Genetically encoded photocrosslinking amino acids for in-cell kinase substrate identification


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Khalil Davis1,Joanna Kelly1,Silvia Martini1,Aaron Borg1,David Mann2,Alan Armstrong2,Peter Parker1
1The Francis Crick Institute,2Imperial College London

Abstract

Background

As the effectors of phosphorylation, protein kinases play a critical role in cellular signalling pathways, and, unsurprisingly, many diseases are associated with aberrant kinase signalling. This is particularly well evidenced in cancer, where kinases represent the most common class of proteins encoded by oncogenes, and as such have grown to become the most targeted family of proteins in cancer drug discovery. However, a significant proportion of human kinome remains largely uncharacterized, with little substrate information, despite the fact that many of these kinases have been shown to be essential for the survival of various cancer cell lines.

Method

We are currently working towards addressing this problem through exploiting genetic code expansion technology to site-specifically incorporate a photocrosslinking amino acid, 3’-azibutyl-N-carbamoyl-lysine (AbK), in the vicinity of the substrate-binding region of kinases in intact cells with the purpose of trapping transient kinase-substrate interactions upon UV irradiation. We are initially applying this methodology to PKCε, a kinase that in previous work from our laboratory has been shown to play an important role in various mitotic checkpoints but where the associated substrates are not fully established.

Results

We have identified a particular site in the PKCε kinase domain, proximal to the substrate-binding site, where mutation to AbK is tolerated, as indicated by expression, priming phosphorylations, and activity. Our initial results of crosslinking between PKCε and potential substrates in cells will be presented.

Conclusion

Techniques to systematically identify kinase substrates in their native cellular environment will be essential if we are to identify new opportunities for therapeutic intervention in cancer and understand kinase signalling in general; we believe that this method may be generally applicable to other kinases, and prove to be useful in gaining insight into kinase signalling.