In situ kinase profiling reveals functionally relevant properties of native kinases

Abstract

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 >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.

Figure 1. The KiNativ^™ Method

(A) Chemical structure of the acylphosphate probe. (B) Schematic of the KiNativ method. Protein lysates are treated with an inhibitor followed by addition of the desthiobiotinylated acylphosphate-nucleotide probe. Following a tryptic digest and streptavidin enrichment, the probe labeled peptides are characterized and quantified using a targeted LC-MS² strategy. (C) Model of an acylphosphate probe bound in the active site of CDK2 (PDB ID 1HCK). Conserved, catalytic lysine residues are illustrated. (D) Signal enhancement through targeted LC-MS² analysis. Signals from MS¹ (or parent) ion signals typically used for quantification in non-targeted runs (upper panel) are compared to extracted MS/MS ions from a targeted LC-MS² run (lower panel).

Figure 2. Dasatinib Inhibits MAP2K5

HeLa cells were treated with varying concentrations of dasatinib for one hour prior to treatment with EGF. Cells lysates were analyzed by a mobility shift western blot for phosphorylation of ERK5, a substrate of MAP2K5. See also Supplemental Figure S4

Figure 3. Cellular Properties of Raf Inhibitors

A375 cells were treated with the indicated inhibitors for 30 minutes. Cells were lysed, and the lysates analyzed using KiNativ. Data are plotted as the ratio of control vs. treated signal for each indicated kinase. Error bars represent the range of duplicate datapoints. Data is representative of three separate duplicate experiments.