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Single- and dual-parameter FRET kinase probes based on pleckstrin

Nature Protocols volume 1, pages 1044–1055 (2006)Cite this article

Abstract

Here we describe protocols for preparing and using fluorescent probes that respond to conformational changes by altered Foerster resonance energy transfer (FRET) efficiencies upon phosphorylation or, in principle, other posttranslational modifications (PTMs). The sensor protein, a truncated version of pleckstrin, is sandwiched between short-wavelength-excitation green fluorescent protein (GFP2) and yellow fluorescent protein (EYFP). As a result of complex conformational changes of the protein upon phosphorylation, the introduction of a second PTM consensus sequence bestows sensitivity to a second modification and yields a dual-parameter probe. The first phase of the protocol lays out the cloning strategy for single- and dual-parameter FRET sensors, including the construction of a versatile platform into which different consensus sequences may be inserted to create diverse probes. Protocols for fluorescence microscopy of the probes in living cells and image processing are also described. Probe preparation takes 7 d; microscopy and image processing take 2 h.

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Figure 1: DNA fragments containing the coding sequences for EYFP, GFP2 and a dummy fragment are ligated together to create pYG.
Figure 2: The pleckstrin coding region of KCP-1 is amplified with primers containing the indicated restriction sites.
Figure 3: Oligonucleotides, in this case encoding the PKA consensus sequence kemptide, are annealed and phosphorylated before ligation with BbsI-opened mKCP-1.
Figure 4: An example trace from the PKA/PKC sensor KCAP-1 in N1E-115 cells.
Figure 5: Influence of expression levels on probe performance.
Figure 6

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References

  1. Zhang, J., Campbell, R.E., Ting, A.Y. & Tsien, R.Y. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).

    Article  CAS  Google Scholar 

  2. Miyawaki, A. Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4, 295–305 (2003).

    Article  CAS  Google Scholar 

  3. Wouters, F.S., Verveer, P.J. & Bastiaens, P.I.H. Imaging biochemistry inside cells. Trends Cell Biol. 11, 203–211 (2001).

    Article  CAS  Google Scholar 

  4. Griesbeck, O. Fluorescent proteins as sensors for cellular functions. Curr. Opin. Neurobiol. 14, 636–641 (2004).

    Article  CAS  Google Scholar 

  5. Hoffmann, C. et al. A FlAsH-based FRET approach to determine G-protein coupled receptor activation in living cells. Nat. Methods 2, 171–176 (2005).

    Article  CAS  Google Scholar 

  6. Rothman, D.M., Shults, M.D. & Imperiali, B. Chemical approaches for investigating phosphorylation in signal transduction networks. Trends Cell Biol. 15, 502–510 (2005).

    Article  CAS  Google Scholar 

  7. Yeh, R.H., Yan, X.W., Cammer, M., Bresnick, A.R. & Lawrence, D.S. Real time visualization of protein kinase activity in living cells. J. Biol. Chem. 277, 11527–11532 (2002).

    Article  CAS  Google Scholar 

  8. Nguyen, A., Rothman, D.M., Stehn, J., Imperiali, B. & Yaffe, M.B. Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function. Nat. Biotechnol. 22, 993–1000 (2004).

    Article  CAS  Google Scholar 

  9. Adams, S.R., Harootunian, A.T., Buechler, Y.J., Taylor, S.S. & Tsien, R.Y. Fluorescence ratio imaging of cyclic-amp in single cells. Nature 349, 694–697 (1991).

    Article  CAS  Google Scholar 

  10. Violin, J.D., Zhang, J., Tsien, R.Y. & Newton, A.C. A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J. Cell Biol. 161, 899–909 (2003).

    Article  CAS  Google Scholar 

  11. Zacharias, D. Sticky caveats in an otherwise glowing report: oligomerizing fluorescent proteins and their use in cell biology. Sci. STKE, PE23 (2002).

  12. Schleifenbaum, A., Stier, G., Gasch, A., Sattler, M. & Schultz, C. Genetically encoded FRET probe for PKC activity based on pleckstrin. J. Am. Chem. Soc. 126, 11786–11787 (2004).

    Article  CAS  Google Scholar 

  13. 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. 128, 24–25 (2006).

    Article  CAS  Google Scholar 

  14. Zimmermann, T., Rietdorf, J., Girod, A., Georget, V. & Pepperkok, R. Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair. FEBS Lett. 531, 245–249 (2002).

    Article  CAS  Google Scholar 

  15. Zhang, J.H., Chung, T.D.Y. & Oldenburg, K.R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screening 4, 67–73 (1999).

    Article  CAS  Google Scholar 

  16. Sittampalam, G.S. et al. Design of signal windows in high throughput screening assays for drug discovery. J. Biomol. Screening 2, 159–169 (1997).

    Article  Google Scholar 

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  1. European Molecular Biology Institute, Meyerhofstraße 1, Heidelberg, 69117, Germany

    Justin Brumbaugh, Andreas Schleifenbaum, Gunter Stier, Michael Sattler & Carsten Schultz

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Correspondence to Carsten Schultz.

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Brumbaugh, J., Schleifenbaum, A., Stier, G. et al. Single- and dual-parameter FRET kinase probes based on pleckstrin. Nat Protoc 1, 1044–1055 (2006). https://doi.org/10.1038/nprot.2006.177

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