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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...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Academic Press
2009
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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. |
format | Text |
id | pubmed-2691924 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | Academic Press |
record_format | MEDLINE/PubMed |
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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