Dissection of Kinase Isoforms through Orthogonal and Chemical Inducible Signaling Cascades
Siqi Zheng1, Xinyuan Fan2, Jie Wang2
1Beijing National Laboratory for Molecular Sciences, Synthetic and Functional Biomolecules Center, Key Laboratory of Bioorganic Chemistry and, Molecular Engineering of the Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Haidian District, Beijing, 100871, China.
Abstract:
Interference from endogenous signaling enzymes represents a major hurdle for building orthogonal signaling cascades inside cells, particularly among closely related isoforms within an enzyme family. Here, we employed a genetically encoded chemical decaging strategy to build orthogonally activated kinase isoforms, with the endogenous counterparts temporally disabled by an extracellularly delivered bacterial effector. This approach eliminated any potential interference from other kinase isoforms as well as 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 closely related kinase isoforms.
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