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

Research output: Contribution to journal › Journal article › Research › peer-review

Standard

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, Serge; Deak, Maria; Sumpton, David; Ford, Rebecca J.; Núñez Galindo, Antonio; Kussmann, Martin; Viollet, Benoit; Steinberg, Gregory R.; Foretz, Marc; Dayon, Loïc; Morrice, Nicholas A.; Sakamoto, Kei.

In: Cellular Signalling, Vol. 27, No. 5, 01.05.2015, p. 978-988.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Ducommun, S, Deak, M, Sumpton, D, Ford, RJ, Núñez Galindo, A, Kussmann, M, Viollet, B, Steinberg, GR, Foretz, M, Dayon, L, Morrice, NA & Sakamoto, K 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, vol. 27, no. 5, pp. 978-988. https://doi.org/10.1016/j.cellsig.2015.02.008

APA

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., & Sakamoto, K. (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), 978-988. https://doi.org/10.1016/j.cellsig.2015.02.008

Vancouver

Ducommun S, Deak M, Sumpton D, Ford RJ, Núñez Galindo A, Kussmann M et al. 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. 2015 May 1;27(5):978-988. https://doi.org/10.1016/j.cellsig.2015.02.008

Author

Ducommun, Serge ; Deak, Maria ; Sumpton, David ; Ford, Rebecca J. ; Núñez Galindo, Antonio ; Kussmann, Martin ; Viollet, Benoit ; Steinberg, Gregory R. ; Foretz, Marc ; Dayon, Loïc ; Morrice, Nicholas A. ; Sakamoto, Kei. / Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets : Identification of mitochondrial fission factor as a new AMPK substrate. In: Cellular Signalling. 2015 ; Vol. 27, No. 5. pp. 978-988.

Bibtex

@article{b620b30566bd4fadbb52b68f9f7e1940,
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.",
keywords = "Cell signalling, Energy metabolism, Mitochondrial dynamics, Mitochondrial fission, Protein kinase",
author = "Serge Ducommun and Maria Deak and David Sumpton and Ford, {Rebecca J.} and {N{\'u}{\~n}ez Galindo}, Antonio and Martin Kussmann and Benoit Viollet and Steinberg, {Gregory R.} and Marc Foretz and Lo{\"i}c Dayon and Morrice, {Nicholas A.} and Kei Sakamoto",
year = "2015",
month = may,
day = "1",
doi = "10.1016/j.cellsig.2015.02.008",
language = "English",
volume = "27",
pages = "978--988",
journal = "Cellular Signalling",
issn = "0898-6568",
publisher = "Elsevier",
number = "5",

}

RIS

TY - JOUR

T1 - Motif affinity and mass spectrometry proteomic approach for the discovery of cellular AMPK targets

T2 - Identification of mitochondrial fission factor as a new AMPK substrate

AU - Ducommun, Serge

AU - Deak, Maria

AU - Sumpton, David

AU - Ford, Rebecca J.

AU - Núñez Galindo, Antonio

AU - Kussmann, Martin

AU - Viollet, Benoit

AU - Steinberg, Gregory R.

AU - Foretz, Marc

AU - Dayon, Loïc

AU - Morrice, Nicholas A.

AU - Sakamoto, Kei

PY - 2015/5/1

Y1 - 2015/5/1

N2 - 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.

AB - 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.

KW - Cell signalling

KW - Energy metabolism

KW - Mitochondrial dynamics

KW - Mitochondrial fission

KW - Protein kinase

UR - http://www.scopus.com/inward/record.url?scp=84925494009&partnerID=8YFLogxK

U2 - 10.1016/j.cellsig.2015.02.008

DO - 10.1016/j.cellsig.2015.02.008

M3 - Journal article

C2 - 25683918

AN - SCOPUS:84925494009

VL - 27

SP - 978

EP - 988

JO - Cellular Signalling

JF - Cellular Signalling

SN - 0898-6568

IS - 5

ER -

ID: 239212721