Abstract
Calcineurin is an evolutionarily conserved, ubiquitously expressed protein phosphatase that serves as a major effector of Ca2+ signals, regulating diverse biological processes such as gene expression, tissue differentiation, immune responses, and neural plasticity. The following method describes how to monitor real-time calcineurin activity in cultured mammalian cells using a fluorescence resonance energy transfer (FRET)-based activity reporter.
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References
Hilioti Z, Cunningham KW (2004) Calcineurin: roles of the Ca2+/calmodulin-dependent protein phosphatase in diverse eukaryotes. Top Curr Genet 5:73–90
Rusnak F, Mertz P (2000) Calcineurin: form and function. Physiol Rev 80(4):1483–1521
Harris CD, Ermak G, Davies KJ (2005) Multiple roles of the DSCR1 (Adapt78 or RCAN1) gene and its protein product calcipressin 1 (or RCAN1) in disease. Cell Mol Life Sci 62(21):2477–2486. doi:10.1007/s00018-005-5085-4
Heineke J, Molkentin JD (2006) Regulation of cardiac hypertrophy by intracellular signalling pathways. Nat Rev Mol Cell Biol 7(8): 589–600. doi:10.1038/nrm1983
Heit JJ (2007) Calcineurin/NFAT signaling in the beta-cell: from diabetes to new therapeutics. Bioessays 29(10):1011–1021. doi:10.1002/bies.20644
Xie CW (2004) Calcium-regulated signaling pathways: role in amyloid beta-induced synaptic dysfunction. Neuromolecular Med 6(1):53–64. doi:10.1385/NMM:6:1:053
Mehta S, Zhang J (2011) Reporting from the field: genetically encoded fluorescent reporters uncover signaling dynamics in living biological systems. Annu Rev Biochem 80: 375–401. doi:10.1146/annurev-biochem-060409-093259
Newman RH, Fosbrink MD, Zhang J (2011) Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chem Rev 111(5):3614–3666. doi:10.1021/cr100002u
Fosbrink M, Aye-Han NN, Cheong R, Levchenko A, Zhang J (2010) Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor. Proc Natl Acad Sci U S A 107(12):5459–5464. doi:10.1073/pnas.0909671107
Fuller BG, Lampson MA, Foley EA, Rosasco-Nitcher S, Le KV, Tobelmann P, Brautigan DL, Stukenberg PT, Kapoor TM (2008) Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient. Nature 453(7198):1132–1136. doi:10.1038/nature06923
Gao X, Zhang J (2008) Spatiotemporal analysis of differential Akt regulation in plasma membrane microdomains. Mol Biol Cell 19(10):4366–4373. doi:10.1091/mbc.E08-05-0449
Kunkel MT, Toker A, Tsien RY, Newton AC (2007) Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter. J Biol Chem 282(9):6733–6742. doi:10.1074/jbc.M608086200
Violin JD, Zhang J, Tsien RY, Newton AC (2003) A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C. J Cell Biol 161(5):899–909. doi:10.1083/jcb.200302125
Wang Y, Botvinick EL, Zhao Y, Berns MW, Usami S, Tsien RY, Chien S (2005) Visualizing the mechanical activation of Src. Nature 434(7036):1040–1045. doi:10.1038/nature03469
Zhang J, Hupfeld CJ, Taylor SS, Olefsky JM, Tsien RY (2005) Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes. Nature 437(7058):569–573. doi:10.1038/nature04140
Newman RH, Zhang J (2008) Visualization of phosphatase activity in living cells with a FRET-based calcineurin activity sensor. Mol Biosyst 4(6):496–501. doi:10.1039/b720034j
Crabtree GR, Olson EN (2002) NFAT signaling: choreographing the social lives of cells. Cell 109(Suppl):S67–S79
Hogan PG, Chen L, Nardone J, Rao A (2003) Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 17(18): 2205–2232
Horsley V, Pavlath GK (2002) NFAT: ubiquitous regulator of cell differentiation and adaptation. J Cell Biol 156(5):771–774
Okamura H, Aramburu J, Garcia-Rodriguez C, Viola JP, Raghavan A, Tahiliani M, Zhang X, Qin J, Hogan PG, Rao A (2000) Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity. Mol Cell 6(3):539–550
Porter CM, Havens MA, Clipstone NA (2000) Identification of amino acid residues and protein kinases involved in the regulation of NFATc subcellular localization. J Biol Chem 275(5):3543–3551
Ananthanarayanan B, Ni Q, Zhang J (2008) Chapter 2: molecular sensors based on fluorescence resonance energy transfer to visualize cellular dynamics. Methods Cell Biol 89:37–57. doi:10.1016/S0091-679X(08)00602-X
Miyawaki A, Tsien RY (2000) Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol 327:472–500
Depry C, Zhang J (2011) Using FRET-based reporters to visualize subcellular dynamics of protein kinase A activity. Methods Mol Biol 756:285–294. doi:10.1007/978-1-61779-160-4_16
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Mehta, S., Zhang, J. (2014). Using a Genetically Encoded FRET-Based Reporter to Visualize Calcineurin Phosphatase Activity in Living Cells. In: Zhang, J., Ni, Q., Newman, R. (eds) Fluorescent Protein-Based Biosensors. Methods in Molecular Biology, vol 1071. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-622-1_11
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DOI: https://doi.org/10.1007/978-1-62703-622-1_11
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