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Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation

Protein phosphorylation and dephosphorylation reactions play key regulatory roles in many fundamental cellular processes. Due to the large number of kinases and phosphatases in the genome, the identification of the specific enzymes responsible for a given site in a given protein is immensely challen...

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Autores principales: Battle, Laura Johnson, Chambers, Timothy C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Journal of Biological Methods 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5726596/
https://www.ncbi.nlm.nih.gov/pubmed/29242808
http://dx.doi.org/10.14440/jbm.2017.199
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author Battle, Laura Johnson
Chambers, Timothy C.
author_facet Battle, Laura Johnson
Chambers, Timothy C.
author_sort Battle, Laura Johnson
collection PubMed
description Protein phosphorylation and dephosphorylation reactions play key regulatory roles in many fundamental cellular processes. Due to the large number of kinases and phosphatases in the genome, the identification of the specific enzymes responsible for a given site in a given protein is immensely challenging. However, because protein kinases and phosphatases recognize local specificity determinants within proteins, it is possible to use small peptides to study the characteristics of site-specific phosphorylation. In addition, phosphorylation usually causes retardation in gel mobility, providing an opportunity to investigate peptide phosphorylation and dephosphorylation by monitoring migration on high resolution peptide gels. In this study, we demonstrate the utility of such a technique using small peptides corresponding to cyclin-dependent kinase-1 (Cdk1)/cyclin B1 sites in two important apoptotic regulatory proteins, Bcl-xL and caspase-9. We show that the mobility of the peptides is retarded following Cdk1-mediated phosphorylation, and that peptide dephosphorylation, catalyzed either by purified phosphatase or by crude cell extracts, is readily observable by increased peptide gel mobility. Furthermore, the procedure can be conducted without the use of radioactive adenosine triphosphate (ATP), and does not require any specialized reagents or apparatus. The method can be used to identify and characterize specific kinase and phosphatases responsible for phosphorylation and dephosphorylation of specific sites in any protein of interest.
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spelling pubmed-57265962017-12-12 Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation Battle, Laura Johnson Chambers, Timothy C. J Biol Methods Article Protein phosphorylation and dephosphorylation reactions play key regulatory roles in many fundamental cellular processes. Due to the large number of kinases and phosphatases in the genome, the identification of the specific enzymes responsible for a given site in a given protein is immensely challenging. However, because protein kinases and phosphatases recognize local specificity determinants within proteins, it is possible to use small peptides to study the characteristics of site-specific phosphorylation. In addition, phosphorylation usually causes retardation in gel mobility, providing an opportunity to investigate peptide phosphorylation and dephosphorylation by monitoring migration on high resolution peptide gels. In this study, we demonstrate the utility of such a technique using small peptides corresponding to cyclin-dependent kinase-1 (Cdk1)/cyclin B1 sites in two important apoptotic regulatory proteins, Bcl-xL and caspase-9. We show that the mobility of the peptides is retarded following Cdk1-mediated phosphorylation, and that peptide dephosphorylation, catalyzed either by purified phosphatase or by crude cell extracts, is readily observable by increased peptide gel mobility. Furthermore, the procedure can be conducted without the use of radioactive adenosine triphosphate (ATP), and does not require any specialized reagents or apparatus. The method can be used to identify and characterize specific kinase and phosphatases responsible for phosphorylation and dephosphorylation of specific sites in any protein of interest. Journal of Biological Methods 2017-08-02 /pmc/articles/PMC5726596/ /pubmed/29242808 http://dx.doi.org/10.14440/jbm.2017.199 Text en © 2013-2018 The Journal of Biological Methods, All rights reserved. https://creativecommons.org/licenses/by/3.0/ This work is licensed under a Creative Commons Attribution 3.0 License.
spellingShingle Article
Battle, Laura Johnson
Chambers, Timothy C.
Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation
title Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation
title_full Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation
title_fullStr Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation
title_full_unstemmed Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation
title_short Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation
title_sort small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5726596/
https://www.ncbi.nlm.nih.gov/pubmed/29242808
http://dx.doi.org/10.14440/jbm.2017.199
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