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

Abstract

Recent estimates of the human proteome suggest that there are ~20,000 protein-coding genes, the protein products of which contain >145,000 phosphorylation sites. Unfortunately, in-depth examination of the human phosphoproteome has far 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 knowledge gaps, 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 regulating cap-dependent translation via phosphorylation of the tumor suppressor 4E-BP1. The discovery of this novel signaling axis sheds new light on the mechanisms by which CDK4/6 inhibitors control cell proliferation and constitutes the first example of successful kinase discovery using an activity-based, kinase-directed probe.