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

S Regot, JJ Hughey, BT Bajar, S Carrasco, MW Covert - Cell, 2014 - cell.com
Cell, 2014cell.com
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 …
Summary
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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