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Enhanced bioluminescent sensor for longitudinal detection of CREB activation in living cells

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Abstract

Cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is associated with memory formation and controls cell survival and proliferation via regulation of downstream gene expression in tumorigenesis. As a transcription factor, CREB binds to cAMP response elements. Phosphorylation of CREB triggers transcriptional activation of CREB downstream genes following the interaction of the kinase-inducible domain (KID) of CREB with the KID interaction domain (KIX) of CREB-binding protein. Nevertheless, because of the lack of single-cell analytical techniques, little is known about spatiotemporal regulation of CREB phosphorylation. To analyze CREB activation in single living cells, we developed genetically encoded bioluminescent sensors using luciferase-fragment complementation: the sensors are designed based on KID–KIX interaction with a single-molecule format. The luminescence intensity of the sensor, designated as CREX (a sensor of CREB activation based on KID (CREB)–KIX interaction), increased by phosphorylation of CREB. Moreover, the luminescence intensity of CREX was sufficient to detect CREB activation in live-cell bioluminescence imaging for single-cell analysis because of the higher sensitivity. CREX sensor is expected to contribute to elucidation of the spatiotemporal regulation of CREB phosphorylation by applying single-cell analysis.

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Authors and Affiliations

  1. Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Natsumi Noda & Takeaki Ozawa

  2. Department of Molecular Neuroscience, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan

    Tetsuya Ishimoto & Hisashi Mori

Authors
  1. Natsumi Noda
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  2. Tetsuya Ishimoto
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  3. Hisashi Mori
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  4. Takeaki Ozawa
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Corresponding author

Correspondence to Takeaki Ozawa.

Additional information

Electronic supplementary information (ESI) available. See DOI: 10.1039/c9pp00249a

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Noda, N., Ishimoto, T., Mori, H. et al. Enhanced bioluminescent sensor for longitudinal detection of CREB activation in living cells. Photochem Photobiol Sci 18, 2740–2747 (2019). https://doi.org/10.1039/c9pp00249a

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  • DOI: https://doi.org/10.1039/c9pp00249a

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