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

  • 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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Literature Cited

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