Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate

Ducommun, S. et al. (2015) Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets: identification of mitochondrial fission factor as a new AMPK substrate. Cellular Signalling, 27(5), pp. 978-988. (doi: 10.1016/j.cellsig.2015.02.008) (PMID:25683918)

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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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Sumpton, Mr David and Morrice, Dr Nicholas
Authors: Ducommun, S., Deak, M., Sumpton, D., Ford, R. J., Núñez Galindo, A., Kussmann, M., Viollet, B., Steinberg, G. R., Foretz, M., Dayon, L., Morrice, N. A., and Sakamoto, K.
College/School:College of Medical Veterinary and Life Sciences > School of Cancer Sciences
Journal Name:Cellular Signalling
Publisher:Elsevier Inc.
ISSN:0898-6568
ISSN (Online):1873-3913
Copyright Holders:Copyright © 2015 The Authors
First Published:First published in Cellular Signalling 27(5):978-988
Publisher Policy:Reproduced under a Creative Commons License

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