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Cyclin-dependent kinase 4 inhibits the translational repressor 4E-BP1 to promote cap-dependent translation during mitosis-G1 transition.

ABSTRACT: Phosphorylation of translational repressor eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) controls the initiation of cap-dependent translation, a type of protein synthesis that is frequently upregulated in human diseases such as cancer. Because of its critical cellular function, it is not surprising that multiple kinases can post-translationally modify 4E-BP1 to drive aberrant cap-dependent translation. We recently reported a site-selective chemoproteomic method for uncovering kinase-substrate interactions, and using this approach, we discovered the cyclin-dependent kinase (CDK)4 as a new 4E-BP1 kinase. Herein, we describe our extension of this work and reveal the role of CDK4 in modulating 4E-BP1 activity in the transition from mitosis to G1, thereby demonstrating a novel role for this kinase in cell cycle regulation.

SUBMITTER: Mitchell DC

PROVIDER: S-EPMC7188594 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Cyclin-dependent kinase 4 inhibits the translational repressor 4E-BP1 to promote cap-dependent translation during mitosis-G1 transition.

Mitchell Dylan C DC Menon Arya A Garner Amanda L AL

FEBS letters 20191231 8

Phosphorylation of translational repressor eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) controls the initiation of cap-dependent translation, a type of protein synthesis that is frequently upregulated in human diseases such as cancer. Because of its critical cellular function, it is not surprising that multiple kinases can post-translationally modify 4E-BP1 to drive aberrant cap-dependent translation. We recently reported a site-selective chemoproteomic method f ...[more]

PMID: 31853978

Similar Datasets

Hyperglycemia mediates a shift from cap-dependent to cap-independent translation via a 4E-BP1-dependent mechanism.

Project description:Diabetes and its associated hyperglycemia induce multiple changes in liver function, yet we know little about the role played by translational control of gene expression in mediating the responses to these conditions. Here, we evaluate the hypothesis that hyperglycemia-induced O-GlcNAcylation of the translational regulatory protein 4E-BP1 alters hepatic gene expression through a process involving the selection of mRNA for translation. In both streptozotocin (STZ)-treated mice and cells in culture exposed to hyperglycemic conditions, expression of 4E-BP1 and its interaction with the mRNA cap-binding protein eIF4E were enhanced in conjunction with downregulation of cap-dependent and concomitant upregulation of cap-independent mRNA translation, as assessed by a bicistronic luciferase reporter assay. Phlorizin treatment of STZ-treated mice lowered blood glucose concentrations and reduced activity of the cap-independent reporter. Notably, the glucose-induced shift from cap-dependent to cap-independent mRNA translation did not occur in cells lacking 4E-BP1. The extensive nature of this shift in translational control of gene expression was revealed using pulsed stable isotope labeling by amino acids in cell culture to identify proteins that undergo altered rates of synthesis in response to hyperglycemia. Taken together, these data provide evidence for a novel mechanism whereby O-GlcNAcylation of 4E-BP1 mediates translational control of hepatic gene expression.

| S-EPMC3712054 | biostudies-literature

A cyclin-dependent kinase, CDK11/p58, represses cap-dependent translation during mitosis.

Project description:During mitosis, translation of most mRNAs is strongly repressed; none of the several explanatory hypotheses suggested can fully explain the molecular basis of this phenomenon. Here we report that cyclin-dependent CDK11/p58-a serine/threonine kinase abundantly expressed during M phase-represses overall translation by phosphorylating a subunit (eIF3F) of the translation factor eIF3 complex that is essential for translation initiation of most mRNAs. Ectopic expression of CDK11/p58 strongly repressed cap-dependent translation, and knockdown of CDK11/p58 nullified the translational repression during M phase. We identified the phosphorylation sites in eIF3F responsible for M phase-specific translational repression by CDK11/p58. Alanine substitutions of CDK11/p58 target sites in eIF3F nullified its effects on cell cycle-dependent translational regulation. The mechanism of translational regulation by the M phase-specific kinase, CDK11/p58, has deep evolutionary roots considering the conservation of CDK11 and its target sites on eIF3F from C. elegans to humans.

| S-EPMC7599166 | biostudies-literature

The Translational Repressor 4E-BP1 Contributes to Diabetes-Induced Visual Dysfunction.

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.

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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

Loss of 4E-BP1 function induces EMT and promotes cancer cell migration and invasion via cap-dependent translational activation of snail.

Project description:The cap-dependent translation is frequently deregulated in a variety of cancers associated with tumor progression. However, the molecular basis of the translation activation for metastatic progression of cancer remains largely elusive. Here, we demonstrate that activation of cap-dependent translation by silencing the translational repressor 4E-BP1 causes cancer epithelial cells to undergo epithelial-mesenchymal transition (EMT), which is associated with selective upregulation of the EMT inducer Snail followed by repression of E-cadherin expression and promotion of cell migratory and invasive capabilities as well as metastasis. Conversely, inhibition of cap-dependent translation by a dominant active mutant 4E-BP1 effectively downregulates Snail expression and suppresses cell migration and invasion. Furthermore, dephosphorylation of 4E-BP1 by mTORC1 inhibition or directly targeting the translation initiation also profoundly attenuates Snail expression and cell motility, whereas knockdown of 4E-BP1 or overexpression of Snail significantly rescues the inhibitory effects. Importantly, 4E-BP1-regulated Snail expression is not associated with its changes in the level of transcription or protein stability. Together, these findings indicate a novel role of 4E-BP1 in the regulation of EMT and cell motility through translational control of Snail expression and activity, and suggest that targeting cap-dependent translation may provide a promising approach for blocking Snail-mediated metastatic potential of cancer.

| S-EPMC4171609 | biostudies-literature

4E-BP1-dependent translation in microglia controls mechanical hypersensitivity

Project description:Spinal microglia play a pivotal role in the development of neuropathic pain. Peripheral nerve injury induces changes in the transcriptional profile of microglia, including increased expression of components of translational machinery. Whether microglial protein synthesis is stimulated following nerve injury and has a functional role in mediating pain hypersensitivity is unknown. Here, we show that nascent protein synthesis is upregulated in spinal microglia following peripheral nerve injury in both male and female mice. Stimulating mRNA translation in microglia, by selectively ablating the translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), promoted the transition of microglia to a reactive state and induced mechanical hypersensitivity in both sexes, whereas spontaneous pain was increased only in males. Conversely, inhibiting microglial translation by expressing a mutant form of 4E-BP1 in microglia attenuated their activation following peripheral nerve injury and alleviated neuropathic pain in both sexes. Thus, stimulating 4E-BP1-dependent translation promotes microglial reactivity and mechanical hypersensitivity, whereas inhibiting it alleviates neuropathic pain.

2025-02-25 | GSE285677 | GEO

4E-BP1-dependent translation in microglia controls mechanical hypersensitivity

Project description:Spinal microglia play a pivotal role in the development of neuropathic pain. Peripheral nerve injury induces changes in the transcriptional profile of microglia, including increased expression of components of translational machinery. Whether microglial protein synthesis is stimulated following nerve injury and has a functional role in mediating pain hypersensitivity is unknown. Here, we show that nascent protein synthesis is upregulated in spinal microglia following peripheral nerve injury. Stimulating mRNA translation in microglia, via selective ablation of the translational repressor, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), promoted the transition of microglia to a reactive state and induced mechanical hypersensitivity. Conversely, inhibiting microglial translation by expressing mutant 4E-BP1 in microglia attenuated their peripheral nerve injury-induced activation and alleviated neuropathic pain. Thus, the stimulation of 4E-BP1-dependent translation promotes microglia reactivity and mechanical hypersensitivity, whereas its inhibition alleviates neuropathic pain.

2024-01-31 | GSE254472 | GEO

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

CDK12 phosphorylates 4E-BP1 to enable mTORC1-dependent translation and mitotic genome stability.

Project description:The RNA polymerase II (RNAPII) C-terminal domain kinase, CDK12, regulates genome stability, expression of DNA repair genes, and cancer cell resistance to chemotherapy and immunotherapy. In addition to its role in mRNA biosynthesis of DNA repair genes, we show here that CDK12 phosphorylates the mRNA 5' cap-binding repressor, 4E-BP1, to promote translation of mTORC1-dependent mRNAs. In particular, we found that phosphorylation of 4E-BP1 by mTORC1 (T37 and T46) facilitates subsequent CDK12 phosphorylation at two Ser-Pro sites (S65 and T70) that control the exchange of 4E-BP1 with eIF4G at the 5' cap of CHK1 and other target mRNAs. RNA immunoprecipitation coupled with deep sequencing (RIP-seq) revealed that CDK12 regulates release of 4E-BP1, and binding of eIF4G, to many mTORC1 target mRNAs, including those needed for MYC transformation. Genome-wide ribosome profiling (Ribo-seq) further identified specific CDK12 "translation-only" target mRNAs, including many mTORC1 target mRNAs as well as many subunits of mitotic and centromere/centrosome complexes. Accordingly, confocal imaging analyses revealed severe chromosome misalignment, bridging, and segregation defects in cells deprived of CDK12 or CCNK. We conclude that the nuclear RNAPII-CTD kinase CDK12 cooperates with mTORC1, and controls a specialized translation network that is essential for mitotic chromosome stability.

| S-EPMC6446539 | biostudies-literature

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis.

Project description:Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G 1/S boundary) or the Cdk1 inhibitor, RO3306 (G 2/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.

| S-EPMC3903713 | biostudies-literature

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