Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E.

Human eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA cap structure and interacts with eIF4G, which serves as a scaffold protein for the assembly of eIF4E and eIF4A to form the eIF4F complex. eIF4E is an important modulator of cell growth and proliferation. It is the least abundant component of the translation initiation machinery and its activity is modulated by phosphorylation and interaction with eIF4E-binding proteins (4E-BPs). One strong candidate for the eIF4E kinase is the recently cloned MAPK-activated protein kinase, Mnk1, which phosphorylates eIF4E on its physiological site Ser209 in vitro. Here we report that Mnk1 is associated with the eIF4F complex via its interaction with the C-terminal region of eIF4G. Moreover, the phosphorylation of an eIF4E mutant lacking eIF4G-binding capability is severely impaired in cells. We propose a model whereby, in addition to its role in eIF4F assembly, eIF4G provides a docking site for Mnk1 to phosphorylate eIF4E. We also show that Mnk1 interacts with the C-terminal region of the translational inhibitor p97, an eIF4G-related protein that does not bind eIF4E, raising the possibility that p97 can block phosphorylation of eIF4E by sequestering Mnk1.     

NMDA receptor activation results in PKA- and ERK-dependent Mnk1 activation and increased eIF4E phosphorylation in hippocampal area CA1

Protein synthesis is essential for the stabilization of glutamate receptor-dependent forms of long-lasting hippocampal synaptic plasticity and for the consolidation of memory, but the signal transduction mechanisms that regulate translation factors during these processes are not well understood. As a first step towards understanding how translation is activated during synaptic plasticity, we investigated how the eukaryotic initiation factor 4E (eIF4E), a rate-limiting mRNA cap-binding protein, and its kinase, Mnk1, are regulated by protein kinase C (PKC), cAMP-dependent protein kinase (PKA) and N-methyl-D-aspartate (NMDA) receptor activation in hippocampal area CA1. We found that treatment of mouse hippocampal slices with either phorbol ester, to activate PKC, or forskolin, to activate PKA, resulted in activation of Mnk1 and increased eIF4E phosphorylation that was dependent on extracellular signal-regulated kinase (ERK). Similarly, brief treatment of hippocampal slices with NMDA resulted in activation of Mnk1 and increased phosphorylation of eIF4E. The NMDA-induced activation of Mnk1 and increased phosphorylation of eIF4E were dependent on PKA and ERK, but not PKC, and were present in synaptoneurosome preparations. Immunohistochemical analysis revealed that the PKA- and ERK-dependent increases in Mnk1 activation induced by NMDA also occurred in dendrites. These findings identify a specific regulatory pathway that can couple NMDA receptor activation to translation initiation factors in the hippocampus, and may represent a mechanism for triggering dendritic protein synthesis during long-term potentiation and long-term memory formation.     

Adenovirus-specific translation by displacement of kinase Mnk1 from cap-initiation complex eIF4F

Translation of cellular mRNAs involves formation of a cap-binding translation initiation complex known as eIF4F, containing phosphorylated cap-binding protein eIF4E, eIF4E kinase Mnk1, eIF4A, poly(A)-binding protein and eIF4G. Adenovirus is shown to prevent cellular translation by displacing Mnk1 from eIF4F, thereby blocking phosphorylation of eIF4E. Over expression of an eIF4E mutant that cannot be phosphorylated by Mnk1 impairs translation of cellular but not viral late mRNAs. Adenovirus 100k protein is shown to bind the C-terminus of eIF4G in vivo and in vitro, the same region bound by Mnk1. In vivo, 100k protein displaces Mnk1 from eIF4G during adenovirus infection, or in transfected cells. Purified 100k protein also evicts Mnk1 from isolated eIF4F complexes in vitro. A mutant adenovirus with a temperature-sensitive 100k protein that cannot inhibit cellular protein synthesis at restrictive temperature no longer blocks Mnk1 binding to eIF4G, or phosphorylation of eIF4E. We describe a mechanism whereby adenovirus selectively inhibits the translation of cellular but not viral mRNAs by displacement of Mnk1 from eIF4G and inhibition of eIF4E phosphorylation.     

Mitotic Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1232 by Cdk1:Cyclin B Inhibits eIF4A Helicase Complex Binding with RNA

During mitosis, global translation is suppressed, while synthesis of proteins with vital mitotic roles must go on. Prior evidence suggests that the mitotic translation shift involves control of initiation. Yet, no signals specifically targeting translation initiation factors during mitosis have been identified. We used phosphoproteomics to investigate the central translation initiation scaffold and “ribosome adaptor,” eukaryotic initiation factor 4G1 (eIF4G1) in interphase or nocodazole-arrested mitotic cells. This approach and kinase inhibition assays, in vitro phosphorylation with recombinant kinase, and kinase depletion-reconstitution experiments revealed that Ser1232 in eIF4G1 is phosphorylated by cyclin-dependent kinase 1 (Cdk1):cyclin B during mitosis. Ser1232 is located in an unstructured region of the C-terminal portion of eIF4G1 that coordinates assembly of the eIF4G/-4A/-4B helicase complex and binding of the mitogen-activated protein kinase (MAPK) signal-integrating kinase, Mnk. Intense phosphorylation of Ser1232 in mitosis strongly enhanced the interactions of eIF4A with HEAT domain 2 of eIF4G and decreased association of eIF4G/-4A with RNA. Our findings implicate phosphorylation of eIF4G1(Ser1232) by Cdk1:cyclin B and its inhibitory effects on eIF4A helicase activity in the mitotic translation initiation shift.     

Phosphorylation of Mnk1 by caspase-activated Pak2/gamma-PAK inhibits phosphorylation and interaction of eIF4G with Mnk

The mitogen-activated protein kinase-interacting kinase 1 (Mnk1) is phosphorylated by caspase-cleaved protein kinase Pak2/gamma-PAK but not by Cdc42-activated Pak2. Phosphorylation of Mnk1 is rapid, reaching 1 mol/mol within 15 min of incubation with Pak2. A kinetic analysis of the phosphorylation of Mnk1 by Pak2 yields a K(m) of 0.6 microm and a V(max) of 14.9 pmol of (32)P/min/microg of Pak2. Two-dimensional tryptic phosphopeptide mapping of Mnk1 phosphorylated by Pak2 yields two distinct phosphopeptides. Analysis of the phosphopeptides by automated microsequencing and manual Edman degradation identified the sites in Mnk1 as Thr(22) and Ser(27). Mnk1, activated by phosphorylation with Erk2, phosphorylates the eukaryotic initiation factor (eIF) 4E and the eIF4G components of eIF4F. Phosphorylation of Mnk1 by Pak2 does not activate Mnk1, as measured with either eIF4E or eIF4F as substrate. Phosphorylation of Erk2-activated Mnk1 by Pak2 has no effect on phosphorylation of eIF4E but reduces phosphorylation of eIF4G by Mnk1 by up to 50%. Phosphorylation of Mnk1 by Pak2 inhibits binding of eIF4G peptides containing the Mnk1 binding site by up to 80%. When 293T cells are subjected to apoptotic induction by hydrogen peroxide, Mnk1 is phosphorylated at both Thr(22) and Ser(27). These results indicate a role for Pak2 in the down-regulation of translation initiation in apoptosis by phosphorylation of Mnk1.     

Structural basis for competitive inhibition of eIF4G-Mnk1 interaction by the adenovirus 100-kilodalton protein

Translation of most cellular mRNAs involves cap binding by the translation initiation complex. Among this complex of proteins are cap-binding protein eIF4E and the eIF4E kinase Mnk1. Cap-dependent mRNA translation generally correlates with Mnk1 phosphorylation of eIF4E when both are bound to eIF4G. During the late phase of adenovirus (Ad) infection translation of cellular mRNA is inhibited, which correlates with displacement of Mnk1 from eIF4G by the viral 100-kDa (100K) protein and dephosphorylation of eIF4E. Here we describe the molecular mechanism for 100K protein displacement of Mnk1 from eIF4G and elucidate a structural basis for eIF4G interaction with Mnk1 and 100K proteins and Ad inhibition of cellular protein synthesis. The eIF4G-binding site is located in an N-terminal 66-amino-acid peptide of 100K which is sufficient to bind eIF4G, displace Mnk1, block eIF4E phosphorylation, and inhibit eIF4F (cap)-dependent cellular mRNA translation. Ad 100K and Mnk1 proteins possess a common eIF4G-binding motif, but 100K protein binds more strongly to eIF4G than does Mnk1. Unlike Mnk1, for which binding to eIF4G is RNA dependent, competitive binding by 100K protein is RNA independent. These data support a model whereby 100K protein blocks cellular protein synthesis by coopting eIF4G and cap-initiation complexes regardless of their association with mRNA and displacing or blocking binding by Mnk1, which occurs only on preassembled complexes, resulting in dephosphorylation of eIF4E.     

Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo

Eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA 5' cap and brings the mRNA into a complex with other protein synthesis initiation factors and ribosomes. The activity of mammalian eIF4E is important for the translation of capped mRNAs and is thought to be regulated by two mechanisms. First, eIF4E is sequestered by binding proteins, such as 4EBP1, in quiescent cells. Mitogens induce the release of eIF4E by stimulating the phosphorylation of 4EBP1. Second, mitogens and stresses induce the phosphorylation of eIF4E at Ser 209, increasing the affinity of eIF4E for capped mRNA and for an associated scaffolding protein, eIF4G. We previously showed that a mitogen- and stress-activated kinase, Mnk1, phosphorylates eIF4E in vitro at the physiological site. Here we show that Mnk1 regulates eIF4E phosphorylation in vivo. Mnk1 binds directly to eIF4G and copurifies with eIF4G and eIF4E. We identified activating phosphorylation sites in Mnk1 and developed dominant-negative and activated mutants. Expression of dominant-negative Mnk1 reduces mitogen-induced eIF4E phosphorylation, while expression of activated Mnk1 increases basal eIF4E phosphorylation. Activated mutant Mnk1 also induces extensive phosphorylation of eIF4E in cells overexpressing 4EBP1. This suggests that phosphorylation of eIF4E is catalyzed by Mnk1 or a very similar kinase in cells and is independent of other mitogenic signals that release eIF4E from 4EBP1.     

Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation

Feeding promotes protein accretion in skeletal muscle through a stimulation of the mRNA translation initiation phase of protein synthesis either secondarily to nutrient-induced rises in insulin or owing to direct effects of nutrients themselves. The present set of experiments establishes the effects of meal feeding on potential signal transduction pathways that may be important in accelerating mRNA translation initiation. Gastrocnemius muscle from male Sprague-Dawley rats trained to consume a meal consisting of rat chow was sampled before, during, and after the meal. Meal feeding enhanced the assembly of the active eIF4G.eIF4E complex, which returned to basal levels within 3 h of removal of food. The increased assembly of the active eIF4G.eIF4E complex was associated with a marked 10-fold rise in phosphorylation of eIF4G(Ser(1108)) and a decreased assembly of inactive 4E-BP1.eIF4E complex. The reduced assembly of 4E-BP1.eIF4E complex was associated with a 75-fold increase in phosphorylation of 4E-BP1 in the gamma-form during feeding. Phosphorylation of S6K1 on Ser(789) was increased by meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than after 1 h. Phosphorylation of mammalian target of rapamycin (mTOR) on Ser(2448) or Ser(2481), an upstream kinase responsible for phosphorylating both S6K1 and 4E-BP1, was increased at all times during meal feeding, although the extent of phosphorylation was greater at 0.5 h after feeding than after 1 h. Phosphorylation of PKB, an upstream kinase responsible for phosphorylating mTOR, was elevated only after 0.5 h of meal feeding for Thr(308), whereas phosphorylation Ser(473) was significantly elevated at only 0.5 and 1 h after initiation of feeding. We conclude from these studies that meal feeding stimulates two signal pathways in skeletal muscle that lead to elevated eIF4G.eIF4E complex assembly through increased phosphorylation of eIF4G and decreased association of 4E-BP1 with eIF4E.     

The mitogen-activated protein kinase signal-integrating kinase Mnk2 is a eukaryotic initiation factor 4E kinase with high levels of basal activity in mammalian cells

The cap-binding translation initiation factor eukaryotic initiation factor 4E (eIF4E) is phosphorylated in vivo at Ser209 in response to a variety of stimuli. In this paper, we show that the mitogen-activated protein kinase (MAPK) signal-integrating kinase Mnk2 phosphorylates eIF4E at this residue. Mnk2 binds to the scaffolding protein eIF4G, and overexpression of Mnk2 results in increased phosphorylation of endogenous eIF4E, showing that it can act as an eIF4E kinase in vivo. We have identified eight phosphorylation sites in Mnk2, of which at least three potential MAPK sites are likely to be essential for Mnk2 activity. In contrast to that of Mnk1, the activity of overexpressed Mnk2 is high under control conditions and could only be reduced substantially by a combination of PD98059 and SB203580, while the activity of endogenous Mnk2 in Swiss 3T3 cells was hardly affected upon treatment with these inhibitors. These compounds did not abolish phosphorylation of eIF4E, implying that Mnk2 may mediate phosphorylation of eIF4E in Swiss 3T3 cells. In vitro phosphorylation studies show that Mnk2 is a significantly better substrate than Mnk1 for extracellular signal-regulated kinase 2 (ERK2), p38MAPKalpha, and p38MAPKbeta. Therefore, the high levels of activity of Mnk2 under several conditions may be explained by efficient activation of Mnk2 by low levels of activity of the upstream kinases. Interestingly, we found that the association of both Mnk1 and Mnk2 with eIF4G increased upon inhibition of the MAPK pathways while activation of ERK resulted in decreased binding to eIF4G. This might reflect a mechanism to ensure rapid, but transient, phosphorylation of eIF4E upon stimulation of the MAPK pathways.     

Regulation of translation during in vitro maturation of bovine oocytes: the role of MAP kinase, eIF4E (cap binding protein) phosphorylation, and eIF4E-BP1

Meiotic maturation of mammalian oocytes (transition from prophase I to metaphase II) is accompanied by complex changes in the protein phosphorylation pattern. At least two major protein kinases are involved in these events; namely, cdc2 kinase and mitogen-activated protein (MAP) kinase, because the inhibition of these kinases arrest mammalian oocytes in the germinal vesicle (GV) stage. We show that during meiotic maturation of bovine oocytes, the translation initiation factor, eIF4E (the cap binding protein), gradually becomes phosphorylated. This substantial phosphorylation begins at the time of germinal vesicle breakdown (GVBD) and continues to the metaphase II stage. The onset of eIF4E phosphorylation occurs in parallel with a significant increase in overall protein synthesis. However, although eIF4E is nearly fully phosphorylated in metaphase II oocytes, protein synthesis reaches only basal levels at this stage, similar to that of prophase I oocytes, in which the factor remains unphosphorylated. We present evidence that a specific repressor of eIF4E, the binding protein 4E-BP1, is present and could be involved in preventing eIF4E function in metaphase II stage oocytes. Recently, two protein kinases, called Mnk1 and Mnk2, have been identified in somatic cells as eIF4E kinases, both of which are substrates of MAP kinase in vivo. In bovine oocytes, a specific inhibitor of cdk kinases, butyrolactone I, arrests oocytes in GV stage and prevents activation of both cdc2 and MAP kinase. Under these conditions, the phosphorylation of eIF4E is also blocked, and its function in initiation of translation is impaired. In contrast, PD 098059, a specific inhibitor of the MAP kinase activation pathway, which inhibits the MAP kinase kinase, called MEK function, leads only to a postponed GVBD, and a delay in MAP kinase and eIF4E phosphorylation. These results indicate that in bovine oocytes, 1) MAP kinase activation is only partially dependent on MEK kinase, 2) MAP kinase is involved in eIF4E phosphorylation, and 3) the abundance of fully phosphorylated eIF4E does not necessarily directly stimulate protein synthesis. A possible MEK kinase-independent pathway of MAP kinase phosphorylation and the role of 4E-BP1 in repressing translation in metaphase II oocytes are discussed.     

Two structurally atypical HEAT domains in the C-terminal portion of human eIF4G support binding to eIF4A and Mnk1

The X-ray structure of the C-terminal region of human eukaryotic translation initiation factor 4G (eIF4G) has been determined at 2.2 A resolution, revealing two atypical HEAT-repeat domains. eIF4G recruits various translation factors and the 40S ribosomal subunit to the mRNA 5' end. In higher eukaryotes, the C terminus of eIF4G (4G/C) supports translational regulation by recruiting eIF4A, an RNA helicase, and Mnk1, the kinase responsible for phosphorylating eIF4E. Structure-guided surface mutagenesis and protein-protein interaction assays were used to identify binding sites for eIF4A and Mnk1 within the HEAT-repeats of 4G/C. p97/DAP5, a translational modulator homologous to eIF4G, lacks an eIF4A binding site in the corresponding region. The second atypical HEAT domain of the 4G/C binds Mnk1 using two conserved aromatic/acidic-box (AA-box) motifs. Within the first AA-box, the aromatic residues contribute to the hydrophobic core of the domain, while the acidic residues form a negatively charged surface feature suitable for electrostatic interactions with basic residues in Mnk1.     

Mnk2 and Mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4E but not for cell growth or development

Mnk1 and Mnk2 are protein kinases that are directly phosphorylated and activated by extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein (MAP) kinases and implicated in the regulation of protein synthesis through their phosphorylation of eukaryotic translation initiation factor 4E (eIF4E) at Ser209. To investigate their physiological functions, we generated mice lacking the Mnk1 or Mnk2 gene or both; the resulting KO mice were viable, fertile, and developed normally. In embryonic fibroblasts prepared from Mnk1-Mnk2 DKO mice, eIF4E was not detectably phosphorylated at Ser209, even when the ERK and/or p38 MAP kinases were activated. Analysis of embryonic fibroblasts from single KO mice revealed that Mnk1 is responsible for the inducible phosphorylation of eIF4E in response to MAP kinase activation, whereas Mnk2 mainly contributes to eIF4E's basal, constitutive phosphorylation. Lipopolysaccharide (LPS)- or insulin-induced upregulation of eIF4E phosphorylation in the spleen, liver, or skeletal muscle was abolished in Mnk1(-/-) mice, whereas the basal eIF4E phosphorylation levels were decreased in Mnk2(-/-) mice. In Mnk1-Mnk2 DKO mice, no phosphorylated eIF4E was detected in any tissue studied, even after LPS or insulin injection. However, neither general protein synthesis nor cap-dependent translation, as assayed by a bicistronic reporter assay system, was affected in Mnk-deficient embryonic fibroblasts, despite the absence of phosphorylated eIF4E. Thus, Mnk1 and Mnk2 are exclusive eIF4E kinases both in cultured fibroblasts and adult tissues, and they regulate inducible and constitutive eIF4E phosphorylation, respectively. These results strongly suggest that eIF4E phosphorylation at Ser209 is not essential for cell growth during development.     

Roles of mitogen-activated protein kinase signal-integrating kinases 1 and 2 in oxidant-mediated eIF4E phosphorylation

Oxidative stress alters cellular metabolic processes including protein synthesis. The eukaryotic initiation factor, eIF4E, acts in the rate-limiting steps of initiation and promotes nuclear export. Phosphorylation of eIF4E by mitogen activated protein kinase signal-integrating kinases 1 and 2 (Mnk) influences the affinity of eIF4E for the 5'-mRNA cap and fosters nuclear export activity. Although phosphorylation of eIF4E on Ser209 is observed following oxidant exposure, the contribution of Mnk isoforms and the significance of phosphorylation remain elusive. Using a Mnk inhibitor and fibroblasts derived from Mnk knockout mice, we demonstrate that that H2O2 enhances eIF4E phosphorylation in cells containing Mnk1. In contrast, cells containing only Mnk2 show little change or a decrease in eIF4E phosphorylation in response to H2O2. H2O2 also shifted eIF4GI protein from the nucleus to the cytoplasm suggesting that the increases in eIF4E phosphorylation may reflect enhanced substrate availability to cytoplasmic Mnk1. In Mnk1(+/+) cells, H2O2 also enhanced eIF4E phosphorylation in the nucleus to a greater degree than in the cytoplasm, an effect not observed in cells containing Mnk2. In response to H2O2, all MEFs showed increased eIF4E:4E-BP1 and 4E-BP2:eIF4E binding and reduced eIF4E:eIF4GI binding. We also observed a dramatic increase in the amount of Mnk1 associated with eIF4E following affinity chromatography. These changes coincided with a smaller reduction in global protein synthesis in response to H2O2 in the DKO cells. These findings suggest that changes in eIF4GI distribution may enhance eIF4E phosphorylation and that the presence of either Mnk1 or 2 or any degree of eIF4E phosphorylation negatively regulates global protein synthesis in response to oxidant stress.     

Diet-dependent effects of the Drosophila Mnk1/Mnk2 homolog Lk6 on growth via eIF4E

The control of cellular growth is tightly linked to the regulation of protein synthesis. A key function in translation initiation is fulfilled by the 5' cap binding eukaryotic initiation factor 4E (eIF4E), and dysregulation of eIF4E is associated with malignant transformation and tumorigenesis . In mammals, the activity of eIF4E is modulated by phosphorylation at Ser209 by mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) , which themselves are activated by ERK and p38 MAPK in response to mitogens, cytokines or cellular stress . Whether phosphorylation of eIF4E at Ser209 exerts a positive or inhibitory effect on translation efficiency has remained controversial. Here we provide a genetic characterization of the Drosophila homolog of Mnk1/2, Lk6. Lk6 function is dispensable under a high protein diet, consistent with the recent finding that mice lacking both Mnk1 and Mnk2 are not growth-impaired . Interestingly, loss of Lk6 function causes a significant growth reduction when the amino acid content in the diet is reduced. Overexpression of Lk6 also results in growth inhibition in an eIF4E-dependent manner. We propose a model of eIF4E regulation that may reconcile the contradictory findings with regard to the role of phosphorylation by Mnk1/2.     

Phosphorylation of eukaryotic initiation factor (eIF) 4E is not required for de novo protein synthesis following recovery from hypertonic stress in human kidney cells

Previous work has suggested that increased phosphorylation of eukaryotic initiation factor (eIF) 4E at Ser-209 in the C-terminal loop of the protein often correlates with increased translation rates. However, the functional consequences of phosphorylation have remained contentious with our understanding of the role of eIF4E phosphorylation in translational control far from complete. To investigate the role for eIF4E phosphorylation in de novo translation, we studied the recovery of human kidney cells from hypertonic stress. Results show that hypertonic shock caused a rapid inhibition of protein synthesis and the disaggregation of polysomes. These changes were associated with the dephosphorylation of eIF4G, eIF4E, 4E-binding protein 1 (4E-BP1), and ribosomal protein S6. In addition, decreased levels of the eIF4F complex and increased association of 4E-BP1 with eIF4E were observed over a similar time course. The return of cells to isotonic medium rapidly promoted the phosphorylation of these initiation factors, increased levels of eIF4F complexes, promoted polysome assembly, and increased rates of translation. However, by using a cell-permeable, specific inhibitor of eIF4E kinase, Mnk1 (CGP57380), we show that de novo initiation of translation and eIF4F complex assembly during this recovery phase did not require eIF4E phosphorylation.     

TGFβ‐induced PI 3 kinase‐dependent Mnk‐1 activation is necessary for Ser‐209 phosphorylation of eIF4E and mesangial cell hypertrophy

Transforming growth factorβ (TGFβ)‐induced canonical signal transduction is involved in glomerular mesangial cell hypertrophy; however, the role played by the noncanonical TGFβ signaling remains largely unexplored. TGFβ time‐dependently stimulated eIF4E phosphorylation at Ser‐209 concomitant with enhanced phosphorylation of Erk1/2 (extracellular signal regulated kinase1/2) and MEK (mitogen‐activated and extracellular signal‐regulated kinase kinase) in mesangial cells. Inhibition of Erk1/2 by MEK inhibitor or by expression of dominant negative Erk2 blocked eIF4E phosphorylation, resulting in attenuation of TGFβ‐induced protein synthesis and mesangial cell hypertrophy. Expression of constitutively active (CA) MEK was sufficient to induce protein synthesis and hypertrophy similar to those induced by TGFβ. Pharmacological or dominant negative inhibition of phosphatidylinositol (PI) 3 kinase decreased MEK/Erk1/2 phosphorylation leading to suppression of eIF4E phosphorylation. Inducible phosphorylation of eIF4E at Ser‐209 is mediated by Mnk‐1 (mitogen‐activated protein kinase signal‐integrating kinase‐1). Both PI 3 kinase and Erk1/2 promoted phosphorylation of Mnk‐1 in response to TGFβ. Dominant negative Mnk‐1 significantly inhibited TGFβ‐stimulated protein synthesis and hypertrophy. Interestingly, inhibition of mTORC1 activity, which blocks dissociation of eIF4E‐4EBP‐1 complex, decreased TGFβ‐stimulated phosphorylation of eIF4E without any effect on Mnk‐1 phosphorylation. Furthermore, mutant eIF4E S209D, which mimics phosphorylated eIF4E, promoted protein synthesis and hypertrophy similar to TGFβ. These results were confirmed using phosphorylation deficient mutant of eIF4E. Together our results highlight a significant role of dissociation of 4EBP‐1‐eIF4E complex for Mnk‐1‐mediated phosphorylation of eIF4E. Moreover, we conclude that TGFβ‐induced noncanonical signaling circuit involving PI 3 kinase‐dependent Mnk‐1‐mediated phosphorylation of eIF4E at Ser‐209 is required to facilitate mesangial cell hypertrophy. J. Cell. Physiol. 228: 1617–1626, 2013. © 2013 Wiley Periodicals, Inc.     

Inhibition of mammalian target of rapamycin induces phosphatidylinositol 3-kinase-dependent and Mnk-mediated eukaryotic translation initiation factor 4E phosphorylation

The initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in initiating translation of mRNAs, including those encoding oncogenic proteins. Therefore, eIF4E is considered a survival protein involved in cell cycle progression, cell transformation, and apoptotic resistance. Phosphorylation of eIF4E (usually at Ser209) increases its binding affinity for the cap of mRNA and may also favor its entry into initiation complexes. Mammalian target of rapamycin (mTOR) inhibitors suppress cap-dependent translation through inhibition of the phosphorylation of eIF4E-binding protein 1. Paradoxically, we have shown that inhibition of mTOR signaling increases eIF4E phosphorylation in human cancer cells. In this study, we focused on revealing the mechanism by which mTOR inhibition increases eIF4E phosphorylation. Silencing of either mTOR or raptor could mimic mTOR inhibitors' effects to increase eIF4E phosphorylation. Moreover, knockdown of mTOR, but not rictor or p70S6K, abrogated rapamycin's ability to increase eIF4E phosphorylation. These results indicate that mTOR inhibitor-induced eIF4E phosphorylation is secondary to mTOR/raptor inhibition and independent of p70S6K. Importantly, mTOR inhibitors lost their ability to increase eIF4E phosphorylation only in cells where both Mnk1 and Mnk2 were knocked out, indicating that mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism. Given that mTOR inhibitors failed to increase Mnk and eIF4E phosphorylation in phosphatidylinositol 3-kinase (PI3K)-deficient cells, we conclude that mTOR inhibition increases eIF4E phosphorylation through a PI3K-dependent and Mnk-mediated mechanism. In addition, we also suggest an effective therapeutic strategy for enhancing mTOR-targeted cancer therapy by cotargeting mTOR signaling and Mnk/eIF4E phosphorylation.