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.

Sodium Channel Inhibitors Reduce DMPK mRNA and Protein.

Myotonic dystrophy type 1 (DM1) is caused by an expanded trinucleotide (CTG)n tract in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene. This results in the aggregation of an expanded mRNA forming toxic intranuclear foci which sequester splicing factors. We believe down-regulation of DMPK mRNA represents a potential, and as yet unexplored, DM1 therapeutic avenue. Consequently, a computational screen for agents which down-regulate DMPK mRNA was undertaken, unexpectedly identifying the sodium channel blockers mexiletine, prilocaine, procainamide, and sparteine as effective suppressors of DMPK mRNA. Analysis of DMPK mRNA in C2C12 myoblasts following treatment with these agents revealed a reduction in the mRNA levels. In vivo analysis of CD1 mice also showed DMPK mRNA and protein down-regulation. The role of DMPK mRNA suppression in the documented efficacy of this class of compounds in DM1 is worthy of further investigation.