Elucidation of intracellular signalling pathways is of great importance for the development of novel drugs and therapeutic strategies in modern medicine. The highly conserved nature of protein kinases restricts the identification of direct and specific protein-protein interactions, especially within a live cell setting. Most classical substrate capture approaches lack specificity between homologous kinase constructs, and many are performed on lysates, rather than living systems.
This thesis describes an improved protein-protein identification approach utilizing the expanded genetic code to install diazirine-, and azide-carrying reactive cross-linkers for covalent binding to substrates for their subsequent identification via proteomics. Using mutant cyclin-dependent kinase 2 (Cdk2) as a model protein, this approach identified 548 covalently-bound proteins with ≥2 unique proteomic peptides observed. These included 431 potential interacting partners and 117 proteins as predicted substrates carrying the structural Cdk2 phosphorylation motif. Proteins captured included orthodox Cdk2 interactors (cyclin E1, p27, cyclin A2), numerous proteins found previously in published Cdk2 substrate literature, and over 500 new potential Cdk2 interactors. Secondary validation of two selected proteins (QARS and ASNS) showed a potential successful phosphorylation of ASNS by activated Cdk2, as seen via an in vitro kinase assay.
Over 500 new Cdk2 interacting partners and substrates have been identified with greater precision and specificity than previous approaches. This work exemplifies the expansion of the unnatural amino acid substrate capture technology to the field of kinases for modular, fast, and specific identification of protein interactors.