title: cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry.
abstract: 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 R2C2 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.
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, U.K.
authors.forename: Dominic P
authors.initials: DP
authors.lastname: Byrne
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, U.K.
authors.forename: Matthias
authors.initials: M
authors.lastname: Vonderach
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, U.K.
authors.forename: Samantha
authors.initials: S
authors.lastname: Ferries
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, U.K.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Brownridge
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, U.K.
authors.forename: Claire E
authors.initials: CE
authors.lastname: Eyers
authors.affiliation: Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, U.K.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Eyers
mesh_terms: D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D015335:Molecular Probes; D010766:Phosphorylation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D015335:Molecular Probes; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: complex; inhibitor; ion mobility; mass spectrometry; protein kinase A; protein structure