Previous studies have shown the potential of using targeted quantification of proteins or post-translational modifications (PTMs) as a highly specific readout for previously characterized biological functions in yeast ( Soste et al., 2014) or human samples ( Abelin et al., 2016). Sensitive and accurate characterization of selected phosphorylation events may be achieved by targeted MS approaches. Furthermore, T-loop phosphorylations often remain undetected or cannot be accurately quantified in large-scale phosphoproteomics studies due to the low abundance of the corresponding peptides. In principle, T-loop phosphorylation can be detected using phospho-specific antibodies, but their availability is limited to a few well-studied kinases. The well-studied functionality of these phosphorylations, in combination with their high level of evolutionary conservation throughout the entire kinome, facilitates the use of T-loop phosphorylation as a direct probe to assess kinase activation states ( Nolen et al., 2004). The majority of protein kinases are regulated through phosphorylation of their activation loop (T-loop). Tackling these limitations requires the analysis of direct markers for kinase activation. Indeed, we and others have utilized such prediction tools to infer kinase activity profiles ( Guo et al., 2011, Zagorac et al., 2018) however, these methods severely suffer in sensitivity due to lack of knowledge on the majority of kinase substrates and thus display a strong bias toward well-studied kinases ( Ding et al., 2011). In combination with targeted mass spectrometry (MS), these methods allow quantification of the expression of >200 kinases however, they do not directly deduce kinase activity.Īlternative approaches to assess kinome activity involve analyzing large-scale phosphoproteomics datasets for over- or underrepresented sequence motifs that can be linked to known kinase substrates or sequence specificities ( Lachmann and Ma'ayan, 2009, Miller et al., 2008). In line with this approach, several studies reported the use of biotin-conjugated acyl-nucleotide probes for the enrichment of kinases via their ATP-binding pocket from complex backgrounds ( Patricelli et al., 2011, Xiao et al., 2014). However, it was later shown that MIBs bind kinases largely independent of their activation status ( Ruprecht et al., 2015). This primed various studies to interpret increased MIB binding affinity as increased kinase activity on a kinome-wide scale ( Stuhlmiller et al., 2015, Stuhlmiller et al., 2017). 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). (2002), an increasing number of studies have focused on the so-called “kinome ” however, robust methods to determine kinase activation on a kinome-wide level are still lacking. Since the complete cataloging of all human kinases by Manning et al. Therefore, the capability to monitor the dynamics of kinase activity is essential to deepen our understanding of cellular function and could greatly impact rational drug design.
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). The sensitivity of our assays is highlighted by the reproducible detection of TNF-α-induced RIPK1 activation and the detection of 46 T-loop phosphorylation sites from a breast tumor needle biopsy. 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. Combining selective phosphopeptide enrichment with robust targeted mass spectrometry, we provide highly specific assays for 248 peptides, covering 221 phosphosites in the T-loop region of 178 human kinases. Here, we describe a 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. Until now, kinase activity has mainly been deduced from either protein expression or substrate phosphorylation levels.
Aberrant kinase activity has been linked to a variety of disorders however, methods to probe kinase activation states in cells have been lacking.
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