A chemical-genetics and nanoparticle enabled approach for in vivo protein kinase analysis

The human kinome contains >500 protein kinases, and regulates up to 30% of all human proteins. Kinase study is currently hindered by a lack of in vivo analysis approaches, mainly due to two factors: our inability to distinguish the kinase reaction of interest from those of other members of the kinome in live cells and the cell impermeability of the ATP analogs. Herein, we aimed to overcome this issue by combining the widely used chemical genetic method developed by Dr. Kevan Shokat and colleagues with nanoparticle-mediated intracellular delivery of the ATP analog. The critical AKT1 protein kinase, which has been successfully studied with the Shokat method, was used as our initial prototype. Briefly, following the Shokat method, enlargement of the ATP binding pocket was performed by mutating the gate-keeper Methionine residue to a Glycine, prompting the mutant AKT1 to preferentially use the bulky ATP analog N6-Benzyl-ATP-γ-S (A*TPγS) and, thus, differentiating AKT1-catalyzed and other phosphorylation events. The (Lipid/Phosphate/Calcium (LPC) nanoparticle was used for efficient intracellular delivery of A*TPγS, overcoming the cell impermeability issue. We demonstrated that the mutant, but not the wild type, AKT1 was able to use the delivered A*TPγS for autophosphorylation as well as phosphorylating its substrates in live cells. Thus, an in vivo protein kinase analysis method has been developed. The strategy should be widely applicable to other protein kinases.