mRNAs, it is an attractive target for drug discovery. The translation of HCV mRNA is initiated from an internal ribosomal entry site (IRES), independent of cap and poly(A) recognition and bypassing eIF4F complex formation. We used mRNA display selection technology combined with a simple and robust cyclization procedure to screen a peptide library of >1013 different sequences and isolate cyclic peptides that bind with high affinity and specificity to HCV IRES RNA. The best peptide binds the IRES with subnanomolar affinity, and a specificity of at least 100-fold relative to binding to several other RNAs of similar length. The peptide specifically inhibits HCV IRES-initiated translation in vitro with no detectable effect on normal cap-dependent translation initiation. An 8-aa cyclic peptide retains most of the activity of the full-length 27-aa bicyclic peptide. These peptides may be useful tools for the study of HCV translation and may have potential for further development as an anti-HCV drug.

L13a Blocks 48S Assembly: Role of a General Initiation Factor in mRNA-Specific Translational Control

Transcript-specific translational control restricts macrophage inflammatory gene expression. The proinflammatory cytokine interferon-gamma induces phosphorylation of ribosomal protein L13a and translocation from the 60S ribosomal subunit to the interferon-gamma-activated inhibitor of translation (GAIT) complex. This complex binds the 3'UTR of ceruloplasmin mRNA and blocks its translation. Here, we elucidate the molecular mechanism underlying repression by L13a. Translation of the GAIT element-containing reporter mRNA is sensitive to L13a-mediated silencing when driven by internal ribosome entry sites (IRESs) that require initiation factor eIF4G, but is resistant to silencing when driven by eIF4F-independent IRESs, demonstrating a critical role for eIF4G. Interaction of L13a with eIF4G blocks 43S recruitment without suppressing eIF4F complex formation. eIF4G attack, e.g., by virus, stress, or caspases, is a well-known mechanism of global inhibition of protein synthesis. However, our studies reveal a unique mechanism in which targeting of eIF4G by mRNA-bound L13a elicits transcript-specific translational repression.

The Translation Repressor 4E-BP2 Is Critical for eIF4F Complex Formation, Synaptic Plasticity, and Memory in the Hippocampus

Long-lasting synaptic plasticity and memory requires mRNA translation, yet little is known as to how this process is regulated. To explore the role that the translation repressor 4E-BP2 plays in hippocampal long-term potentiation (LTP) and learning and memory, we examined 4E-BP2 knock-out mice. Interestingly, genetic elimination of 4E-BP2 converted early-phase LTP to late-phase LTP (L-LTP) in the Schaffer collateral pathway, likely as a result of increased eIF4F complex formation and translation initiation. A critical limit for activity-induced translation was revealed in the 4E-BP2 knock-out mice because L-LTP elicited by traditional stimulation paradigms was obstructed. Moreover, the 4E-BP2 knock-out mice also exhibited impaired spatial learning and memory and conditioned fear-associative memory deficits. These results suggest a crucial role for proper regulation of the eIF4F complex by 4E-BP2 during LTP and learning and memory in the mouse hippocampus.

N-acetyl-cysteine abolishes hydrogen peroxide-induced modification of eukaryotic initiation factor 4F activity via distinct signalling pathways

During the oxidative stress generated by hydrogen peroxide (H 2O 2) in nerve growth factor (NGF)-differentiated PC12 cells, eIF4E binding protein (4E-BP1) and initiation factor 4E (eIF4E) phosphorylated levels decrease significantly, and an enhancement of the association of 4E-BP1 to eIF4E, which in turn decreases eIF4F formation is observed. The treatment with N-acetyl-cysteine (NAC) completely abolishes the H 2O 2-induced decrease in eIF4E phosphorylated levels, whereas the decrease in 4E-BP1 phosphorylated levels and eIF4F activity inhibition are significantly but not fully reversed. Rapamycin, the mammalian target of rapamycin (FRAP/mTOR) inhibitor, prevents the effect of NAC on H 2O 2-induced eIF4F complex formation inhibition. Besides the inhibitor induces a similar decrease in 4E-BP1 phosphorylated levels to that promote by H 2O 2. However, rapamycin has no effect on the NAC-induced recovery in phosphorylated eIF4E levels. Neither the MAP kinase inhibitors, PD98056 and SB203580, or the protein phosphatase 2A inhibitor, okadaic acid, mimic NAC effect on the H 2O 2-induced eIF4E dephosphorylation. Altogether our findings suggest that the effects caused by oxidative stress on eIF4s factors depends on two MAP kinase-independent signal transduction pathways, being at least one of them rapamycin-dependent.

Rapamycin-sensitive induction of eukaryotic initiation factor 4F in regenerating mouse liver.

Following acute injuries that diminish functional liver mass, the remaining hepatocytes substantially increase overall protein synthesis to meet increased metabolic demands and to allow for compensatory liver growth. Previous studies have not clearly defined the mechanisms that promote protein synthesis in the regenerating liver. In the current study, we examined the regulation of key proteins involved in translation initiation following 70% partial hepatectomy (PH) in mice. PH promoted the assembly of eukaryotic initiation factor (eIF) 4F complexes consisting of eIF4E, eIF4G, eIF4A1, and poly-A binding protein. eIF4F complex formation after PH occurred without detectable changes in eIF4E-binding protein 1 (4E-BP1) phosphorylation or its binding eIF4E. The amount of serine 1108-phosphorylated eIF4G (but not Ser209-phosphorylated eIF4E) was induced following PH. These effects were antagonized by treatment with rapamycin, indicating that target of rapamycin (TOR) activity is required for eIF4F assembly in the regenerating liver. Rapamycin inhibited the induction of cyclin D1, a known eIF4F-sensitive gene, at the level of protein expression but not messenger RNA (mRNA) expression. In conclusion, increased translation initiation mediated by the mRNA cap-binding complex eIF4F contributes to the induction of protein synthesis during compensatory liver growth. Further study of factors that regulate translation initiation may provide insight into mechanisms that govern metabolic homeostasis and regeneration in response to liver injury.

Caerulein-induced acute pancreatitis inhibits protein synthesis through effects on eIF2B and eIF4F.

Acute pancreatitis (AP) has been shown in some studies to inhibit total protein synthesis in the pancreas, whereas in other studies, protein synthesis was not affected. Previous in vitro work has shown that high concentrations of cholecystokinin both inhibit protein synthesis and inhibit the activity of the guanine nucleotide exchange factor eukaryotic initiation factor (eIF)2B by increasing the phosphorylation of eIF2alpha. We therefore evaluated in C57BL/6 mice the effects of caerulein-induced AP on pancreatic protein synthesis, eIF2B activity and other protein translation regulatory mechanisms. Repetitive hourly injections of caerulein were administered at 50 microg/kg ip. Pancreatic protein synthesis was reduced 10 min after the initial caerulein administration and was further inhibited after three and five hourly injections. Caerulein inhibited the two major regulatory points of translation initiation: the activity of the guanine nucleotide exchange factor eIF2B (with an increase of eIF2alpha phosphorylation) and the formation of the eIF4F complex due, in part, to degradation of eIF4G. This inhibition was not accounted for by changes in the upstream stimulatory pathway, because caerulein activated Akt as well as phosphorylating the downstream effectors of mTOR, 4E-BP1, and ribosomal protein S6. Caerulein also decreased the phosphorylation of the eukaryotic elongation factor 2, implying that this translation factor was not inhibited in AP. Thus the inhibition of pancreatic protein synthesis in this model of AP most likely results from the inhibition of translation initiation as a result of increased eIF2alpha phosphorylation, reduction of eIF2B activity, and the inhibition of eIF4F complex formation.

Increased levels of the translation initiation factor eIF4E in differentiating epithelial lung tumor cell lines

Rates of eukaryotic protein synthesis and proliferation are dependent upon the availability of eIF4F, the cap-binding translation initiation complex that guides the ribosome onto the mRNA. One possible rate-limiting factor in eIF4F complex formation is the availability of eIF4E, which interacts specifically with the mRNA cap structure. As such, it has a potential role in the selective translation of growth-related mRNAs, with overexpression of eIF4E resulting in aberrant cell growth and transformation. A number of studies suggest that eIF4E may play a role in cellular differentiation as well as proliferation. We have previously reported that post-transcriptional regulation is involved in the induction of keratins in epithelial lung tumor cell lines exposed to the differentiation-modulating agent, bromo-deoxyuridine (BrdU). Here, we demonstrate that these BrdU-treated lung cells express elevated levels of eIF4E protein and enhanced phosphorylation of eIF4E. Overexpression of eIF4E by cDNA transfection in the poorly differentiated, keratin-negative human lung cell line, DLKP, was found to promote a flattened, more epithelial appearance to these cells, coupled with the induction of simple keratins (keratins 8 and 18). In contrast, levels of eIF4E expression were found to decrease during BrdU-induced differentiation of the leukemic cell line, HL-60, suggesting that there are cell-type differences in the response to BrdU and in the requirement for eIF4E during differentiation.

Possible mechanisms involved in the down-regulation of translation during transient global ischaemia in the rat brain.

The striking correlation between neuronal vulnerability and down-regulation of translation suggests that this cellular process plays a critical part in the cascade of pathogenetic events leading to ischaemic cell death. There is compelling evidence supporting the idea that inhibition of translation is exerted at the polypeptide chain initiation step, and the present study explores the possible mechanism/s implicated. Incomplete forebrain ischaemia (30 min) was induced in rats by using the four-vessel occlusion model. Eukaryotic initiation factor (eIF)2, eIF4E and eIF4E-binding protein (4E-BP1) phosphorylation levels, eIF4F complex formation, as well as eIF2B and ribosomal protein S6 kinase (p70(S6K)) activities, were determined in different subcellular fractions from the cortex and the hippocampus [the CA1-subfield and the remaining hippocampus (RH)], at several post-ischaemic times. Increased phosphorylation of the alpha subunit of eIF2 (eIF2 alpha) and eIF2B inhibition paralleled the inhibition of translation in the hippocampus, but they normalized to control values, including the CA1-subfield, after 4--6 h of reperfusion. eIF4E and 4E-BP1 were significantly dephosphorylated during ischaemia and total eIF4E levels decreased during reperfusion both in the cortex and hippocampus, with values normalizing after 4 h of reperfusion only in the cortex. Conversely, p70(S6K) activity, which was inhibited in both regions during ischaemia, recovered to control values earlier in the hippocampus than in the cortex. eIF4F complex formation diminished both in the cortex and the hippocampus during ischaemia and reperfusion, and it was lower in the CA1-subfield than in the RH, roughly paralleling the observed decrease in eIF4E and eIF4G levels. Our findings are consistent with a potential role for eIF4E, 4E-BP1 and eIF4G in the down-regulation of translation during ischaemia. eIF2 alpha, eIF2B, eIF4G and p70(S6K) are positively implicated in the translational inhibition induced at early reperfusion, whereas eIF4F complex formation is likely to contribute to the persistent inhibition of translation observed at longer reperfusion times.

Possible mechanisms involved in the down-regulation of translation during transient global ischaemia in the rat brain

The striking correlation between neuronal vulnerability and down-regulation of translation suggests that this cellular process plays a critical part in the cascade of pathogenetic events leading to ischaemic cell death. There is compelling evidence supporting the idea that inhibition of translation is exerted at the polypeptide chain initiation step, and the present study explores the possible mechanism/s implicated. Incomplete forebrain ischaemia (30min) was induced in rats by using the four-vessel occlusion model. Eukaryotic initiation factor (eIF)2, eIF4E and eIF4E-binding protein (4E-BP1) phosphorylation levels, eIF4F complex formation, as well as eIF2B and ribosomal protein S6 kinase (p70S6K) activities, were determined in different subcellular fractions from the cortex and the hippocampus [the CA1-subfield and the remaining hippocampus (RH)], at several post-ischaemic times. Increased phosphorylation of the α subunit of eIF2 (eIF2α) and eIF2B inhibition paralleled the inhibition of translation in the hippocampus, but they normalized to control values, including the CA1-subfield, after 4–6h of reperfusion. eIF4E and 4E-BP1 were significantly dephosphorylated during ischaemia and total eIF4E levels decreased during reperfusion both in the cortex and hippocampus, with values normalizing after 4h of reperfusion only in the cortex. Conversely, p70S6K activity, which was inhibited in both regions during ischaemia, recovered to control values earlier in the hippocampus than in the cortex. eIF4F complex formation diminished both in the cortex and the hippocampus during ischaemia and reperfusion, and it was lower in the CA1-subfield than in the RH, roughly paralleling the observed decrease in eIF4E and eIF4G levels. Our findings are consistent with a potential role for eIF4E, 4E-BP1 and eIF4G in the down-regulation of translation during ischaemia. eIF2α, eIF2B, eIF4G and p70S6K are positively implicated in the translational inhibition induced at early reperfusion, whereas eIF4F complex formation is likely to contribute to the persistent inhibition of translation observed at longer reperfusion times.

Modifications of eukaryotic initiation factor 4F (eIF4F) in adult cardiocytes by adenoviral gene transfer: differential effects on eIF4F activity and total protein synthesis rates.

In adult feline cardiocytes, increases in eukaryotic initiation factor 4F (eIF4F) activity are correlated with accelerated rates of total protein synthesis produced in response to increased load. Adenoviral gene transfer was employed to increase either eIF4F complex formation or the phosphorylation of eIF4E on Ser-209. To simulate load,cardiocytes were electrically stimulated to contract (2 Hz,5 ms pulses). Non-stimulated cardiocytes were used as controls.Adenovirus-mediated overexpression of wild-type eIF4E increased the total eIF4E pool by 120-140% above endogenous levels after 24 h and produced a corresponding increase in eIF4F content.However, it did not accelerate total protein synthesis rates inquiescent cardiocytes; neither did it potentiate the increase produced by contraction. To modify the affinity of eIF4F, cardiocytes were infected with a mutant (eIF4E/W56F) with a decreased binding affinity for the mRNA cap. Overexpression of eIF4E/W56F increased the quantity of eIF4F but the rate of total protein synthesis was decreased inquiescent and contracting cardiocytes. Overexpression of a mutant that blocked eIF4E phosphorylation (eIF4E/S209A) increased the quantity ofeIF4F without any significant effect on total protein synthesis rates in quiescent or contracting cardiocytes. Overexpression of the eIF4Ekinase Mnk-1 increased eIF4E phosphorylation without a corresponding increase in eIF4F complex formation or in the rate of total protein synthesis. We conclude the following: (1) eIF4F assembly is increased by raising eIF4E levels via adenoviral gene transfer; (2) the capbinding affinity of eIF4F is a rate-limiting determinant for total protein synthesis rates; and (3) increases in the quantity of eIF4Falone or in eIF4E phosphorylation are not sufficient to accelerate total protein synthesis rates.

Modifications of eukaryotic initiation factor 4F (eIF4F) in adult cardiocytes by adenoviral gene transfer: differential effects on eIF4F activity and total protein synthesis rates

In adult feline cardiocytes, increases in eukaryotic initiation factor 4F (eIF4F) activity are correlated with accelerated rates of total protein synthesis produced in response to increased load. Adenoviral gene transfer was employed to increase either eIF4F complex formation or the phosphorylation of eIF4E on Ser-209. To simulate load, cardiocytes were electrically stimulated to contract (2Hz, 5ms pulses). Non-stimulated cardiocytes were used as controls. Adenovirus-mediated overexpression of wild-type eIF4E increased the total eIF4E pool by 120–140% above endogenous levels after 24h and produced a corresponding increase in eIF4F content. However, it did not accelerate total protein synthesis rates in quiescent cardiocytes; neither did it potentiate the increase produced by contraction. To modify the affinity of eIF4F, cardiocytes were infected with a mutant (eIF4E/W56F) with a decreased binding affinity for the mRNA cap. Overexpression of eIF4E/W56F increased the quantity of eIF4F but the rate of total protein synthesis was decreased in quiescent and contracting cardiocytes. Overexpression of a mutant that blocked eIF4E phosphorylation (eIF4E/S209A) increased the quantity of eIF4F without any significant effect on total protein synthesis rates in quiescent or contracting cardiocytes. Overexpression of the eIF4E kinase Mnk-1 increased eIF4E phosphorylation without a corresponding increase in eIF4F complex formation or in the rate of total protein synthesis. We conclude the following: (1) eIF4F assembly is increased by raising eIF4E levels via adenoviral gene transfer; (2) the cap binding affinity of eIF4F is a rate-limiting determinant for total protein synthesis rates; and (3) increases in the quantity of eIF4F alone or in eIF4E phosphorylation are not sufficient to accelerate total protein synthesis rates.

Feeding stimulates protein synthesis in muscle and liver of neonatal pigs through an mTOR-dependent process.

Protein synthesis is repressed in both skeletal muscle and liver after a short-term fast and is rapidly stimulated in response to feeding. Previous studies in rats and pigs have shown that the feeding-induced stimulation of protein synthesis is associated with activation of the 70-kDa ribosomal protein S6 kinase (S6K1) as well as enhanced binding of eukaryotic initiation factor eIF4E to eIF4G to form the active eIF4F complex. In cells in culture, hormones and nutrients regulate both of these events through a protein kinase termed the mammalian target of rapamycin (mTOR). In the present study, the involvement of mTOR in the feeding-induced stimulation of protein synthesis in skeletal muscle and liver was examined. Pigs at 7 days of age were fasted for 18 h, and then one-half of the animals were fed. In addition, one-half of the animals in each group were administered rapamycin (0.75 mg/kg) 2 h before feeding. The results reveal that treating 18-h fasted pigs with rapamycin, a specific inhibitor of mTOR, before feeding prevented the activation of S6K1 and the changes in eIF4F complex formation observed in skeletal muscle and liver after feeding. Rapamycin also ablated the feeding-induced stimulation of protein synthesis in liver. In contrast, in skeletal muscle, rapamycin attenuated, but did not prevent, the stimulation of protein synthesis in response to feeding. The results suggest that feeding stimulates hepatic protein synthesis through an mTOR-dependent process involving enhanced eIF4F complex formation and activation of S6K1. However, in skeletal muscle, these two processes may account for only part of the stimulation of protein synthesis, and thus additional steps may be involved in the response.

Distinct Signalling Pathways Mediate Insulin and Phorbol Ester-stimulated Eukaryotic Initiation Factor 4F Assembly and Protein Synthesis in HEK 293 Cells

Stimulation of serum-starved human embryonic kidney (HEK) 293 cells with either the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), or insulin resulted in increases in the phosphorylation of 4E-BP1 and p70 S6 kinase, eIF4F assembly, and protein synthesis. All these effects were blocked by rapamycin, a specific inhibitor of mTOR. Phosphatidylinositol 3-kinase and protein kinase B were activated by insulin but not by TPA. Therefore TPA can induce eIF4F assembly, protein synthesis, and the phosphorylation of p70 S6 kinase and 4E-BP1 independently of both phosphatidylinositol 3-kinase and protein kinase B. Using two structurally unrelated inhibitors of MEK (PD098059 and U0126), we provide evidence that Erk activation is important in TPA stimulation of eIF4F assembly and the phosphorylation of p70 S6 kinase and 4E-BP1 and that basal MEK activity is important for basal, insulin, and TPA-stimulated protein synthesis. Transient transfection of constitutively active mitogen-activated protein kinase interacting kinase 1 (the eIF4E kinase) indicated that inhibition of protein synthesis and eIF4F assembly by PD098059 is not through inhibition of eIF4E phosphorylation but of other signals emanating from MEK. This report also provides evidence that increased eIF4E phosphorylation alone does not affect the assembly of the eIF4F complex or general protein synthesis.

Role of load in regulating eIF-4F complex formation in adult feline cardiocytes.

This study examined whether cardiocyte load increases eIF-4F complex formation. To increase load in vitro, adult feline cardiocytes were electrically stimulated to contract (1 Hz, 5-ms pulses). eIF-4F complex formation, measured by eIF-4G association with eIF-4E, increased 57 +/- 16% after 4 h of contraction compared with controls. eIF-4F complex formation did not increase on electrical stimulation with 2,3-butanedione monoxime (BDM), an inhibitor of active tension. Both insulin and phorbol ester increased eIF-4F complex formation, but these increases were unaffected by BDM. Insulin caused a shift of eIF-4E binding proteins (4E-BPs) into their hyperphosphorylated gamma-isoforms and dissociation of 4E-BPs from eIF-4E. Rapamycin inhibited 4E-BP phosphorylation in response to insulin but had no effect on eIF-4F complex formation. Electrically stimulated contraction caused a partial shift of 4E-BP1 and 4E-BP2 into the gamma-isoforms, but it had no effect on 4E-BP association with eIF-4E. Rapamycin blocked the increase in eIF-4F complex formation in electrically stimulated cardiocytes and depressed contractility. These data indicate that cardiocyte load causes a tension-dependent increase in eIF-4F complex formation that does not require dissociation of 4E-BPs from eIF-4E.