High-sensitivity Measurements of Multiple Kinase Activities in Live Single Cells

Cell. 2014 Jun 19;157(7):1724-34. doi: 10.1016/j.cell.2014.04.039.

Abstract

Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells. Our technology converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy. Our reporters reproduce kinase activity for multiple types of kinases and allow for calculation of active kinase concentrations via a mathematical model. Using this technology, we made several experimental observations that had previously been technicallyunfeasible, including stimulus-dependent patterns of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) activation. We also measured JNK, p38, and ERK activities simultaneously, finding that p38 regulates the peak number, but not the intensity, of ERK fluctuations. Our approach opens the possibility of analyzing a wide range of kinase-mediated processes in individual cells.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biosensing Techniques / methods*
  • JNK Mitogen-Activated Protein Kinases / chemistry
  • JNK Mitogen-Activated Protein Kinases / metabolism
  • Mice
  • Molecular Sequence Data
  • Phosphotransferases / metabolism*
  • Sequence Alignment
  • Single-Cell Analysis

Substances

  • Phosphotransferases
  • JNK Mitogen-Activated Protein Kinases