Chemoproteomic Profiling Uncovers CDK4-Mediated Phosphorylation of the Translational Suppressor 4E-BP1.
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ABSTRACT:
SUBMITTER: Mitchell DC
PROVIDER: S-EPMC6642002 | biostudies-literature | 2019 Jul
REPOSITORIES: biostudies-literature
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Project description:Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) inhibits cap-dependent translation in eukaryotes by competing with eIF4G for an interaction with eIF4E. Phosphorylation at Ser-83 of 4E-BP1 occurs during mitosis through the activity of cyclin-dependent kinase 1 (CDK1)/cyclin B rather than through canonical mTOR kinase activity. Here, we investigated the interaction of eIF4E with 4E-BP1 or eIF4G during interphase and mitosis. We observed that 4E-BP1 and eIF4G bind eIF4E at similar levels during interphase and mitosis. The most highly phosphorylated mitotic 4E-BP1 isoform (δ) did not interact with eIF4E, whereas a distinct 4E-BP1 phospho-isoform, EB-γ, phosphorylated at Thr-70, Ser-83, and Ser-101, bound to eIF4E during mitosis. Two-dimensional gel electrophoretic analysis corroborated the identity of the phosphorylation marks on the eIF4E-bound 4E-BP1 isoforms and uncovered a population of phosphorylated 4E-BP1 molecules lacking Thr-37/Thr-46-priming phosphorylation. Moreover, proximity ligation assays for phospho-4E-BP1 and eIF4E revealed different in situ interactions during interphase and mitosis. The eIF4E:eIF4G interaction was not inhibited but rather increased in mitotic cells, consistent with active translation initiation during mitosis. Phosphodefective substitution of 4E-BP1 at Ser-83 did not change global translation or individual mRNA translation profiles as measured by single-cell nascent protein synthesis and eIF4G RNA immunoprecipitation sequencing. Mitotic 5'-terminal oligopyrimidine RNA translation was active and, unlike interphase translation, resistant to mTOR inhibition. Our findings reveal the phosphorylation profiles of 4E-BP1 isoforms and their interactions with eIF4E throughout the cell cycle and indicate that 4E-BP1 does not specifically inhibit translation initiation during mitosis.
| S-EPMC6682726 | biostudies-literature
Project description:Ribonucleotide reductase (RNR), which is a heterodimeric tetramer composed of RRM1 and RRM2 subunits, is the rate-limiting enzyme in the synthesis of deoxyribonucleoside triphosphates (dNTPs) and essential for both DNA replication and the repair of DNA damage. The activity of RNR is coordinated with the cell cycle and regulated by fluctuations in the level of the RRM2 subunit. Multiple cancer types, including Ewing sarcoma tumors, are sensitive to inhibitors of RNR or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. Here, we show that the expression of the RRM2 protein is dependent on active protein synthesis and that 4E-BP1, a repressor of cap-dependent protein translation, specifically regulates the level of the RRM2 protein. Furthermore, inhibition of mTORC1/2, but not mTORC1, activates 4E-BP1, inhibits protein synthesis, and reduces the level of the RRM2 protein in multiple sarcoma cell lines. This effect of mTORC1/2 inhibitors on protein synthesis and RRM2 levels was rescued in cell lines with the CRISPR/Cas9-mediated knockout of 4E-BP1. In addition, the inducible expression of a mutant 4E-BP1 protein that cannot be phosphorylated by mTOR blocked protein synthesis and inhibited the growth of Ewing sarcoma cells in vitro and in vivo in a xenograft. Overall, these results provide insight into the multifaceted regulation of RRM2 protein levels and identify a regulatory link between protein translation and DNA replication.
| S-EPMC7856031 | biostudies-literature
Project description:Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr(69)-phosphorylated alone, Thr(69)- and Thr(36)/Thr(45)-phosphorylated, all these plus Ser(64) phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr(36)/Thr(45) phosphorylation alone was detected without Thr(69) phosphorylation, and neither was Ser(64) phosphorylation without Thr(36)/Thr(45)/Thr(69) phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr(69), Thr(36)/Thr(45), and Ser(64) residues, with 4E-BP1 remaining phosphorylated at Thr(69) alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr(36)/Thr(45) and Ser(64), in addition to Thr(69). Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr(69) is phosphorylated first followed by Thr(36)/Thr(45) phosphorylation, and Ser(64) is phosphorylated last. Thr(69) phosphorylation alone allows binding to eIF4E, and subsequent Thr(36)/Thr(45) phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.
| S-EPMC2966049 | biostudies-literature
Project description:Eukaryotic translation initiation factor 4E (eIF4E)–binding protein 1 (4E-BP1) inhibits cap-dependent translation in eukaryotes by competing with eIF4G for an interaction with eIF4E. Phos-phorylation at Ser-83 of 4E-BP1 occurs during mitosis through the activity of cyclin-dependent kinase 1 (CDK1)/cyclin B rather than through canonical mTOR kinase activity. Here, we investi-gated the interaction of eIF4E with 4E-BP1 or eIF4G during interphase and mitosis. We observed that 4E-BP1 and eIF4G bind eIF4E at similar levels during interphase and mitosis. The most highly phosphorylated mitotic 4E-BP1 isoform (δ) did not interact with eIF4E, whereas a distinct 4E-BP1 phospho-isoform, EB-γ—phosphorylated at Thr-70, Ser-83, and Ser-101—bound to eIF4E during mitosis. Two-dimensional gel electropho-retic analysis corroborated the identity of the phosphorylation marks on the eIF4E-bound 4E-BP1 isoforms and uncovered a population of phosphorylated 4E-BP1 molecules lacking Thr-37/Thr-46–priming phosphorylation. Moreover, proximity ligation assays for phospho–4E-BP1 and eIF4E revealed different in situ interactions during interphase and mitosis. The eIF4E:eIF4G interaction was not inhibited, but rather increased in mitotic cells, consistent with active translation initiation during mitosis. Phospho-defective substitution of 4E-BP1 at Ser-83 did not change global translation or individual mRNA translation profiles as measured by single-cell nascent protein synthesis and eIF4G RNA-immunoprecipitation sequencing. Mitotic 5’-terminal oligopyrimidine RNA translation was active and, unlike interphase translation, resistant to mTOR inhibition. Our findings reveal the phosphorylation profiles of 4E-BP1 isoforms and their interactions with eIF4E throughout the cell cycle and indicate that 4E-BP1 does not specifically inhibit translation initiation during mitosis.
2019-06-14 | GSE131668 | GEO
Project description:PurposeThe translational repressor 4E-BP1 interacts with the mRNA cap-binding protein eIF4E and thereby promotes cap-independent translation of mRNAs encoding proteins that contribute to diabetic retinopathy. Interaction of 4E-BP1 with eIF4E is enhanced in the retina of diabetic rodents, at least in part, as a result of elevated 4E-BP1 protein expression. In the present study, we examined the role of 4E-BP1 in diabetes-induced visual dysfunction, as well as the mechanism whereby hyperglycemia promotes 4E-BP1 expression.MethodsNondiabetic and diabetic wild-type and 4E-BP1/2 knockout mice were evaluated for visual function using a virtual optomotor test (Optomotry). Retinas were harvested from nondiabetic and type 1 diabetic mice and analyzed for protein abundance and posttranslational modifications. Similar analyses were performed on cells in culture exposed to hyperglycemic conditions or an O-GlcNAcase inhibitor (Thiamet G [TMG]).ResultsDiabetes-induced visual dysfunction was delayed in mice deficient of 4E-BP1/2 as compared to controls. 4E-BP1 protein expression was enhanced by hyperglycemia in the retina of diabetic rodents and by hyperglycemic conditions in retinal cells in culture. A similar elevation in 4E-BP1 expression was observed with TMG. The rate of 4E-BP1 degradation was significantly prolonged by either hyperglycemic conditions or TMG. A PEST motif in the C-terminus of 4E-BP1 regulated polyubiquitination, turnover, and binding of an E3 ubiquitin ligase complex containing CUL3.ConclusionsThe findings support a model whereby elevated 4E-BP1 expression observed in the retina of diabetic rodents is the result of O-GlcNAcylation of 4E-BP1 within its PEST motif.
| S-EPMC4811182 | biostudies-literature
Project description:4E-BP1 is a tumor suppressor regulating cap-dependent translation that is in turn controlled by mechanistic target of rapamycin (mTOR) or cyclin-dependent kinase 1 (CDK1) phosphorylation. 4E-BP1 serine 82 (S82) is phosphorylated by CDK1, but not mTOR, and the consequences of this mitosis-specific phosphorylation are unknown. Knock-in mice were generated with a single 4E-BP1 S82 alanine (S82A) substitution leaving other phosphorylation sites intact. S82A mice were fertile and exhibited no gross developmental or behavioral abnormalities, but the homozygotes developed diffuse and severe polycystic liver and kidney disease with aging, and lymphoid malignancies after irradiation. Sublethal irradiation caused immature T-cell lymphoma only in S82A mice while S82A homozygous mice have normal T-cell hematopoiesis before irradiation. Whole genome sequencing identified PTEN mutations in S82A lymphoma and impaired PTEN expression was verified in S82A lymphomas derived cell lines. Our study suggests that the absence of 4E-BP1S82 phosphorylation, a subtle change in 4E-BP1 phosphorylation, might predispose to polycystic proliferative disease and lymphoma under certain stressful circumstances, such as aging and irradiation.
| S-EPMC10162543 | biostudies-literature
Project description:Pancreatic ductal adenocarcinoma (PDAC) relies on hyper-activated protein synthesis. Consistently, human and mouse PDAC lose expression of the translational repressor and mTOR target 4E-BP1. Using genome-wide polysome-profiling, we here explore mRNAs whose translational efficiencies depend on the mTOR/4E-BP1 axis in Miapaca-2 cells. This was performed by isolating cytoplasmic and efficiently translated (heavy polysome-associated) mRNAs from MiaPaca-2 cells upon PP242-mediated mTOR inhibition
2019-09-18 | GSE137553 | GEO
Project description:TSC1 or TSC2 mutations cause Tuberous Sclerosis Complex (TSC), and lead to mechanistic target of rapamycin (mTOR) hyperactivation evidenced by hyperphosphorylation of ribosomal S6 protein and 4-elongation factor binding protein 1 (4E-BP1). Amino acid (AA) levels modulate mTOR-dependent S6 and 4E-BP1 phosphorylation in non-neural cells, but this has not been comprehensively investigated in neurons. The effects of AA levels on mTOR signaling and S6 and 4E-BP1 phosphorylation were analyzed in Tsc2 and Depdc5 (a distinct mTOR regulatory gene associated with epilepsy) CRISPR-edited Neuro2a (N2a) cells and differentiated neurons. Tsc2 or Depdc5 knockout (KO) led to S6 and 4E-BP1 hyperphosphorylation and cell soma enlargement, but while Tsc2 KO N2a cells exhibited reduced S6 phosphorylation (Ser240/244) and cell soma size after incubation in AA free (AAF) media, Depdc5 KO cells did not. Using a CFP/YFP FRET-biosensor coupled to 4E-BP1, we assayed 4E-BP1 phosphorylation in living N2a cells and differentiated neurons following Tsc2 or Depdc5 KO. AAF conditions reduced 4E-BP1 phosphorylation in Tsc2 KO N2a cells but had no effect in Depdc5 KO cells. Rapamycin blocked S6 protein phosphorylation but had no effect on 4E-BP1 phosphorylation, following either Tsc2 or Depdc5 KO. Confocal imaging demonstrated that AAF media promoted movement of mTOR off the lysosome, functionally inactivating mTOR, in Tsc2 KO but not Depdc5 KO cells, demonstrating that AA levels modulate lysosomal mTOR localization and account, in part, for differential effects of AAF conditions following Tsc2 versus Depdc5 KO. AA levels and rapamycin differentially modulate S6 and 4E-BP1 phosphorylation and mTOR lysosomal localization in neurons following Tsc2 KO versus Depdc5 KO. Neuronal mTOR signaling in mTOR-associated epilepsies may have distinct responses to mTOR inhibitors and to levels of cellular amino acids.
| S-EPMC8170708 | biostudies-literature
Project description:Protein synthesis is critical for circadian clock function, but little is known of how translational regulation controls the master pacemaker in mammals, the suprachiasmatic nucleus (SCN). Here we demonstrate that the pivotal translational repressor, the eukaryotic translational initiation factor 4E binding protein 1 (4E-BP1), is rhythmically regulated via the mechanistic target of rapamycin (mTOR) signaling in the SCN and preferentially represses vasoactive intestinal peptide (Vip) mRNA translation. Knockout (KO) of Eif4ebp1 (gene encoding 4E-BP1) leads to upregulation of VIP and higher amplitude of molecular rhythms in the SCN. Consequently, the 4E-BP1 null mice exhibit accelerated re-entrainment to a shifted light/dark cycle and are more resistant to the rhythm-disruptive effects of constant light. Conversely, in Mtor(+/-) mice VIP expression is decreased and susceptibility to the effects of constant light is increased. These results reveal a key role for mTOR/4E-BP1-mediated translational control in regulating entrainment and synchrony of the master clock.
| S-EPMC3856944 | biostudies-literature
Project description:ObjectiveCyclin-dependent kinase 1 (CDK1)/cyclin B1 phosphorylates many of the same substrates as mTORC1 (a key regulator of glucose metabolism), including the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Only mitotic CDK1 phosphorylates 4E-BP1 at residue S82 in mice (S83 in humans), in addition to the common 4E-BP1 phospho-acceptor sites phosphorylated by both CDK1 and mTORC1. We examined glucose metabolism in mice having a single aspartate phosphomimetic amino acid knock in substitution at the 4E-BP1 serine 82 (4E-BP1S82D) mimicking constitutive CDK1 phosphorylation.MethodsKnock-in homozygous 4E-BP1S82D and 4E-BP1S82A C57Bl/6N mice were assessed for glucose tolerance testing (GTT) and metabolic cage analysis on regular and on high-fat chow diets. Gastrocnemius tissues from 4E-BP1S82D and WT mice were subject to Reverse Phase Protein Array analysis. Since the bone marrow is one of the few tissues typically having cycling cells that transit mitosis, reciprocal bone-marrow transplants were performed between male 4E-BP1S82D and WT mice, followed by metabolic assessment, to determine the role of actively cycling cells on glucose homeostasis.ResultsHomozygous knock-in 4E-BP1S82D mice showed glucose intolerance that was markedly accentuated with a diabetogenic high-fat diet (p = 0.004). In contrast, homozygous mice with the unphosphorylatable alanine substitution (4E-BP1S82A) had normal glucose tolerance. Protein profiling of lean muscle tissues, largely arrested in G0, did not show protein expression or signaling changes that could account for these results. Reciprocal bone-marrow transplantation between 4E-BP1S82D and wild-type littermates revealed a trend for wild-type mice with 4E-BP1S82D marrow engraftment on high-fat diets to become hyperglycemic after glucose challenge.Conclusions4E-BP1S82D is a single amino acid substitution that induces glucose intolerance in mice. These findings indicate that glucose metabolism may be regulated by CDK1 4E-BP1 phosphorylation independent from mTOR and point towards an unexpected role for cycling cells that transit mitosis in diabetic glucose control.
| S-EPMC10004604 | biostudies-literature