Dataset Information

Chemoproteomic Profiling Uncovers CDK4-Mediated Phosphorylation of the Translational Suppressor 4E-BP1.

ABSTRACT: Recent estimates of the human proteome suggest there are ?20,000 protein-coding genes, the protein products of which contain >145,000 phosphosites. Unfortunately, in-depth examination of the human phosphoproteome has outpaced the ability to annotate the kinases that mediate these post-translational modifications. To obtain actionable information about phosphorylation-driven signaling cascades, it is essential to identify the kinases responsible for phosphorylating sites that differ across disease states. To fill in these gaps we have developed an unbiased, chemoproteomic approach for identifying high-confidence kinase-substrate interactions with phosphosite specificity. Using this assay, we uncovered the role of cyclin-dependent kinase 4 (CDK4), a clinically validated kinase important for cell-cycle progression, in regulating cap-dependent translation via phosphorylation of the tumor suppressor 4E-BP1. The discovery of this signaling axis sheds light on the mechanisms by which CDK4/6 inhibitors control cell proliferation and constitutes a successful example of kinase discovery using an activity-based, kinase-directed probe.

SUBMITTER: Mitchell DC

PROVIDER: S-EPMC6642002 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Chemoproteomic Profiling Uncovers CDK4-Mediated Phosphorylation of the Translational Suppressor 4E-BP1.

Mitchell Dylan C DC Menon Arya A Garner Amanda L AL

Cell chemical biology 20190502 7

Recent estimates of the human proteome suggest there are ∼20,000 protein-coding genes, the protein products of which contain >145,000 phosphosites. Unfortunately, in-depth examination of the human phosphoproteome has outpaced the ability to annotate the kinases that mediate these post-translational modifications. To obtain actionable information about phosphorylation-driven signaling cascades, it is essential to identify the kinases responsible for phosphorylating sites that differ across diseas ...[more]

PMID: 31056462

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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.

Dataset Information

Chemoproteomic Profiling Uncovers CDK4-Mediated Phosphorylation of the Translational Suppressor 4E-BP1.

Publications

Chemoproteomic Profiling Uncovers CDK4-Mediated Phosphorylation of the Translational Suppressor 4E-BP1.

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