title: Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate.
abstract: AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Although it is best known for its effects on carbohydrate and lipid metabolism, AMPK is implicated in diverse cellular processes, including mitochondrial biogenesis, autophagy, and cell growth and proliferation. To further our understanding of energy homeostasis through AMPK-dependent processes, the design and application of approaches to identify and characterise novel AMPK substrates are invaluable. Here, we report an affinity proteomicstrategy for the discovery and validation of AMPK targets using an antibody to isolate proteins containing the phospho-AMPK substrate recognition motif from hepatocytes that had been treated with pharmacological AMPK activators. We identified 57 proteins that were uniquely enriched in the activator-treated hepatocytes, but were absent in hepatocytes lacking AMPK. We focused on two candidates, cingulin and mitochondrial fission factor (MFF), and further characterised/validated them as AMPK-dependent targets by immunoblotting with phosphorylation site-specific antibodies. A small-molecule AMPK activator caused transient phosphorylation of endogenous cingulin at S137 in intestinal Caco2 cells. Multiple splice-variants of MFF appear to express in hepatocytes and we identified a common AMPK-dependent phospho-site (S129) in all the 3 predominant variants spanning the mass range and a short variant-specific site (S146). Collectively, our proteomic-based approach using a phospho-AMPK substrate antibody in combination with genetic models and selective AMPK activators will provide a powerful and reliable platform for identifying novel AMPK-dependent cellular targets.
authors: Nestlé Institute of Health Sciences SA, EPFL Innovation Park, bâtiment G/H, 1015 Lausanne, Switzerland; School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.
authors: Serge
authors: S
authors: Ducommun
authors: Nestlé Institute of Health Sciences SA, EPFL Innovation Park, bâtiment G/H, 1015 Lausanne, Switzerland.
authors: Maria
authors: M
authors: Deak
authors: Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, UK.
authors: David
authors: D
authors: Sumpton
authors: Department of Medicine, Division of Endocrinology and Metabolism, McMaster University, Hamilton, Ontario, Canada.
authors: Rebecca J
authors: RJ
authors: Ford
authors: Nestlé Institute of Health Sciences SA, EPFL Innovation Park, bâtiment G/H, 1015 Lausanne, Switzerland.
authors: Antonio
authors: A
authors: Núñez Galindo
authors: Nestlé Institute of Health Sciences SA, EPFL Innovation Park, bâtiment G/H, 1015 Lausanne, Switzerland; School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.
authors: Martin
authors: M
authors: Kussmann
authors: Inserm, U1016, Institut Cochin, Paris, France; CNRS, UMR 8104, Paris, France; Université Paris Descartes, Sorbonne Paris cité, Paris, France.
authors: Benoit
authors: B
authors: Viollet
authors: Department of Medicine, Division of Endocrinology and Metabolism, McMaster University, Hamilton, Ontario, Canada.
authors: Gregory R
authors: GR
authors: Steinberg
authors: Inserm, U1016, Institut Cochin, Paris, France; CNRS, UMR 8104, Paris, France; Université Paris Descartes, Sorbonne Paris cité, Paris, France.
authors: Marc
authors: M
authors: Foretz
authors: Nestlé Institute of Health Sciences SA, EPFL Innovation Park, bâtiment G/H, 1015 Lausanne, Switzerland.
authors: Loïc
authors: L
authors: Dayon
authors: Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, UK.
authors: Nicholas A
authors: NA
authors: Morrice
authors: Nestlé Institute of Health Sciences SA, EPFL Innovation Park, bâtiment G/H, 1015 Lausanne, Switzerland; School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland. Electronic address: Kei.Sakamoto@rd.nestle.com.
authors: Kei
authors: K
authors: Sakamoto
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000032:analysis / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D018938:Caco-2 Cells; D002478:Cells, Cultured; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D024101:Mitochondrial Proteins / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D040901:Proteomics
chemical_list: C489227:Cgn protein, mouse; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; C000603499:mitochondrial fission factor, mouse; D055372:AMP-Activated Protein Kinases
keywords: Cell signalling; Energy metabolism; Mitochondrial dynamics; Mitochondrial fission; Protein kinase