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Review
. 2008 Jun 27;363(1500):2143-51.
doi: 10.1098/rstb.2008.2267.

Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Förster resonance energy transfer

Kazuhiro Aoki  1 , Etsuko Kiyokawa, Takeshi Nakamura, Michiyuki Matsuda
Affiliations
Review

Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Förster resonance energy transfer

Kazuhiro Aoki et al. Philos Trans R Soc Lond B Biol Sci. .

Abstract

Fluorescence probes based on the principle of Förster resonance energy transfer (FRET) have shed new light on our understanding of signal transduction cascades. Among them, unimolecular FRET probes containing fluorescence proteins are rapidly increasing in number because these genetically encoded probes can be easily loaded into living cells and allow simple acquisition of FRET images. We have developed probes for small GTPases, tyrosine kinases, serine-threonine kinases and phosphoinositides. Images obtained with these probes have revealed that membrane protrusions such as nascent lamellipodia or neurites provide an active signalling platform in the growth factor-stimulated cells.

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Figures

Figure 1
Figure 1
FRET probes for protein kinases. (a) Structure and mode of action of Picchu, a probe for tyrosine phosphorylation of the CrkII adaptor protein. The SH2 domain of CrkII binds intramolecularly to its Tyr221, when it is phosphorylated. (b) Non-covalent binding of the Picchu probe to EGF receptor. Lower panels show the FRET image of a Cos cell expressing Picchu or Picchu-Z before and after stimulation with EGF. (c) Akind, a probe for Akt. CFP is inserted between the PH domain and the catalytic domain. Akt recruitment to the plasma membrane via the PH domain is followed by phosphorylation and conformational change. (d) Miu2, a probe for ERK. ERK liberated from MEK adopts an open active conformation. Lower panels show the FRET image of cells expressing Akind, Prin-c-Raf and Miu2 before and after stimulation with growth factor.
Figure 2
Figure 2
FRET probes for small GTPases. (a) Structure of effector-type probes. The binding of the cognate GTPase to the effector dissociates the association of CFP and YFP. (b) Archetypal structure of Raichu-type unimolecular FRET probes. (c) Localization of active Ras, RalA and Rap1 in Cos cells as visualized by Raichu probes. (d) Localized activity of Rho-family GTPases in HeLa cells. The white arrows indicate the direction of migration.
Figure 3
Figure 3
FRET probes for phosphoinositides. (a) Structure of Fllip-type probes for phosphoinositides. The binding of the lipid-binding domain to the inner surface of the cell membrane fixes the probe to the membrane, resulting in a decrease in the distance between CFP and YFP. (b) Localization of (i) PIP3 (Pippi-PI(3,4,5)P3) and (ii) PI(3,4)P2 (Pippi-PI(3,4)P2) in Cos cells before and after EGF stimulation.
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References

    1. Awais M, Sato M, Lee X, Umezawa Y. A fluorescent indicator to visualize activities of the androgen receptor ligands in single living cells. Angew. Chem. Int. Ed. Engl. 2006;45:2707–2712. doi:10.1002/anie.200503185 - DOI - PubMed
    1. Bastiaens P.I, Squire A. Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol. 1999;9:48–52. doi:10.1016/S0962-8924(98)01410-X - DOI - PubMed
    1. Bos J.L. Ras-like GTPases. Biochim. Biophys. Acta. 1997;1333:M19–M31. - PubMed
    1. Braun D.C, Garfield S.H, Blumberg P.M. Analysis by fluorescence resonance energy transfer of the interaction between ligands and protein kinase Cdelta in the intact cell. J. Biol. Chem. 2005;280:8164–8171. doi:10.1074/jbc.M413896200 - DOI - PubMed
    1. Brumbaugh J, Schleifenbaum A, Gasch A, Sattler M, Schultz C. A dual parameter FRET probe for measuring PKC and PKA activity in living cells. J. Am. Chem. Soc. 2006;128:24–25. doi:10.1021/ja0562200 - DOI - PubMed

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