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.

Abstract:The highly efficient hydroacylation reaction of aldehydes with azodicarboxylates has been carried out in the ionic liquid,1-n-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide,. The products were readily separated by extraction from the reaction medium and the ionic liquid could be recycled up to 8 times and the yields of the reactions were not affected. Compared to conventional solvents, high yields were achieved with aliphatic saturated aldehydes, and the reaction can be conducted under normal to mild conditions without the use of a catalyst.

Kinase-catalyzed protein phosphorylation is an important post-translational modification that regulates a variety of cellular functions. Identification of the many substrates of a specific kinase is critical to fully characterize cell biology. Unfortunately, kinase–substrate interactions are often transient, which makes their identification challenging. Here, the transient kinase–substrate complex was stabilized by covalent crosslinking using γ-phosphate modified ATP analogs. Building upon prior use of an ATP-aryl azide photocrosslinking analog, we report here the creation of an ATP-benzophenone photocrosslinking analog. ATP-benzophenone displayed a higher conversion percentage but more diffuse crosslinking compared to the ATP-aryl azide analog. A docking study was also performed to rationalize the conversion and crosslinking data. In total, the photocrosslinking ATP analogs produced stable kinase–substrate complexes that are suitable for future applications characterizing cell signaling pathways.

Kinase-catalyzed protein phosphorylation is an important biochemical process involved in cellular functions. We recently discovered that kinases promiscuously accept γ-modified ATP analogs as cosubstrates and used several ATP analogs as tools for studying protein phosphorylation. Herein, we explore the structural requirements of γ-modified ATP analogs for kinase compatibility. To understand the influence of linker length and composition, a series of ATP analogs was synthesized and the efficiency of kinase-catalyzed labeling was determined by quantitative mass spectrometry. This study on factors influencing kinase cosubstrate promiscuity will enable design of ATP analogs for a variety of kinase-catalyzed labeling reactions.

Three novel chiral ionic liquids (CILs) containing two chiral centers in the side chain bonded to the 2-position of the imidazolium cation and different anions have been synthesized, characterized and used as chiral solvents for asymmetric Baylis-Hillman (BH) reactions; good yields and fair enantioselectivities were obtained.