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Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale

AMP-activated protein kinase (AMPK) is responsible for sensing of the cell’s energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the γ2 regulatory subunit have been show...

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Detalles Bibliográficos
Autores principales: Renz, Bernhard, Davies, Joanna K., Carling, David, Watkins, Hugh, Redwood, Charles
Formato: Texto
Lenguaje:English
Publicado: Academic Press 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2691924/
https://www.ncbi.nlm.nih.gov/pubmed/19269329
http://dx.doi.org/10.1016/j.pep.2009.02.016
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author Renz, Bernhard
Davies, Joanna K.
Carling, David
Watkins, Hugh
Redwood, Charles
author_facet Renz, Bernhard
Davies, Joanna K.
Carling, David
Watkins, Hugh
Redwood, Charles
author_sort Renz, Bernhard
collection PubMed
description AMP-activated protein kinase (AMPK) is responsible for sensing of the cell’s energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the γ2 regulatory subunit have been shown to cause hypertrophic cardiomyopathy (HCM) with conduction abnormalities. As part of a study to examine the role of AMPK in the heart, we tested whether specific domains of the thick filament component cardiac myosin binding protein-C (cMyBP-C) were good in vitro AMPK substrates. The commercially available pET28a expression vector was used to generate a recombinant form of the cMyBP-C C8 domain as a fusion protein with a hexahistidine tag. In vitro phosphorylation with activated kinase showed that the purified fusion protein was a good AMPK substrate, phosphorylated at a similar rate to the control SAMS peptide and with phosphate incorporation specifically in serine residues. However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector. As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.
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spelling pubmed-26919242009-06-11 Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale Renz, Bernhard Davies, Joanna K. Carling, David Watkins, Hugh Redwood, Charles Protein Expr Purif Article AMP-activated protein kinase (AMPK) is responsible for sensing of the cell’s energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the γ2 regulatory subunit have been shown to cause hypertrophic cardiomyopathy (HCM) with conduction abnormalities. As part of a study to examine the role of AMPK in the heart, we tested whether specific domains of the thick filament component cardiac myosin binding protein-C (cMyBP-C) were good in vitro AMPK substrates. The commercially available pET28a expression vector was used to generate a recombinant form of the cMyBP-C C8 domain as a fusion protein with a hexahistidine tag. In vitro phosphorylation with activated kinase showed that the purified fusion protein was a good AMPK substrate, phosphorylated at a similar rate to the control SAMS peptide and with phosphate incorporation specifically in serine residues. However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector. As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present. Academic Press 2009-08 /pmc/articles/PMC2691924/ /pubmed/19269329 http://dx.doi.org/10.1016/j.pep.2009.02.016 Text en © 2009 Elsevier Inc. https://creativecommons.org/licenses/by-nc-nd/3.0/ Open Access under CC BY-NC-ND 3.0 (https://creativecommons.org/licenses/by-nc-nd/3.0/) license
spellingShingle Article
Renz, Bernhard
Davies, Joanna K.
Carling, David
Watkins, Hugh
Redwood, Charles
Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale
title Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale
title_full Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale
title_fullStr Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale
title_full_unstemmed Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale
title_short Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale
title_sort determination of amp-activated protein kinase phosphorylation sites in recombinant protein expressed using the pet28a vector: a cautionary tale
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2691924/
https://www.ncbi.nlm.nih.gov/pubmed/19269329
http://dx.doi.org/10.1016/j.pep.2009.02.016
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