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

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.