AbstractThe human kinome contains >500 protein kinases, and regulates up to 30% of the proteome. Kinase study is currently hindered by a lack of in vivo analysis approaches due to two factors: our inability to distinguish the kinase reaction of interest from those of other kinases in live cells and the cell impermeability of the ATP analogs. Herein, we tackled 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 method, was used as our initial prototype. Briefly, enlargement of the ATP binding pocket, by mutating the gate-keeper Methionine residue to a Glycine, prompted 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/calcium/phosphate (LCP) nanoparticle was used for efficient intracellular delivery of A*TPγS, overcoming the cell impermeability issue. The mutant, but not wild-type, AKT1 used the delivered A*TPγS for autophosphorylation and 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.
A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking
