Spatiotemporal Dynamics of Kinase Signaling Visualized by Targeted Reporters

Maya T. Kunkel1, Alexandra C. Newton1

1 University of California at San Diego, La Jolla, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch090106
Online Posting Date:  December, 2009
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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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Table 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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Materials

Basic Protocol 1: Imaging Kinase Activity Reporters to Measure Kinase Signaling in Live Cells

  Materials
  • Cells of interest: e.g., HeLa cells
  • Cell culture medium (e.g., DMEM)
  • DNA encoding kinase activity reporter (see )
  • Transfection reagent (e.g., FuGENE 6 from Roche)
  • Hanks' Balanced Salt Solution (HBSS), without Ca2+ and Mg2+ (e.g., Cellgro), but supplemented with 1 mM Ca2+ on the day of the imaging experiment
  • Activator of kinase signaling path: e.g., histamine or phorbol dibutyrate (PdBu)
  • Kinase inhibitor or inhibitor of kinase signaling pathway, as control: e.g., Gö 6983 (Calbiochem)
  • Control construct: phospho‐acceptor mutant (see )
  • Sterile 35‐mm glass‐bottom culture dishes (MatTek)
  • Imaging setup (see )
  • Data acquisition software (e.g., Metafluor from Molecular Devices and Microsoft Excel)

Support Protocol 1: Determining the Dynamic Range of a Fluorescent Reporter

  • Phosphatase inhibitors that have been determined to inhibit phosphatases that act on the reporter's substrate sequence, e.g., calyculin A (Calbiochem)
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Figures

  •   FigureFigure 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.
  •   FigureFigure 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.
  •   FigureFigure 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., ).
  •   FigureFigure 4. Targeting kinase activity reporters to subcellular regions. Images of targeted reporters utilizing some the targeting sequences from Table .

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Literature Cited

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