3 years ago

[ASAP] Identification of Kinases and Interactors of p53 Using Kinase-Catalyzed Cross-Linking and Immunoprecipitation

[ASAP] Identification of Kinases and Interactors of p53 Using Kinase-Catalyzed Cross-Linking and Immunoprecipitation
Satish Garre, Aparni K. Gamage, Todd R. Faner, Pavithra Dedigama-Arachchige, Mary Kay H. Pflum
Kinase enzymes phosphorylate protein substrates in a highly ordered manner to control cell signaling. Unregulated kinase activity is associated with a variety of disease states, most notably cancer, making the characterization of kinase activity in cells critical to understand disease formation. However, the paucity of available tools has prevented a full mapping of the substrates and interacting proteins of kinases involved in cellular function. Recently we developed kinase-catalyzed cross-linking to covalently connect substrate and kinase in a phosphorylation-dependent manner. Here, we report a new method combining kinase-catalyzed cross-linking and immunoprecipitation (K-CLIP) to identify kinase-substrate pairs and kinase-associated proteins. K-CLIP was applied to the substrate p53, which is robustly phosphorylated. Both known and unknown kinases of p53 were isolated from cell lysates using K-CLIP. In follow-up validation studies, MRCKbeta was identified as a new p53 kinase. Beyond kinases, a variety of p53 and kinase-associated proteins were also identified using K-CLIP, which provided a snapshot of cellular interactions. The K-CLIP method represents an immediately useful chemical tool to identify kinase-substrate pairs and multiprotein complexes in cells, which will embolden cell signaling research and enhance our understanding of kinase activity in normal and disease states.

Publisher URL: http://dx.doi.org/10.1021/jacs.8b10160

DOI: 10.1021/jacs.8b10160

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