Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphoproteins in normal and diseased states is critical to fully characterize cell signalling. Towards phosphoprotein analysis tools, our lab reported kinase-catalyzed labeling where γ-phosphate modified ATP analogs are utilized by kinases to label peptides or protein substrates with a functional tag. In particular, the ATP-biotin analog was developed for kinase-catalyzed biotinylation. However, kinase-catalyzed labeling has been tested rigorously with only a few kinases, preventing use of ATP-biotin as a general tool. Here, biotinylation experiments, gel or HPLC-based quantification, and kinetic measurements indicated that twenty-five kinases throughout the kinome tree accepted ATP-biotin as a cosubstrate. With this rigorous characterization of ATP-biotin compatibility, kinase-catalyzed labeling is now immediately useful for studying phosphoproteins and characterizing the role of phosphorylation in various biological events.

ATP analogs have been powerful tools in the study of kinase-catalyzed phosphorylation. However, the cell impermeability of ATP analogs has largely limited their use to in vitro lysate-based experiments. Here we report the first cell permeable ATP analog, ATP-polyamine-biotin (APB). APB promoted biotin labeling of kinase substrates in live cells. APB has future application in phosphoprotein purification and analysis. More generally, these studies provide a foundation for development of additional cell permeable ATP analogs for cell signaling research.

Kinases mediate cell signaling pathways by catalyzing protein phosphorylation. Irregularities in kinase activity are directly associated with disease conditions. Therefore, methods to identify substrates of a particular kinase are needed to understand signaling cascades in normal and diseased states. Photocrosslinking ATP analogs provide powerful tools to study kinases by covalently linking kinases with substrates. However, the involvement of UV light and nonspecific reactivity of current ATP-photocrosslinkers challenge kinasesubstrate identification. We report here an affinity-based crosslinking ATP analog, ATP-methylacrylamide (ATP-MAc), that contains a cysteine-reactive acrylamide crosslinking group, which avoids the UV irradiation and non-specific reactivity of prior analogs. Using in vitro kinase assays, ATP-MAc acts as a kinase co-substrate and covalently crosslinks only kinases containing cysteines in the active site. ATP-MAc was also able to crosslink cellular proteins in lysates, documenting compatibility with cell-based studies.

ATPanalogues have been powerfulcompounds for the study of kinase-catalyzed phosphorylation. However,t he cell impermeability of ATPanalogues has largely limited their use to in vitro lysate-based experiments.H erein, we report the first cell-permeable ATPa nalogue,A TP-polyamine-biotin (APB). APB is shown to promote biotin labeling of kinase substrates in live cells and has future applications in phosphoprotein purification and analysis.More generally,these studies provideafoundation for the development of additional cellpermeable ATPanalogues for cell-signaling research.

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