Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 140, Issue 47, Pages 16299-16310Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.8b10160
Keywords
-
Categories
Funding
- National Institute of General Medical Sciences of the National Institutes of Health [R01GM079529]
- NIH Center Grant [P30 ES020957]
- NIH Cancer Center Support Grant [P30 CA022453]
- Wayne State University
- NIH Shared Instrumentation Grant [S10 OD010700]
Ask authors/readers for more resources
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 pS3 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available