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

Abstract

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 technically unfeasible, 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.

Akt signaling dynamics in individual cells

“…Many signaling pathways that have been examined at the single cell level, including the NFκβ and Erk pathways, are activated transiently in response to most signaling stimuli (Albeck et al, 2013;Regot et al, 2014;Tay et al, 2010). In our experiments, we find that several growth factors persistently activate Akt signaling, as judged by the continual cytoplasmic localization of the FoxO1-clover reporter (Figs 2-5).…”

“…8D). To place our observations in context with published studies using live-cell imaging (Regot et al, 2014;Tay et al, 2010), at each time point we also measured the ratio of nuclear to cytoplasmic fluorescence (denoted N/C), including when cytoplasmic and nuclear fluorescence intensities were identical (N/C=1). Although this varied among different cells, it typically occurred by ∼15 min after addition of PI-103 ( Fig.…”

“…Recently, new biological sensors have been developed that translocate between the nucleus and the cytoplasm in response to a stimulus. These molecules typically maintain high signal-to-noise ratios, provide robust readouts in response to changes in signaling activity and are generally easier to use than FRET reporters (Hao et al, 2013;Regot et al, 2014;Spencer et al, 2013). Here, we have developed and characterized a translocation reporter for Akt kinase activity based on the transcription factor FoxO1.…”

“…Translocation reporters of this type have been developed for CDK2, JNK, Erk (ERK1 and ERK2, also known as MAPK3 and MAPK1) and the p38 mitogen-activated protein kinase (MAPK) families (Regot et al, 2014;Spencer et al, 2013), for the kinases that are upstream of the transcription factors NFAT1 and NFAT4 (also known as NFATC2 and NFATC3) (Yissachar et al, 2013), p53 (Batchelor et al, 2011;Purvis et al, 2012), and for subunits of NFκβ (Nelson et al, 2004;Tay et al, 2010). Results using these reporters have shown that signaling pathways encode stimuli into a variety of different output patterns.…”

Pierced Lasso Topology Controls Function in Leptin

“…MCF 10A human cell line containing the leptin receptor ( Figure S5) was obtained from ATCC (Manassas, VA, USA) and maintained as monolayers under standard tissue culture conditions following established protocols. 29 A genetically encoded biosensor for kinase activity, Erk kinase translocation reporter, 30 was transferred to a PiggyBac custom destination vector using Gateway Cloning Technology. The expression vector was transfected into a MCF 10A cell line together with transposaseexpressing helper plasmid (kind gift from Dr. John Albeck, UC Davis) using a 3:1 ratio of FuGene HD (Promega) to DNA and allowed to incubate overnight.…”