In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native Kinases

Abstract

Summary Protein 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 more than 200 kinases in native cell proteomes and reveal new 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 upon 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.

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-Based Strategy for Proteome-Wide Thermodynamic Analysis of Protein-Ligand Binding Interactions

“…With the exception of a recent study that reported the discovery of over 800 ATP-interacting proteins in several different breast cancer cell lines using a specially designed ATP probe in combination with a targeted mass spectrometrybased approach (35), the number of protein hits identified in the current study is comparable to the 100 -200 ATP interacting proteins identified in several recent chemical proteomics studies, which also relied on specially designed ATP probes to characterize ATP-interacting proteins (30,31,36,37). The current study is the largest proteome-wide profile of an ATP-interactome using an energetics-based method, to date.…”

CC-223, a Potent and Selective Inhibitor of mTOR Kinase: In Vitro and In Vivo Characterization

“…Upon follow-up IC 50 value determination, only two were inhibited by CC-223 with IC 50 values below 1 mmol/L; FLT4 (0.651 mmol/L) and cFMS (0.028 mmol/L). The exquisite kinase selectivity of CC-223 was confirmed upon evaluation in cellular systems using ActivX KiNavtiv profiling (17,18). Other than mTOR kinase, no kinase target was identified when HCT 116 or A549 cells were treated for 1 hour with 1 mmol/L CC-223 and assayed for kinase activity.…”

Interleukin 1/Toll-like Receptor-induced Autophosphorylation Activates Interleukin 1 Receptor-associated Kinase 4 and Controls Cytokine Induction in a Cell Type-specific Manner

“…tated use of micromolar concentrations for our studies. To test target occupancy in the context of the cell, we subjected cells treated with 10 M compound 1 to analysis by ActivX technology in the KiNativ panel (23). We observed that the compound has excellent selectivity against 170 kinases in the panel with 90% occupancy against IRAK4 and only 55% occupancy against IRAK1.…”