CDK6 activity in a recurring convergent kinase network motif

Abstract

More than 500 kinases phosphorylate [~]15% of all human proteins. The architecture of the phosphorylation network has been studied extensively, for instance to compile global interaction maps, trace signal transduction pathways or identify chemical intervention points. In contrast, systematic investigation of local motifs is rare but may provide a complementary understanding of network and kinase function. Here, we report on the occurrence, topology and experimental analysis of convergent kinase-substrate relationships (cKSRs) in which [≥]two kinases phosphorylate the same substrate. Through bioinformatics analysis we found that human cKSRs are frequent and involve >80% of all kinases and >24% of all substrates. We show that cKSRs occur over a wide range of stoichiometries harnessing converging kinases that are in many instances recruited from the same family subgroup and co-expressed. Experimentally, we demonstrate at the prototypical convergent CDK4/6 kinase pair how multiple inputs at the major tumor suppressor retinoblastoma protein (RB) can hamper in situ analysis of individual kinases. We hypothesized that overexpression of one kinase combined with a CDK4/6 inhibitor can dissect convergence. In breast cancer cells that express high levels of CDK4, we confirmed this hypothesis and developed a high throughput compatible assay that sensitively quantifies genetically modified CDK6 variants and small molecule or protein inhibitors. Collectively, our work provides deeper insights into the phosphorylation network through the identification and analysis of kinase convergence.