Delineating the Biology of Translational Repressor 4E-BP1

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Eukaryotic translation initiation factor (eIF4E)-binding protein 1 (4E-BP1) is an intrinsically disordered protein that functions as the gate-keeper of cap-dependent mRNA translation, the process by which mRNA transcripts containing a m7G cap at their 5’ terminus are converted into protein. As cap-containing mRNAs predominantly encode for growth and survival factors, the role of 4E-BP1 in cell biology is significant and aberrant 4E-BP1 activity has been linked to cancer, neurodegenerative diseases and metabolic disorders among others. 4E-BP1 is regulated by phosphorylation: hypophosphorylated 4E-BP1 binds strongly to eIF4E, the m7G cap-binding translation initiation factor, to inhibit translation, while hyperphosphorylated 4E-BP1 releases eIF4E to initiate translation. To date, the only kinase known to affect 4E-BP1 phosphorylation is mechanistic target of rapamycin complex 1 (mTORC1); however, reports have demonstrated that other unknown kinases can also phosphorylate 4E-BP1 to stimulate cap-dependent translation, particularly in cases of mTORC1 inhibition. In order to identify these kinases, we have developed an unbiased, chemoproteomic approach for identifying high confidence kinase-substrate interactions with phosphosite specificity. Using this assay, we have uncovered the role of cyclin- dependent kinase 4 (CDK4), a clinically validated kinase important for cell cycle progression, in driving cap- dependent translation via phosphorylation of 4E-BP1. Importantly, this work constitutes the first example of successful kinase discovery using an activity-based, kinase-directed probe. As 4E-BP1 is phosphorylated at as many as 13 unique sites, we hypothesize that many other kinases signal to and regulate 4E-BP1. Additionally, despite the critical role of 4E-BP1 phosphorylation in protein synthesis, few studies have been disclosed regarding the biological function of each of its phosphorylated serine and threonine residues, including its orphan sites known to be unaffected by mTORC1. To fill in these knowledge gaps, the Specific Aims of this proposal are as follows: (1) To determine the molecular details of CDK4-mediated 4E-BP1 hyperphosphorylation; (2) To determine the functional and mechanistic significance of CDK4-mediated 4E-BP1 hyperphosphorylation; and (3) To identify and validate additional kinases acting on 4E-BP1 using chemoproteomics. Through these studies, we will not only further enhance our knowledge of 4E-BP1-mediated translational regulation, but also illuminate new druggable targets for treatment of the many diseases associated with aberrant cap-dependent translation.