An ultrasensitive fiveplex activity assay for cellular kinases

Sci Rep. 2019 Dec 19;9(1):19409. doi: 10.1038/s41598-019-55998-8.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biocatalysis
  • Enterovirus / physiology
  • Enzyme Assays / methods*
  • Genes, Reporter
  • HT29 Cells
  • High-Throughput Screening Assays
  • Humans
  • Insulin / pharmacology
  • Peptides / metabolism
  • Phosphorylation
  • Protein Kinases / chemistry
  • Protein Kinases / metabolism*
  • Reproducibility of Results
  • Substrate Specificity
  • Tumor Necrosis Factor-alpha / pharmacology


  • Insulin
  • Peptides
  • Tumor Necrosis Factor-alpha
  • Protein Kinases