title: A fluorescent reporter of AMPK activity and cellular energy stress.
abstract: AMP-activated protein kinase (AMPK) is activated when the AMP/ATP ratio in cells is elevated due to energy stress. Here, we describe a biosensor, AMPKAR, that exhibits enhanced fluorescence resonance energy transfer (FRET) in response to phosphorylation by AMPK, allowing spatiotemporal monitoring of AMPK activity in single cells. We show that this reporter responds to a variety of stimuli that are known to induce energy stress and that the response is dependent on AMPK α1 and α2 and on the upstream kinase LKB1. Interestingly, we found that AMPK activation is confined to the cytosol in response to energy stress but can be observed in both the cytosol and nucleus in response to calcium elevation. Finally, using this probe with U2OS cells in a microfluidic device, we observed a very high cell-to-cell variability in the amplitude and time course of AMPK activation and recovery in response to pulses of glucose deprivation.
authors: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA.
authors: Peiling
authors: P
authors: Tsou
authors: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA.
authors: Bin
authors: B
authors: Zheng
authors: BioMEMS Resource Center, Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors: Chia-Hsien
authors: CH
authors: Hsu
authors: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA.
authors: Atsuo T
authors: AT
authors: Sasaki
authors: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA. Electronic address: lewis_cantley@hms.harvard.edu.
authors: Lewis C
authors: LC
authors: Cantley
mesh_terms: D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000378:metabolism; D045744:Cell Line, Tumor; D004734:Energy Metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D046210:Microfluidic Analytical Techniques; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism
chemical_list: C526181:PRKAA2 protein, human; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D000091162:AMP-Activated Protein Kinase Kinases; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human; D002118:Calcium