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

Nikolaus A Watson,Marcos Cámara-Donoso,Onur Sen,Conor Lawless,Tyrell N Cartwright,Toru Hirota,Jonathan M G Higgins,Kosuke Sako,Hiroshi Kimura

doi:10.1038/s41467-020-15428-0

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-kinome-wide 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.

Highlights

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
B1-Cdk[1] target sites on Inner Centromere Protein (INCENP), but was distinct from that of INCENP TSSph (Fig. 4d) which is generated by Aurora B activity throughout mitosis, including on the central spindle in anaphase[60]. These results show that Cyclin B-Cdk[1] is the primary direct kinase for INCENP S446 in cells, and that kinase inhibitor profiling to identify kinases (KiPIK) can identify the kinases required for the generation of orphan phosphorylation sites
For a residue in the Chromosomal Passenger Complex protein INCENP (S446) that sits within a consensus motif commonly phosphorylated in mitosis by unknown kinase(s)[4,57], KiPIK unambiguously identified Cyclin-dependent kinases as candidates, and Cyclin B1-Cdk[1] was the top hit

Summary

Introduction

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. The second category includes techniques such as kinome-wide RNAi, CRISPR/Cas[9] or overexpression screens Such screens are tremendously useful to biologists, but they often identify pathways or networks of kinases that are indirectly required for phosphorylation of a particular substrate rather than (or in addition to) the direct kinase[17,18,19,20]. These indirect effects can make it difficult to identify the direct kinase for a particular phosphorylation site. If RNAi for a kinase impedes cell cycle progression, it is difficult to determine whether its effect on a substrate is direct[21]

Methods

Results

Conclusion