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Sensors (Basel, Switzerland)
Jan 10, 2014;14 (1): 1140-54.

Optimization of ERK activity biosensors for both ratiometric and lifetime FRET measurements.

Pauline Vandame, Corentin Spriet, Franck Riquet, Dave Trinel, Katia Cailliau-Maggio, Jean-François Bodart
Author Information
  1. Pauline Vandame: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France. pauline.vandame@ed.univ-lille1.fr.
  2. Corentin Spriet: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France. corentin.spriet@iri.univ-lille1.fr.
  3. Franck Riquet: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France. franck.riquet@iri.univ-lille1.fr.
  4. Dave Trinel: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France. dave.trinel@iri.univ-lille1.fr.
  5. Katia Cailliau-Maggio: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France.
  6. Jean-François Bodart: Laboratoire de Régulation des Signaux de division, EA4479, Institut Fédératif de Recherche (IFR) 147, Site de Recherche Intégré en Cancérologie (SIRIC) ONCOLILLE, University Lille1, Villeneuve d'Ascq F-59655, France. Jean-Francois.Bodart@univ-lille1.fr.
PMID: 24434874 DOI: 10.3390/s140101140

Abstract

Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes. There is a constant need for better tools to analyze kinases in vivo, and to detect even the slightest variations of their activities. Here we report the optimization of the previous ERK activity reporters, EKAR and EKAREV. Those tools are constituted by two fluorophores adapted for FRET experiments, which are flanking a specific substrate of ERK, and a domain able to recognize and bind this substrate when phosphorylated. The latter phosphorylation allows a conformational change of the biosensor and thus a FRET signal. We improved those biosensors with modifications of: (i) fluorophores and (ii) linkers between substrate and binding domain, resulting in new versions that exhibit broader dynamic ranges upon EGF stimulation when FRET experiments are carried out by fluorescence lifetime and ratiometric measurements. Herein, we characterize those new biosensors and discuss their observed differences that depend on their fluorescence properties.

References

  1. Nat Cell Biol. 2007 Mar;9(3):324-30 [PMID: 17310240]
  2. Nat Methods. 2010 Feb;7(2):137-9 [PMID: 20081836]
  3. Mol Biol Cell. 2011 Dec;22(23):4647-56 [PMID: 21976697]
  4. Mol Biosyst. 2011 Jan;7(1):52-8 [PMID: 20838685]
  5. Growth Factors. 2006 Mar;24(1):21-44 [PMID: 16393692]
  6. Cell Cycle. 2006 Apr;5(7):759-65 [PMID: 16582626]
  7. Cell Signal. 2007 Aug;19(8):1621-32 [PMID: 17553668]
  8. J Cell Biochem. 2010 Apr 1;109(5):850-7 [PMID: 20082320]
  9. Biochem Biophys Res Commun. 2006 Sep 22;348(2):716-21 [PMID: 16895723]
  10. Anal Chem. 2007 Mar 15;79(6):2570-5 [PMID: 17261026]
  11. J Biol Chem. 2006 Mar 31;281(13):8917-26 [PMID: 16418172]
  12. Sci Signal. 2013 Jul 23;6(285):rs12 [PMID: 23882122]
  13. Cell. 2007 Mar 23;128(6):1119-32 [PMID: 17382881]
  14. Science. 1998 May 8;280(5365):895-8 [PMID: 9572732]
  15. Nat Protoc. 2011 Nov 03;6(12):1835-46 [PMID: 22051797]
  16. Prog Mol Biol Transl Sci. 2013;113:145-216 [PMID: 23244791]
  17. PLoS One. 2011 Apr 29;6(4):e19170 [PMID: 21559477]
  18. Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19264-9 [PMID: 19033456]
  19. Liver Int. 2004 Oct;24(5):432-6 [PMID: 15482339]
  20. Biochemistry. 2006 May 30;45(21):6570-80 [PMID: 16716067]

MeSH Term

Biosensing Techniques
Fluorescence Resonance Energy Transfer
Fluorescent Dyes
Phosphorylation
Signal Transduction

Chemicals

Fluorescent Dyes
Journal Article Research Support, Non-U.S. Gov't

Word Cloud

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