High-Throughput Assessment of Kinome-Wide Activation States

Thierry Schmidlin,Kelly E Stecker,Albert J R Heck,Stamatia Rontogianni,Bart L Van Den Eshof,Daniel S Peeper,Charlotte A G H Van Gelder,A.F Maarten Altelaar,Kristel Kemper,Esther H Lips,Maartje Van Den Biggelaar

doi:10.2139/ssrn.3275288

Abstract

Protein kinases play a pivotal role in cellular communication and aberrant kinase activity has been linked to a variety of disorders. Methods to directly probe kinase activation states in cells have been lacking and until now kinase activity has mainly been extracted from either kinase protein expression levels or data on substrate phosphorylation. Here, we describe a novel strategy to directly infer kinase activation through targeted quantification of t-loop phosphorylation, which serves as a critical activation switch in a majority of protein kinases. Combining selective phosphopeptide enrichment with robust targeted mass spectrometry (MS), we provide highly specific assays for 248 peptides, covering 221 phosphorylation sites in the t-loop region of 178 human kinases. Using these assays, we monitored the activation of 63 kinases through 73 t-loop phosphosites across different cell types, primary cells and patient-derived tissue material. The sensitivity of our assays is highlighted by the reproducible detection of TNFα induced RIPK1 activation, which has thus far only been reported using a protein expression system in combination with an in vitro kinase assay. Moreover, our methodology enables the detection of 46 t-loop phosphorylation sites directly from a breast tumor 14G needle biopsy.

Highlights

Kinases are key regulators of inter- and intracellular communication, and their inhibitors are critical in targeted therapy and precision medicine (Blume-Jensen and Hunter, 2001; Garay and Gray, 2012; Lahiry et al, 2010)
Initial attempts to measure global kinase activation states exploited the interaction of kinases with immobilized unspecific multiplexed inhibitor beads (MIBs), which demonstrated altered binding affinities upon enzymatic activation (Bantscheff et al, 2007)
Our method currently encompasses robust molecular assays to accurately quantify 221 phosphorylation sites in the their activation loop (T-loop) region of 178 kinases (Figure 1A; Table S1). This number is primarily based on accessibility of the T-loop phosphopeptides by trypsin and can be expanded by the use of alternative proteases

Summary

Introduction

Kinases are key regulators of inter- and intracellular communication, and their inhibitors are critical in targeted therapy and precision medicine (Blume-Jensen and Hunter, 2001; Garay and Gray, 2012; Lahiry et al, 2010). Initial attempts to measure global kinase activation states exploited the interaction of kinases with immobilized unspecific multiplexed inhibitor beads (MIBs), which demonstrated altered binding affinities upon enzymatic activation (Bantscheff et al, 2007). This primed various studies to interpret increased MIB binding affinity as increased kinase activity on a kinome-wide scale (Stuhlmiller et al, 2015, 2017). It was later shown that MIBs bind kinases largely independent of their activation status (Ruprecht et al, 2015). In combination with targeted mass spectrometry (MS), these methods allow quantification of the expression of >200 kinases; they do not directly deduce kinase activity

Methods

Results

Discussion

Conclusion