5 years ago

Dissection of Kinase Isoforms through Orthogonal and Chemical Inducible Signaling Cascades

Dissection of Kinase Isoforms through Orthogonal and Chemical Inducible Signaling Cascades
Peng R. Chen, Siqi Zheng, Jie Wang, Jingyi Zhao, Xinyuan Fan
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. Unpicking a cascade: Orthogonal and inducible kinase cascades engineered through chemical decaging in living cells is reported. An orthogonal MEK/Erk pathway can be constructed by employing a genetically-encoded chemical decaging strategy to specifically activate an individual MEK isoform while its endogenous counterpart can be temporally disabled by an extracellularly delivered effector protein.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/cbic.201700255

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