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

Abstract

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 cellulo 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 crosslinking to covalently connect substrate and kinase in a phosphorylation-dependent manner. Here, we report a new method combining kinase-catalyzed crosslinking 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 multi-proteins complexes in cells, which will embolden cell signaling research and enhance our understanding of kinase activity in normal and disease states.

Quest for the Crypto‐phosphoproteome

“…Development in bioinformatics and structural biology will also help find homologs of currently known kinases. For known phosphorylated substrates, cross‐linking of substrate‐kinase pairs may help find the kinase [72] …”

“…For known phosphorylated substrates, cross-linking of substrate-kinase pairs may help find the kinase. [72]…”

Mass Spectrometry-Based Discovery of <i>in vitro</i> Kinome Substrates

“…To identify more physiologically relevant substrates, combinations of an in vitro assay and in vivo experiments have been developed. [42][43][44] In addition, puri cation of the kinase-substrate complex 45) and the photocrosslinking of the kinase and substrate using ATP analog 46) also enables the identi cation of more signi cant substrates by focusing on the interacting substrate proteins.…”

Kinase‐Catalyzed Crosslinking and Immunoprecipitation (K‐CLIP) to Explore Kinase‐Substrate Pairs

“…Because p53 is robustly phosphorylated by many known kinases (Kruse & Gu, 2009; Maclaine & Hupp, 2009), p53 was an ideal substrate model to establish the method. K‐CLIP combined with LC‐MS/MS analysis successfully identified two known (DNA‐PK and PKR) kinases and one unknown (MRCKβ) kinase of p53 (Garre et al., 2018). In addition, K‐CLIP followed by SDS‐PAGE separation and Western blot analysis confirmed known kinase‐substrate pairs (Garre et al., 2018).…”

“…( F ) Due to the transient nature of kinase‐substrate interactions, a significant amount of substrate will diffuse away from the kinase before UV irradiation and crosslinking. ( G ) Upon UV irradiation after diffusion, the arylazide group will crosslink the substrate to nearby proteins, which will include associated proteins (Garre et al., 2018).…”