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Curr Protoc Chem Biol. 2019 Mar;11(1):e60. doi: 10.1002/cpch.60. Epub 2019 Jan 7.

In Vivo Identification of Protein Kinase Substrates by Kinase-Oriented Substrate Screening (KIOSS).

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Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Japan.


Protein phosphorylation plays a critical role in the regulation of cellular function. Information on protein phosphorylation and the responsible kinases is important for understanding intracellular signaling. A method for in vivo screening of kinase substrates named KIOSS (kinase-oriented substrate screening) has been developed. This protocol provides a method that utilizes phosphoprotein-binding modules such as 14-3-3 protein, the pin1-WW domain, and the chek2-FHA domain as biological filters to successfully enrich phosphorylated proteins related to intracellular signaling rather than housekeeping and/or structural proteins. More than 1000 substrate candidates for PKA, PKC, MAPK, and Rho-kinase in HeLa cells, as well as phosphorylation downstream of D1R, NMDAR, adenosine A2a receptor, PKA, PKC, MAPK, and Rho-kinase in mouse brain slice cultures have been identified by this method. An online database named KANPHOS (Kinase-Associated Neural Phospho-Signaling) provides the phosphorylation signals identified by these studies, as well as those previously reported in the literature.


interactome; kinase; mass spectrometry; phosphoproteomics; phosphorylation

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