Unraveling Kinase-Substrate Relationships through Enzymology and Chemical Biology

Abstract

Protein post-translational modifications (PTMs) are important events that expand the complexity of the human proteome. Over 2/3 of all encoded proteins have been shown to be phosphorylated by a class of enzymes called protein kinases. Since phosphorylation affects aspects such as enzyme activity, protein-protein interactions, and cellular localization, aberrant phosphorylation is often implicated in abnormal cell growth and metabolism, giving rise to many diseases including cancer and cardiovascular disorders. Therefore, understanding how kinases recognize and phosphorylate their substrates can provide insights into novel therapeutic development. This dissertation outlines my work on deciphering kinase-substrate relationships through enzymology and chemical biology methods. Starting with a project that focuses on a specific phosphorylation reaction of Akt by the mTORC2 complex, Chapter 2 summarizes our efforts in dissecting the kinetic parameters underlying Akt Ser473 phosphorylation. We prepared stoichiometric mTORC2 complex and semisynthetic Akt proteins to perform enzymatic characterizations using our newly developed quantitative western blot-based assay. In vitro and in cellulo data suggested that mTORC2 directly phosphorylates Akt at Ser473. Biochemical experiments and crosslinking mass spectrometry provided clues to a mechanism through which distal interactions drive mTORC2 substrate recognition. In Chapter 3, I present the structural basis of a long-range recruitment mechanism for mTORC2 substrate recognition. Employing a bisubstrate inhibitor approach, we successfully obtained a first cryo-EM structure of the mTORC2-Akt co-complex. We identified two major interfaces between Akt and the mTORC2 complex that are critical for substrate recruitment: (1) between mSin1 CRIM domain and Akt kinase domain N-lobe, and (2) between mSin1 N-terminus and Akt kinase domain C-lobe. We validated these interfaces in biochemical and cellular assays and suggest this mechanism to be conserved across other canonical mTORC2 substrates. In Chapter 4, I discuss a separate project in which we aim to devise a chemical rescue-based approach to study Ser/Thr kinases, their substrates and precise roles in signaling pathways. We introduced an inactivating mutation into a model kinase, CK2α, and identified small molecule rescue agents that could be supplemented to restore catalytic activity in vitro. This approach appears generalizable, as we have successfully applied chemical rescue to a number of kinases from different families in purified systems. We propose that, with further optimization, chemical rescue can be used in live cells to delineate the physiological functions of protein kinases. In summary, my projects present a range of enzymology and chemical biology approaches that enhance our understanding of kinase-substrate relationships, which could be the key to uncovering disease biology and discovering novel therapeutics.