Spatiotemporal Dynamics of Kinase Signaling Visualized by Targeted Reporters
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- Abstract
- Table of Contents
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- Literature Cited
Abstract
The advent of genetically encoded FRET?based kinase activity reporters has ushered in a new era of signal transduction research. Such reporters allow the direct monitoring of kinase activity in live cells at specific locations, providing unprecedented information on the spatiotemporal dynamics of kinase signaling. Specifically, FRET?sensitive conformational changes in the reporters following phosphorylation serve as a direct readout of kinase activity. These genetically encoded reporters allow not only temporal resolution of kinase activity, but also spatial resolution: by fusing appropriate targeting sequences, reporters can be positioned at specific subcellular locations. Herein is presented a strategy to generate and target kinase activity reporters to discrete intracellular regions to measure kinase signaling in live cells. Curr. Protoc. Chem Biol. 1:17?28. © 2009 by John Wiley & Sons, Inc.
Keywords: kinase activity reporter; imaging; FRET; localized kinase signaling
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Strategic Planning
- Basic Protocol 1: Imaging Kinase Activity Reporters to Measure Kinase Signaling in Live Cells
- Support Protocol 1: Determining the Dynamic Range of a Fluorescent Reporter
- Commentary
- Literature Cited
- Figures
- Tables
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PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: Imaging Kinase Activity Reporters to Measure Kinase Signaling in Live Cells
Materials
Support Protocol 1: Determining the Dynamic Range of a Fluorescent Reporter
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Figure 1. Schematic diagram showing the modular structure of kinase activity reporters. Kinase activity reporters consist of a FRET donor (e.g., CFP), a phosphoamino acid binding domain (PBD), a consensus phosphorylation sequence (substrate peptide), and a FRET acceptor (e.g., YFP). The FRET pair are at a distance and orientation with respect to one another that changes once the substrate peptide is phosphorylated (circle with P) by the kinase; following phosphorylation, the PBD binds to the phosphorylated sequence, resulting in a conformational change that alters FRET between the FRET donor and FRET acceptor. Depending on the reporter construct, phosphorylation can cause an increase (A ) or decrease (B ) in FRET. View Image -
Figure 2. Schematic depicting a method to calibrate reporter concentration in cells. A drop of purified fluorophore of known concentration (gray color) is placed in a wedge generated using coverslips. A series of images is acquired across the length of the wedge to correlate the intensity of signal with the height at each point in the wedge. From this, and knowing the estimated thickness of the cell type used, one can estimate the intensity expected for reporter concentration within a cell. View Image -
Figure 3. Quantitation of the basal, stimulated, and phosphatase‐suppressed PKC activities at specific regions, showing the range of the C Kinase Activity Reporter (CKAR) at each cellular location. The reporter has a consistent maximal range (20% FRET ratio change) at plasma membrane (PM), Golgi, cytosol, and nucleus, but a reduced range at the outer membrane of mitochondria. (figure from Gallegos et al., ). View Image -
Figure 4. Targeting kinase activity reporters to subcellular regions. Images of targeted reporters utilizing some the targeting sequences from Table . View Image
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Literature Cited
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