cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry
cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Portland Press Ltd.
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095912/ https://www.ncbi.nlm.nih.gov/pubmed/27444646 http://dx.doi.org/10.1042/BCJ20160648 |
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author | Byrne, Dominic P. Vonderach, Matthias Ferries, Samantha Brownridge, Philip J. Eyers, Claire E. Eyers, Patrick A. |
author_facet | Byrne, Dominic P. Vonderach, Matthias Ferries, Samantha Brownridge, Philip J. Eyers, Claire E. Eyers, Patrick A. |
author_sort | Byrne, Dominic P. |
collection | PubMed |
description | cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro. We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R(2)C(2) holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling. |
format | Online Article Text |
id | pubmed-5095912 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Portland Press Ltd. |
record_format | MEDLINE/PubMed |
spelling | pubmed-50959122016-11-08 cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry Byrne, Dominic P. Vonderach, Matthias Ferries, Samantha Brownridge, Philip J. Eyers, Claire E. Eyers, Patrick A. Biochem J Research Articles cAMP-dependent protein kinase (PKA) is an archetypal biological signaling module and a model for understanding the regulation of protein kinases. In the present study, we combine biochemistry with differential scanning fluorimetry (DSF) and ion mobility–mass spectrometry (IM–MS) to evaluate effects of phosphorylation and structure on the ligand binding, dynamics and stability of components of heteromeric PKA protein complexes in vitro. We uncover dynamic, conformationally distinct populations of the PKA catalytic subunit with distinct structural stability and susceptibility to the physiological protein inhibitor PKI. Native MS of reconstituted PKA R(2)C(2) holoenzymes reveals variable subunit stoichiometry and holoenzyme ablation by PKI binding. Finally, we find that although a ‘kinase-dead’ PKA catalytic domain cannot bind to ATP in solution, it interacts with several prominent chemical kinase inhibitors. These data demonstrate the combined power of IM–MS and DSF to probe PKA dynamics and regulation, techniques that can be employed to evaluate other protein-ligand complexes, with broad implications for cellular signaling. Portland Press Ltd. 2016-10-01 2016-09-27 /pmc/articles/PMC5095912/ /pubmed/27444646 http://dx.doi.org/10.1042/BCJ20160648 Text en © 2016 The Author(s) https://creativecommons.org/licenses/by/4.0/This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0) . |
spellingShingle | Research Articles Byrne, Dominic P. Vonderach, Matthias Ferries, Samantha Brownridge, Philip J. Eyers, Claire E. Eyers, Patrick A. cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry |
title | cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry |
title_full | cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry |
title_fullStr | cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry |
title_full_unstemmed | cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry |
title_short | cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry |
title_sort | camp-dependent protein kinase (pka) complexes probed by complementary differential scanning fluorimetry and ion mobility–mass spectrometry |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095912/ https://www.ncbi.nlm.nih.gov/pubmed/27444646 http://dx.doi.org/10.1042/BCJ20160648 |
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