Dissection of Kinase Isoforms through Orthogonal and Chemical Inducible Signaling Cascades

The interference from endogenous signaling enzymes represents a major hurdle for building orthogonal signaling cascade inside cells, particularly among the close related isoforms within an enzyme family. Here we employed the genetically-encoded chemical decaging strategy to build orthogonal-activated kinase isoforms, with the endogenous counterparts temporally disabled by an extracellular-delivered bacterial effector. This approach eliminated the potential interference from other kinase isoforms as well as the endogenous kinases, which allowed the specific, gain-of-function report of mitogen activated protein kinase kinase 1 (MEK1) activity as opposed to MEK2 with high temporal resolution. Our study dissected the distinct enzymatic activity, feedback regulation and signal outputs between these close related kinase isoforms.