Label Transfer Reagents for the Study of Protein Kinase Complexes

Protein kinases are heavily involved in the regulation of many intracellular processes. The specificity of these kinases is often regulated by their interactions with other proteins. Consequently, we have sought to develop new tools to elucidate the identities and spatial relationships of these protein kinase complexes. We proposed a novel label transfer reagent (LTR) which has three components: a protein kinase inhibitor to direct the LTR to the kinase of interest; a photocrosslinker; and a label to use as a chemical handle for visualization or purification of labeled proteins. We have synthesized and tested several LTRs. They retain the specificity and potency of the kinase inhibitor portion, and effectively label known activators and substrates of the kinases. They are highly selective, with no discernible labeling of other highly abundant proteins. Moreover, we can use these LTRs to study kinase complexes expressed in mammalian cells, expanding the use of the LTRs beyond proteins expressible in bacteria. These LTRs promise to be useful tools for validating proposed kinase binding partners and for identifying the spatial relationships of protein kinase complex members.