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

Regot, Sergi; Hughey, Jacob J.; Bajar, Bryce T.; Carrasco, Silvia; Covert, Markus W.

doi:10.1016/j.cell.2014.04.039

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

Published Date:

Jun 19 2014

Publisher's site:

http://dx.doi.org/10.1016/j.cell.2014.04.039 New Site Icon

Source:

Cell. 2014; 157(7):1724-1734.

[PDF-3.43 MB]

Details:

Personal Authors:

Regot, Sergi ; Hughey, Jacob J. ; Bajar, Bryce T. ; Carrasco, Silvia ; Covert, Markus W. ;

Keywords:

[+]

Pubmed ID:

24949979

Pubmed Central ID:

PMC4097317

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.

Document Type:

Journal Article

Collection(s):

CDC Public Access

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

Supporting Files:

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