In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native Kinases

SummaryProtein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.

► Chemoproteomics method allows quantitative profiling of >150 kinases per cell type ► Reports binding constants for ATP, Staurosporine, and five advanced kinase inhibitors ► Identifies MAP2K5 as a novel target of dasatinib and verifies this result in cellular assays ► Native kinase profiling capable of detecting inhibitor induced kinase activation ► Differences were observed between recombinant and native Raf kinase inhibition ► Native Raf kinase binding correlates well with in vivo activity of Raf inhibitors