An ultrasensitive fiveplex activity assay for cellular kinases

Abstract Protein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 µg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt–IKK–JNK–MEK–MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.