Binding Of 4E-BP1 Research Articles

4E‐BP1/2 Deletion Enhances mRNA Cap‐binding Complex Assembly and Protein Synthesis in Immobilized Skeletal Muscle But is Not Sufficient to Prevent Muscle Atrophy

Skeletal muscle atrophy in long‐term care patients is positively correlated with worse health outcomes and patient mortality. Immobilization‐induced muscle atrophy is due to an imbalance between protein synthesis and protein degradation. The imbalance favors degradation due to anabolic resistance within the muscle. Targeting regulators of protein synthesis to increase mRNA translation under immobilized conditions may improve the protein synthesis/degradation imbalance and ameliorate immobilization‐induced skeletal muscle atrophy. 4E‐BP1 and 4E‐BP2 (eukaryotic initiation factor 4E (eIF4E) binding proteins 1 and 2) are important regulators of mRNA translation initiation that bind to eIF4E and prevents its binding to the scaffold protein eIF4G, an essential and rate‐limiting step of cap‐dependent mRNA translation. Previous studies have implicated eIF4E and its binding proteins in disuse‐induced repression of muscle protein synthesis. Consequently, we hypothesize that eliminating 4E‐BP1/2 may enhance mRNA translation and protect against disuse‐induced muscle atrophy.The hindlimb muscles of 8–9‐week‐old C57Bl/6 mixed‐sex wild‐type (WT) (20.25±2.39 g; n=19) and global 4E‐BP1/4E‐BP2 double knockout (DKO) (20.55±2.65 g; n=19) mice were compared after 3 days of unilateral hindlimb immobilization. Each mouse had one hindlimb immobilized in the plantarflexed position while the contralateral nonimmobilized hindlimb served as a control. Food and water were provided to the mice ad libitum. The hindlimb muscles were removed after a 4‐6 h fast, and the weights were compared between both genotypes for both the immobilized and free limbs. The gastrocnemius muscles were homogenized, eIF4E was immunoprecipitated, and eIF4E, eIF4G, and 4E‐BP1 in the immunoprecipitated sample was assessed by western blot analysis. In the immobilized gastrocnemius of the DKO mice the amount of eIF4G associated with eIF4E was higher than in control mice (p=0.001). In addition, mRNA translation, as assessed in a separate study by the incorporation of puromycin (0.04 μmol puromycin/g of body mass) into protein in the gastrocnemius, was also higher in muscle of the immobilized leg of DKO (n=24) compared to control mice (n=26) (17.1% increase, p=0.012). Surprisingly, eIF4E binding protein deficiency did not prevent muscle atrophy, as the mass of the gastrocnemius, plantaris, and soleus muscles was reduced to a similar extent in the immobilized compared to the control leg of both DKO and wildtype mice. To assess a possible role for upregulation of protein degradation in disuse‐induced muscle atrophy, expression of the E3 ubiquitin ligase, MAFbx‐1 (muscle atrophy f‐box 1) was also assessed. MAFbx‐1 expression was elevated in the gastrocnemius of the immobilized compared to the contralateral non‐immobilized limb, though no significant difference was found between genotypes. More research into other components of the mRNA cap binding complex, e.g., eIF4A, and additional biomarkers of protein degradative pathways may reveal more details on the mechanism of immobilization‐induced muscle atrophy.

A biphenyl inhibitor of eIF4E targeting an internal binding site enables the design of cell-permeable PROTAC-degraders.

The eukaryotic translation initiation factor 4E (eIF4E) is the master regulator of cap-dependent protein synthesis. Overexpression of eIF4E is implicated in diseases such as cancer, where dysregulation of oncogenic protein translation is frequently observed. eIF4E has been an attractive target for cancer treatment. Here we report a high-resolution X-ray crystal structure of eIF4E in complex with a novel inhibitor (i4EG-BiP) that targets an internal binding site, in contrast to the previously described inhibitor, 4EGI-1, which binds to the surface. We demonstrate that i4EG-BiP is able to displace the scaffold protein eIF4G and inhibit the proliferation of cancer cells. We provide insights into how i4EG-BiP is able to inhibit cap-dependent translation by increasing the eIF4E-4E-BP1 interaction while diminishing the interaction of eIF4E with eIF4G. Leveraging structural details, we designed proteolysis targeted chimeras (PROTACs) derived from 4EGI-1 and i4EG-BiP and characterized these on biochemical and cellular levels. We were able to design PROTACs capable of binding eIF4E and successfully engaging Cereblon, which targets proteins for proteolysis. However, these initial PROTACs did not successfully stimulate degradation of eIF4E, possibly due to competitive effects from 4E-BP1 binding. Our results highlight challenges of targeted proteasomal degradation of eIF4E that must be addressed by future efforts.

Inhibition of mTORC1 by lncRNA H19 via disrupting 4E-BP1/Raptor interaction in pituitary tumours

Aberrant expression of long noncoding RNA H19 has been associated with tumour progression, but the underlying molecular tumourigenesis mechanisms remain largely unknown. Here, we report that H19 expression is frequently downregulated in human primary pituitary adenomas and is negatively correlated with tumour progression. Consistently, upregulation of H19 expression inhibits pituitary tumour cell proliferation in vitro and tumour growth in vivo. Importantly, we uncover a function of H19, which controls cell/tumour growth through inhibiting function of mTORC1 but not mTORC2. Mechanistically, we show that H19 could block mTORC1-mediated 4E-BP1 phosphorylation without affecting S6K1 activation. At the molecular level, H19 interacted with 4E-BP1 at the TOS motif and competitively inhibited 4E-BP1 binding to Raptor. Finally, we demonstrate that H19 is more effective than cabergoline treatment in the suppression of pituitary tumours. Together, our study uncovered the role of H19-mTOR-4E-BP1 axis in pituitary tumour growth regulation that may be a potential therapeutic target for human pituitary tumours.

Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine

Although amino acids are known regulators of translation, the unique contributions of specific amino acids are not well understood. We compared effects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profiling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no significant effect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E. Leucine starvation suppressed polysome formation and was the only tested condition that caused a significant decrease in 4EBP1 phosphorylation or increase in 4EBP1 binding to eIF4E, but effects of leucine starvation were not replicated by overexpressing nonphosphorylatable 4EBP1. This suggests the binding of 4EBP1 to eIF4E may not by itself explain the suppression of mRNA translation under conditions of leucine starvation. Ribosome profiling suggested that leucine deprivation may primarily inhibit ribosome loading, whereas methionine deprivation may primarily impair start site recognition. These data underscore our lack of a full understanding of how mRNA translation is regulated and point to a unique regulatory role of methionine status on translation initiation that is not dependent upon eIF2 phosphorylation.

1H, 13C, and 15N backbone chemical shift assignments of 4E-BP144-87 and 4E-BP144-87 bound to eIF4E.

The eukaryotic translational initiation factor 4G (eIF4G) interacts with the cap-binding protein eIF4E through a consensus binding motif, Y(X)4LΦ (where X is any amino acid and Φ is a hydrophobic residue). 4E binding proteins (4E-BPs), which also contain a Y(X)4LΦ motif, regulate the eIF4E/eIF4G interaction. The non- or minimally-phosphorylated form of 4E-BP1 binds eIF4E, preventing eIF4E from interacting with eIF4G, thus inhibiting translation initiation. 4EGI-1, a small molecule inhibitor of the eIF4E/eIF4G interaction that is under investigation as a novel anti-cancer drug, has a dual activity; it disrupts the eIF4E/eIF4G interaction and stabilizes the binding of 4E-BP1 to eIF4E. Here, we report the complete backbone NMR resonance assignment of an unliganded 4E-BP1 fragment (4E-BP144-87). We also report the near complete backbone assignment of the same fragment in complex to eIF4E/m7GTP (excluding the assignment of the last C-terminus residue, D87). The chemical shift data constitute a prerequisite to understanding the mechanism of action of translation initiation inhibitors, including 4EGI-1, that modulate the eIF4E/4E-BP1 interaction.

Mesenchymal stromal cells inhibit CD25 expression via the mTOR pathway to potentiate T-cell suppression

Mesenchymal stromal cells (MSCs) are known to suppress T-cell activation and proliferation. Several studies have reported that MSCs suppress CD25 expression in T cells. However, the molecular mechanism underlying MSC-mediated suppression of CD25 expression has not been fully examined. Here, we investigated the mTOR pathway, which is involved in CD25 expression in T cells. We showed that MSCs inhibited CD25 expression, which was restored in the presence of an inducible nitric oxide synthase (iNOS) inhibitor. Since CD25 mRNA expression was not inhibited, we focused on determining whether MSCs modulated components of the mTOR pathway in T cells. MSCs increased the phosphorylation of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK) and decreased the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). In addition, the expression of 4E-BP1 increased dramatically in the presence of MSCs. An m7GTP pull-down assay showed increased binding of 4E-BP1 to the 5′ cap-binding eukaryotic translation initiation factor 4E (eIF4E) complex in the presence of MSCs, which resulted in inhibition of mRNA translation. Treatment with 4EGI-1, a synthetic inhibitor of mRNA translation, also reduced CD25 expression in T cells. Polysome analysis confirmed decreased CD25 mRNA in the polysome-rich fraction in the presence of MSCs. Taken together, our results showed that nitric oxide, produced by MSCs, inhibits CD25 translation through regulation of the LKB1-AMPK-mTOR pathway to suppress T cells.

Kavalactone yangonin induces autophagy and sensitizes bladder cancer cells to flavokawain A and docetaxel via inhibition of the mTOR pathway.

Consumption of kava (Piper methysticum Forst) has been linked to reduced cancer risk in the South Pacific Islands. Kavalactones are major bioactive components in kava root extracts, which have recently demonstrated anti-cancer activities. However, molecular mechanisms of kavalactones' anti-cancer action remain largely unknown. We have identified two kavalactones, yangonin and 5′ 6'-dehydrokawain, as potent inducers of autophagic cell death in bladder cancer cells. The effect of yangonin inducing autophagy is associated with increased expression of beclin and ATG5. In addition, yangonin increases the expression of LKB1 and decreases the phosphorylation of Akt, PRAS40, rpS6, p70S6K and 4E-BP1, leading to increased binding of 4E-BP1 to m7 GTP. The growth inhibitory effects of yangonin were attenuated inTSC1 or LKB1 knockout mouse embryonic fibroblasts, suggesting that TSC1 and LKB1 expression may contribute to optimal growth inhibition by yangonin. Furthermore, yangonin reduces the viability of bladder cancer cell lines derived from different stages of human bladder cancer, and acts synergistically with apoptosis-inducing agents such as docetaxel and flavokawain A. Our results support a novel anti-bladder cancer mechanism by yangonin and further studies are needed to assess the potential use of yangonin for bladder cancer prevention and treatment

4E-BP1 as an oncotarget

The cap-dependent mRNA translation process that enables post-transcriptional control of gene expression and protein synthesis is tightly regulated in eukaryotes. Deregulation of this process, particularly in the formation of the eIF4F translation initiation complex—comprising the eIF4E mRNA cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase—is associated with cancer development and progression. It is widely believed that translation of certain key oncogenic mRNAs is strongly dependent on the level of eIF4E. Consequently, expression of these oncogenic mRNAs is preferentially and dis-proportionately affected by eIF4E availability and is sensitive to alteration of its level. Free eIF4E level can be increased substantially in cancer cells by a number of mechanisms, including elevated eIF4E expression, decreased expression of eIF4E inhibitory binding proteins, 4E-BPs, and release of eIF4E from 4E-BPs by inactivating phosphorylation of 4E-BPs results from the oncogenic activation of PI3K/AKT and RAS/RAF/MEK/ERK signaling pathways. Our recent studies have revealed that 4E-BP1 is a critical regulatory node that integrates oncogenic signals of the P3K/AKT and RAS/RAF/MEK/ERK pathways for cancer progression and metastasis [1-3]. Mutations in genes that encode components of these two pathways occur at high frequency in cancer. Moreover, the AKT and ERK pathways are often concurrently activated by separate mutations in many human tumors. For instance, KRAS and PIK3CA mutation occur simultaneously in colorectal carcinoma; BRAF and PIK3CA mutation in thyroid carcinoma; and BRAF and PTEN mutation occur simultaneously in melanoma. A number of small molecule inhibitors targeting components of these two pathways have been tested for the treatment of cancer in preclinical and clinical studies, but have shown only limited activity as a single agent. Our work demonstrated that in tumors such as colorectal cancer (CRC) with coexistent activation of both PI3K/AKT and RAS/ERK pathways, drug resistance, following inhibition of either pathway, is associated with redundant activation of eIF4E-initiated cap-dependent translation through convergent phosphorylation of the translational repressor 4E-BP1 mediated by the AKT and ERK pathways. We found that combined inhibition of both pathways is required to maximally inhibit 4E-BP1 phosphorylation and subsequent induction of 4E-BP1 binding to eIF4E to prevent cap-dependent translation of oncogenic mRNAs (e.g. survivin) and ultimately achieve antitumor activity. Furthermore, we showed that a genetically-modified non-phosphorylated 4E-BP1 mutant that binds constitutively to eIF4E and represses eIF4E-dependent oncogene expression exerts similar inhibitory effects on tumor growth and metastasis as the combined pharmacological inhibition of AKT and ERK pathways, whereas loss of 4E-BP1 expression induces epithelial-mesenchymal transition through translational control of snail expression and its activity to promote metastatic progression of CRC and reduce CRC dependence on AKT/ERK signaling for translation, survival and motility. Collectively, these findings support the conclusion that deregulation of 4E-BP1 phosphorylation-mediated assembly of eIF4F translation initiation complex causes CRC resistance to its upstream kinase targeted therapies and thus maintain the transformed phenotype for CRC progression and metastasis. Of notice, several lines of evidence have also established incomplete inhibition of 4E-BP1 phosphorylation, amplification of eIF4E or increased eIF4E/4E-BP ratio as a mechanism of primary resistance to anti-PI3K/AKT/mTOR and anti-BRAF/MEK/ERK pathway inhibitors in a variety of cancers [4-6]. Our data and others strongly suggest that 4E-BP1 phosphorylation is an important biomarker and therapeutic target in cancer. It is well know that mTOR kinase complex 1 (mTORC1) phosphorylates 4E-BP1, which in turn, activates cap-dependent translation and promotes protein synthesis. Our recent studies demonstrated that the effects of AKT and ERK activation on translational regulation of CRC progression are largely mediated by mTORC1 [2], suggesting that targeting mTOR is an attractive strategy for cancer therapy. Nevertheless, incomplete inhibition of 4E-BP1 phosphorylation and a concomitant activation of AKT via loss of a negative feedback mechanism by mTOR inhibitors, including rapalogs and mTOR kinase inhibitors, have been implicated in reduction of their therapeutic efficacy. Our most recent findings revealed a critical role of PRAS40, a mTORC1 inhibitor, as a key integrator of mTORC1 and AKT signaling for 4E-BP1-mediated translational regulation of tumor progression, and indicated that the redundant phosphorylation of PRAS40 on Ser183 and Thr246 sites by both mTORC1 and AKT signaling is a novel mechanistic basis for the acquired resistance to mTOR inhibitors in cancer cells [7]. Our findings further suggest that directly targeting the convergence of multiple oncogenic signals on eIF4F translation initiation complex may provide a promising strategy for improving cancer therapy. Several translation initiation inhibitors, including eIF4E antisense-oligonucleotides and the eIF4A inhibitor silvestrol, have recently produced encouraging anti-tumor effects with limited toxicity. Future works require additional clinical correlative studies and await the development of clinically effective translation initiation inhibitors for cancer treatment, either as single agents or as part of combination therapy.

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1

The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T37/T46, followed by T70 and S65. Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14- to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, (54)YXXXXLΦ(60) (motif 1) but lack known phosphorylation sites. We report here a 2.1-Å crystal structure of mouse eIF4E in complex with m(7)GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP150-84). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S65 and T70). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1-mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. The binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.

Regulated in Development and DNA Damage 1 Is Necessary for Hyperglycemia-induced Vascular Endothelial Growth Factor Expression in the Retina of Diabetic Rodents

Vascular endothelial growth factor (VEGF) is considered a major role player in the pathogenesis of diabetic retinopathy, yet the mechanisms regulating its expression are not fully understood. Our laboratory previously demonstrated that diabetes-induced VEGF expression in the retina was dependent on the repressor of mRNA translation 4E-BP1. Interaction of 4E-BP1 with the cap-binding protein eIF4E regulates protein expression by controlling the selection of mRNAs for translation. The process is regulated by the master kinase mTOR in complex 1 (mTORC1), which phosphorylates 4E-BP1, thus promoting its disassociation from eIF4E. In the present study, we investigated the role of the Akt/mTORC1 repressor REDD1 (regulated in development and DNA damage) in diabetes-induced VEGF expression. REDD1 expression was induced by hyperglycemia in the retina of diabetic rodents and by hyperglycemic conditions in Müller cells concomitant with increased VEGF expression. In Müller cells, hyperglycemic conditions attenuated global rates of protein synthesis and cap-dependent mRNA translation concomitant with up-regulated cap-independent VEGF mRNA translation, as assessed by a bicistronic luciferase reporter assay. Hyperglycemic conditions also attenuated mTORC1 signaling and enhanced 4E-BP1 binding to eIF4E. Furthermore, ectopic expression of REDD1 in Müller cells was sufficient to promote both increased 4E-BP1 binding to eIF4E and VEGF expression. Whereas the retina of wild-type mice exhibited increased expression of VEGF and tumor necrosis factor alpha (TNF-α) 4 weeks after streptozotocin administration, the retina of REDD1 knock-out mice failed to do so. Overall, the results demonstrate that REDD1 contributes to the pathogenesis of diabetes in the retina by mediating the pathogenic effects of hyperglycemia.

MNK1 pathway activity maintains protein synthesis in rapalog-treated gliomas

High levels of mammalian target of rapamycin complex 1 (mTORC1) activity in malignant gliomas promote tumor progression, suggesting that targeting mTORC1 has potential as a therapeutic strategy. Remarkably, clinical trials in patients with glioma revealed that rapamycin analogs (rapalogs) have limited efficacy, indicating activation of resistance mechanisms. Targeted depletion of MAPK-interacting Ser/Thr kinase 1 (MNK1) sensitizes glioma cells to the mTORC1 inhibitor rapamycin through an indistinct mechanism. Here, we analyzed how MNK1 and mTORC1 signaling pathways regulate the assembly of translation initiation complexes, using the cap analog m7GTP to enrich for initiation complexes in glioma cells followed by mass spectrometry-based quantitative proteomics. Association of eukaryotic translation initiation factor 4E (eIF4E) with eIF4E-binding protein 1 (4EBP1) was regulated by the mTORC1 pathway, whereas pharmacological blocking of MNK activity by CGP57380 or MNK1 knockdown, along with mTORC1 inhibition by RAD001, increased 4EBP1 binding to eIF4E. Furthermore, combined MNK1 and mTORC1 inhibition profoundly inhibited 4EBP1 phosphorylation at Ser65, protein synthesis and proliferation in glioma cells, and reduced tumor growth in an orthotopic glioblastoma (GBM) mouse model. Immunohistochemical analysis of GBM samples revealed increased 4EBP1 phosphorylation. Taken together, our data indicate that rapalog-activated MNK1 signaling promotes glioma growth through regulation of 4EBP1 and indicate a molecular cross-talk between the mTORC1 and MNK1 pathways that has potential to be exploited therapeutically.

Casein Kinase 1ϵ Promotes Cell Proliferation by Regulating mRNA Translation

Deregulation of translation initiation factors contributes to many pathogenic conditions, including cancer. Here, we report the definition of a novel regulatory pathway for translational initiation with possible therapeutic import in cancer. Specifically, we found that casein kinase 1ε (CK1ε) is highly expressed in breast tumors and plays a critical role in cancer cell proliferation by controlling mRNA translation. Eukaryotic translation initiation factor eIF4E, an essential component of the translation initiation complex eIF4F, is downregulated by binding the negative-acting factor 4E-BP1. We found that genetic or pharmacologic inhibition of CK1ε attenuated 4E-BP1 phosphorylation, thereby increasing 4E-BP1 binding to eIF4E and inhibiting mRNA translation. Mechanistic investigations showed that CK1ε interacted with and phosphorylated 4E-BP1 at two novel sites T41 and T50, which were essential for 4E-BP1 inactivation along with increased mRNA translation and cell proliferation. In summary, our work identified CK1ε as a pivotal regulator of mRNA translation and cell proliferation that acts by inhibiting 4E-BP1 function. As CK1ε is highly expressed in breast tumors, these findings offer an initial rationale to explore CK1ε blockade as a therapeutic strategy to treat cancers driven by deregulated mRNA translation.

Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E, and the mRNA 5′ cap

Initiation is the rate-limiting step during mRNA 5′ cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.

Total 4EBP1 Is Elevated in Liver of Rats in Response to Low Sulfur Amino Acid Intake

Translation initiation is known to be regulated by the binding of eukaryotic initiation factor 4E (eIF4E) by binding proteins (4EBPs), and there is evidence that amino acid deprivation and other cellular stresses upregulate 4EBP1 expression. To pursue the question of whether diets limited in an essential amino acid lead to induction of 4EBP1 expression in vivo, diets that varied in methionine and cystine content were fed to rats for 7 days, and 4EBP1 mRNA and protein levels and 4EBP1 phosphorylation state were determined. Total 4EBP1 mRNA and protein abundance increased in liver of rats with severely deficient intakes of sulfur amino acids (0.23% or 0.11% methionine without cystine) but not in animals with a less restricted intake of sulfur amino acids (0.11% methionine plus 0.35% cystine) but a similarly restricted intake of total diet (53 to 62% of control). The amount of 4EBP1 binding activity (α + β forms) was elevated in liver of rats fed sulfur amino acid-deficient diets, whereas the hyperphosphorylation of 4EBP1 was not affected by dietary treatment. Results suggest that changes in total 4EBP1 expression should be considered when examining mechanisms that attenuate protein synthesis during amino acid deficiency states.

Effects of leucine and its metabolite β‐hydroxy‐β‐methylbutyrate on human skeletal muscle protein metabolism

Maintenance of skeletal muscle mass is contingent upon the dynamic equilibrium (fasted losses–fed gains) in protein turnover. Of all nutrients, the single amino acid leucine (Leu) possesses the most marked anabolic characteristics in acting as a trigger element for the initiation of protein synthesis. While the mechanisms by which Leu is ‘sensed’ have been the subject of great scrutiny, as a branched-chain amino acid, Leu can be catabolized within muscle, thus posing the possibility that metabolites of Leu could be involved in mediating the anabolic effect(s) of Leu. Our objective was to measure muscle protein anabolism in response to Leu and its metabolite HMB. Using [1,2-13C2]Leu and [2H5]phenylalanine tracers, and GC-MS/GC-C-IRMS we studied the effect of HMB or Leu alone on MPS (by tracer incorporation into myofibrils), and for HMB we also measured muscle proteolysis (by arteriovenous (A–V) dilution). Orally consumed 3.42 g free-acid (FA-HMB) HMB (providing 2.42 g of pure HMB) exhibited rapid bioavailability in plasma and muscle and, similarly to 3.42 g Leu, stimulated muscle protein synthesis (MPS; HMB +70%vs. Leu +110%). While HMB and Leu both increased anabolic signalling (mechanistic target of rapamycin; mTOR), this was more pronounced with Leu (i.e. p70S6K1 signalling ≤90 min vs. ≤30 min for HMB). HMB consumption also attenuated muscle protein breakdown (MPB; −57%) in an insulin-independent manner. We conclude that exogenous HMB induces acute muscle anabolism (increased MPS and reduced MPB) albeit perhaps via distinct, and/or additional mechanism(s) to Leu.

Hypothalamic mTORC1 Signaling Controls Sympathetic Nerve Activity and Arterial Pressure and Mediates Leptin Effects

The fundamental importance of the hypothalamus in the regulation of autonomic and cardiovascular functions is well established. However, the molecular processes involved are not well understood. Here, we show that the mammalian (or mechanistic) target of rapamycin (mTOR) signaling in the hypothalamus is tied to the activity of the sympathetic nervous system and to cardiovascular function. Modulation of mTOR complex 1 (mTORC1) signaling caused dramatic changes in sympathetic traffic, blood flow, and arterial pressure. Our data also demonstrate the importance of hypothalamic mTORC1 signaling in transducing the sympathetic and cardiovascular actions of leptin. Moreover, we show that the PI3K pathway links the leptin receptor to mTORC1 signaling and that changes in its activity impact sympathetic traffic and arterial pressure. These findings establish mTORC1 activity in the hypothalamus as a key determinant of sympathetic and cardiovascular regulation and suggest that dysregulated hypothalamic mTORC1 activity may influence the development of cardiovascular diseases.

Rapamycin-insensitive mTORC1 activity controls eIF4E:4E-BP1 binding

The recent development of mammalian target of rapamycin (mTOR) kinase domain inhibitors and genetic dissection of rapamycin-sensitive and -insensitive mTOR protein complexes (mTORC1 and mTORC2) have revealed that phosphorylation of the mTOR substrate 4E-BP1 on amino acids Thr37 and/or Thr46 represents a rapamycin-insensitive activity of mTORC1. Despite numerous previous reports utilizing serine (Ser)-to-alanine (Ala) and threonine (Thr)-to-Ala phosphorylation site mutants of 4E-BP1 to assess which post-translational modification(s) directly regulate binding to eIF4E, an ambiguous understanding persists. This manuscript demonstrates that the initial, rapamycin-insensitive phosphorylation event at Thr46 is sufficient to prevent eIF4E:4E-BP1 binding. This finding is relevant, particularly as mTOR kinase domain inhibitors continue to be assessed for clinical efficacy, since it clarifies a difference between the action of these second-generation mTOR inhibitors and those of rapamycin analogues.

Rapamycin-insensitive mTORC1 activity controls eIF4E:4E-BP1 binding

Rapamycin-insensitive mTORC1 activity controls eIF4E:4E-BP1 binding

Translational Homeostasis via the mRNA Cap-Binding Protein, eIF4E

Translational control of gene expression plays a key role in many biological processes. Consequently, the activity of the translation apparatus is under tight homeostatic control. eIF4E, the mRNA 5' cap-binding protein, facilitates cap-dependent translation and is a major target for translational control. eIF4E activity is controlled by a family of repressor proteins, termed 4E-binding proteins (4E-BPs). Here, we describe the surprising finding that despite the importance of eIF4E for translation, a drastic knockdown of eIF4E caused only minor reduction in translation. This conundrum can be explained by the finding that 4E-BP1 is degraded in eIF4E-knockdown cells. Hypophosphorylated 4E-BP1, which binds to eIF4E, is degraded, whereas hyperphosphorylated 4E-BP1 is refractory to degradation. We identified the KLHL25-CUL3 complex as the E3 ubiquitin ligase, which targets hypophosphorylated 4E-BP1. Thus, the activity of eIF4E is under homeostatic control via the regulation of the levels of its repressor protein 4E-BP1 through ubiquitination.

Abstract 5727: Competitive inhibition of mTOR results in tumor specific radiosensitization

Abstract Translational control of gene expression has been implicated as a component of the cellular response to ionizing radiation (IR). The mammalian target of rapamycin (mTOR) is a critical kinase in the regulation of translation. mTOR exists in two distinct complexes: mTORC1 and mTORC2. mTORC1 regulates 4E-BP1 and p70-s6k, whereas mTORC2 regulates several AGC kinases including AKT. Most studies of mTOR have focused on the use of the allosteric inhibitor rapamycin and its analogs, which incompletely inhibit mTORC1 and do not inhibit mTORC2. In contrast, newly developed competitive mTOR inhibitors (e.g. PP242) inhibit mTORC1 output more completely and inhibit mTORC2. In the study described here, we compared the effects of rapamycin and PP242 on cellular radiosensitivity using a clonogenic assay. In two tumor cell lines originating from different histologies, U251 and MDA-MB-231, PP242 treatment 1h before irradiation increased radiosensitivity with DEFs (dose enhancement factors at 0.1 surviving fraction) of 1.52 and 1.34 respectively, whereas rapamycin had no effect. However, PP242 had no effect on the radiosensitivity of the normal human fibroblast cell line, MRC9. PP242 treatment of U251 cells resulted in a dose-dependent increase in radiosensitivity. Furthermore, addition of PP242 immediately, 1, or 6h after IR resulted in increased radiosensitivity with DEFs of 1.60, 1.50, and 1.26, respectively. To investigate the mechanism of this radiosensitization U251 cells were treated with PP242, irradiated (2Gy) and stained for γH2AX, a marker of DNA double strand breaks (DSBs). PP242 exposure significantly delayed the dispersal of radiation-induced γH2AX foci, consistent with an inhibition of DSB repair. Under conditions of clonogenic survival, exposure of U251 or MDA-MB-231 cells to 2Gy resulted in no detectable increase in mTOR output. PP242 treatment of both tumor lines resulted in complete inhibition of mTORC1/2 output whereas rapamycin only partially inhibited mTORC1 and did not inhibit mTORC2. m7-GTP batch chromatography showed PP242 induced a decrease in cap-bound eIF4G and an increase in cap-bound 4E-BP1, while rapamycin slightly increased 4E-BP1 binding and had no effect on eIF4G binding. Consistent with the effects on cap-complex formation, PP242 treatment caused a considerably greater decrease in polysome abundance than rapamycin. These results suggest that competitive inhibitors of mTOR, in contrast to rapalogs, induce tumor selective radiosensitization. Moreover, the mechanism appears to involve the inhibition of DSB repair and the suppression of translation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5727. doi:1538-7445.AM2012-5727