2023-11-202023-11-202023-08https://hdl.handle.net/2346/96791Protein kinases form the backbone of the cellular signaling networks, and their aberrancy is a major underpinning of many diseases. The human kinome contains >500 protein kinases and they act by transferring the γ-phosphate group of the ATP molecules covalently onto their substrates to activate or repress their activities. The issue is that kinase studies are hindered by a lack of in vivo analysis approaches due to two factors: the inability to distinguish the kinase reaction of interest from other kinases and the cell impermeability of the ATP analogs. This project focuses on AKT1 and AKT2, two prominent protein kinases and therapeutic targets for multiple diseases as our initial model to develop a system for kinase-substrate relationship analysis and kinase activity measurement, in cells. AKT1 and AKT2 are a part of the PI3K-AKT/PKB-PTEN signaling module that integrates signals for cell growth, cell cycle, metabolism and many other cellular processes. In summary, we addressed these issues by combining chemical genetics with nanoparticle mediated intracellular delivery of the ATP analog. Enlargement of the ATP binding pocket, by mutating the gate-keeper Methionine residue to a Glycine, prompted the mutant AKT1 and AKT2 to preferentially use the bulky ATP analog Trinitrophenol-N6-Benzyl-ATP (TNP-A*TP) and, thus, differentiating AKT1 and AKT2 phosphorylation events. The lipid/calcium/phosphate (LCP) nanoparticle was used for efficient intracellular delivery of TNP-A*TP, overcoming the cell impermeability issue. Only mutant, not wildtype, kinases are able to use the TNP-A*TP analog. This approach will help to improve the understanding of the PI3K-AKT/PKB-PTEN signaling pathway.Application/pdfenColorectal cancerProtein kinasesDissertationAccess is not restricted.