Kinase inhibition profiles as a tool to identify kinases for specific phosphorylation sites

Abstract

There are thousands of known cellular phosphorylation sites, but the paucity of ways to identify kinases for particular phosphorylation events remains a major roadblock for understanding kinase signaling. To address this, we here develop a generally applicable method that exploits the large number of kinase inhibitors that have been profiled on near-kinomewide panels of protein kinases. The inhibition profile for each kinase provides a fingerprint that allows identification of unknown kinases acting on target phosphosites in cell extracts. We validate the method on diverse known kinase-phosphosite pairs, including histone kinases, EGFR autophosphorylation, and Integrin β1 phosphorylation by Src-family kinases. We also use our approach to identify the previously unknown kinases responsible for phosphorylation of INCENP at a site within a commonly phosphorylated motif in mitosis (a non-canonical target of Cyclin B-Cdk1), and of BCL9L at S915 (PKA). We show that the method has clear advantages over in silico and genetic screening.

Signaling Pathways in Cancer: Therapeutic Targets, Combinatorial Treatments, and New Developments

“…Endogenous protein kinases that bind to these inhibitors are captured and analyzed by mass spectrometry to provide a global kinome representation. Phosphoproteomics-based methods have also been used to determine the activation states of kinases [ 266 ] and to establish new kinase–phosphosite relationships [ 267 ]. Comprehensive use of proteomics approaches for cancer therapeutics can be found in specialized reviews [ 268 , 269 ].…”

Micro-Meta App: an interactive software tool to facilitate the collection of microscopy metadata based on community-driven specifications

“…In response to significant interest we observed in the community and after beta testing, microscope custodians at several light microscopy facilities (Supplemental Table III) volunteered to serve as case studies on the use of Micro-Meta App to document both microscope instrumentation and example published image datasets produced in microscopy platforms [59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78] . The application was utilized by microscope custodians at 16 sites to document one of their microscopes alongside the settings utilized for published images based on the 4DN-BINA-OME model Core + Basic Extension 37,38,51 (Figure 5 and Supplemental Figures 2-16).…”

Dissecting the roles of Haspin and VRK1 in Histone H3 threonine-3 phosphorylation during mitosis

“…Recombinant kinases may lack cofactors (such as post-translational modifications or activating subunits) that are required for activity towards their substrates. To identify H3T3 kinases in a more physiological environment, and in an unbiased fashion, we made use of a new kinase screening methodology known as KiPIK (Kinase inhibitor Profiling to Identify Kinases) that uses cell extracts as a source of kinases (48). The method compares the pattern of inhibition of the unknown kinase activity in cell extracts with the inhibition profiles of known protein kinases determined in vitro using small molecule kinase inhibitors.…”

“…To prepare mitotic cell extract, HeLa cells were treated with 300 nM nocodazole for 15 h, collected by shake off , and lysed at 4°C in 50 (ThermoFisher), at 37°C for 30 min, in a total volume of 35 µl/well. An expanded custom library of inhibitors similar to that described previously (48) was used (Supplementary Table 1).…”