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...

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Autores principales: Byrne, Dominic P., Vonderach, Matthias, Ferries, Samantha, Brownridge, Philip J., Eyers, Claire E., Eyers, Patrick A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2016
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.
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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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