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AMPK pp 255-272 | Cite as

Cellular Application of Genetically Encoded Sensors and Impeders of AMPK

  • Takafumi Miyamoto
  • Elmer Rho
  • Allen Kim
  • Takanari Inoue
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1732)

Abstract

Unraveling the spatiotemporal dynamics of 5'-AMP-activated protein kinase (AMPK) signaling is necessary to bridge the gap between nutrient signaling and downstream function. Three genetically encoded Förster Resonance Energy Transfer (FRET)-based AMPK biosensors are available yielding insight into how AMPK-derived signal propagates throughout a cell in response to particular inputs. These findings, together with accumulating evidence obtained from biochemical techniques, promise to give a holistic understanding of the AMPK signaling. In this protocol, we describe the procedures and materials required for imaging intracellular AMPK activity in an organelle-specific manner, with a focus on ABKAR, a FRET-based biosensor. In addition, we introduce a novel AMPK inhibitor peptide that allows us to inhibit AMPK activity at specific subcellular compartments.

Key words

Organelle-specific Kinase activity monitoring Biosensor Förster resonance energy transfer (FRET) Compartmentalization Signaling dynamics Inhibitor peptide 

Notes

Acknowledgments

We are grateful to Robert DeRose and Daniel Frigo for constructive comments. This work was supported in part by the US National Institutes of Health (NIH) grant to T.I. (DK102910).

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Takafumi Miyamoto
    • 1
  • Elmer Rho
    • 2
  • Allen Kim
    • 2
  • Takanari Inoue
    • 2
  1. 1.Department of Internal Medicine (Endocrinology and Metabolism), Faculty of MedicineUniversity of TsukubaIbarakiJapan
  2. 2.Cell Biology and Biomedical Engineering, School of MedicineJohns Hopkins UniversityBaltimoreUSA

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