High-sensitivity measurements of multiple kinase activities in live single cells

Details

• Alternative Title:
  Cell
• Personal Author:
  Regot, Sergi ; Hughey, Jacob J. ; Bajar, Bryce T. ; Carrasco, Silvia ; Covert, Markus W.
• Description:
  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.
• Subjects:
  Amino Acid Sequence Animals Article Biosensing Techniques JNK Mitogen-Activated Protein Kinases Mice Molecular Sequence Data Phosphotransferases Sequence Alignment Single-Cell Analysis
• Source:
  Cell. 2014; 157(7):1724-1734.
• Pubmed ID:
  24949979
• Pubmed Central ID:
  PMC4097317
• Document Type:
  Journal Article
• Funding:
  5DP1LM01150-05/DP/NCCDPHP CDC HHS/United States ; DP1 LM011510/LM/NLM NIH HHS/United States ; DP1 OD006413/OD/NIH HHS/United States ; P50 GM107615/GM/NIGMS NIH HHS/United States ; P50GM107615/GM/NIGMS NIH HHS/United States ; R21 5R21AI104305-02/AI/NIAID NIH HHS/United States ; R21 AI104305/AI/NIAID NIH HHS/United States
• Volume:
  157
• Issue:
  7
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:c6f3da728b0c758bb594f67bc5718257bf22b326e98a245df98c6f9975e6f614
• Download URL:
  https://stacks.cdc.gov/view/cdc/30035/cdc_30035_DS1.pdf
• File Type:
  [PDF - 3.43 MB ]