TY - JOUR
T1 - High-sensitivity measurements of multiple kinase activities in live single cells
AU - Regot, Sergi
AU - Hughey, Jacob J.
AU - Bajar, Bryce T.
AU - Carrasco, Silvia
AU - Covert, Markus W.
N1 - Funding Information:
We thank S. Collins and members of the Covert lab, in particular D. Macklin, for helpful discussions and comments on the manuscript. We thank Dr. K. Aoki for providing the FRET-based JNKAR plasmid. We also gratefully acknowledge a Human Frontier Science Program (HFSP) postdoctoral fellowship (LT000529/2012-L) to S.R., as well as an NIH R21 (5R21AI104305-02), an NIH Pioneer Award (5DP1LM01150-05), the Stanford Center for Systems Biology (NIH P50GM107615), and an Allen Distinguished Investigator Award to M.W.C. S.R., J.J.H., and M.W.C. are listed as inventors in a patent application for KTR technology.
PY - 2014/6/19
Y1 - 2014/6/19
N2 - 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.
AB - 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.
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U2 - 10.1016/j.cell.2014.04.039
DO - 10.1016/j.cell.2014.04.039
M3 - Article
C2 - 24949979
AN - SCOPUS:84903217674
SN - 0092-8674
VL - 157
SP - 1724
EP - 1734
JO - Cell
JF - Cell
IS - 7
ER -