Development of a Chemoproteomic Approach to Study Kinase-Substrate Interactions and the Discovery of CDK4 as a 4E-BP1 Kinase

Abstract

An instrumental aspect of cancer progression is the acquired deregulation of the cellular processes that regulate growth and proliferation. Most of these processes are, at some level, controlled by a kinase-catalyzed change in the phosphorylation status of key signaling intermediates. Given the role of kinases in regulating every key cellular process, kinase inhibitors have emerged as an important class of anticancer therapeutics. However, as our knowledge of the phosphoproteome grows, our understanding of the identified phosphorylation sites remains limited. However, the ability to fully deconvolute kinase signaling networks is hampered by the transient nature of kinase-substrate interactions, a property that renders traditional methods of identifying novel protein- protein interactions ineffectual. Thus, new tools are needed with which to identify kinases that act on phosphorylation sites known to contribute to the pathophysiology of cancer. Cap-dependent translation (CDT) is a critical cellular process that enables the expression of mRNA transcripts encoding for growth and survival proteins, many of which contribute to malignancy. Consequently, regulation of CDT is typically maintained at different levels by numerous signaling cascades. However, these pathways ultimately converge to alter the phosphorylation status of the translational suppressor eukaryotic initiation factor 4E binding protein (4E-BP1). 4E-BP1 serves as a gate-keeper of CDT by competing with translation initiation factors for binding to the m7G-cap-binding protein eIF4E. In cancers, oncogenic signaling results in hyperphosphorylation of 4E-BP1, eliminating the negative repression on eIF4E, leading to constitutive CDT. This has provided rationale for the development of mechanistic Target Of Rapamycin (mTOR) inhibitors as cancer therapeutics, as mTOR complex 1 (mTORC1) is thought to be responsible for the ultimate regulation of 4E-BP1 phosphorylation. However, resistance to allosteric and active site mTOR inhibitors is a hallmark of many cancers, which has decreased the clinical efficacy of mTOR-targeted therapeutics. Moreover, 4E-BP1 contains several uncharacterized phosphorylation sites, the function and regulation of which are unknown. These findings have resulted in speculation about the possibility of uncharacterized 4E-BP1 kinases that promote 4E-BP1 hyperphosphorylation, CDT, and tumorigenesis. To facilitate the identification of kinases capable of acting on phosphorylation sites of interest, a kinase crosslinking assay (termed PhAXA) was developed. When coupled to LC-MS/MS analysis, PhAXA yields high confidence kinase-substrate interactions with phosphosite specificity. Proof-of-concept studies demonstrated the broad applicability of this approach by validating known relationships for three distinct kinase-substrate pairs. This chemoproteomic pipeline was then used to uncover the role of CDK4 in regulating 4E-BP1 phosphorylation. Dissection of this relationship revealed that CDK4 activity maintains CDT under conditions of mTORC1 inhibition in CDK4/6 inhibitor-sensitive cell lines. Moreover, Ser101, an orphan, understudied 4E-BP1 phosphorylation site, was identified and validated as a CDK4 substrate. Evaluation of this event provided a mechanism by which inhibition of CDK4 regulates translation of the oncoprotein c-Myc, which drives a host of known cancers. The discovery of this signaling axis has uncovered a novel function of clinically approved CDK4/6 inhibitors, and sheds light on the mechanism by which these drugs synergize with mTORC1-targeted therapies. The successful implementation of this approach to uncover new biology should result in wide- spread adaptation of the PhAXA platform for elucidating novel drug targets for the treatment of cancers and other kinase-driven diseases.