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.
Highlights
Protein phosphorylation is a reversible post-translational modification (PTM) catalyzed by the protein kinase-mediated enzymatic transfer of ATP γ-phosphate to an appropriate side chain in a substrate [1]
circular dichroism (CD) confirmed similar secondary structures in both WT and K72H PKA catalytic (C) subunits (PKAc) proteins when analyzed in the presence and absence of ATP and Mg2+ ions, the latter inducing no major changes in the spectra
Our comprehensive sequence coverage revealed a total of 11 sites of phosphorylation in recombinant WT and 11 phosphosites on R133A PKAc, all of which were absent from K72H PKAc preparations (Table 1 and Supplementary Material), confirming that they are autophosphorylation sites
Summary
Protein phosphorylation is a reversible post-translational modification (PTM) catalyzed by the protein kinase-mediated enzymatic transfer of ATP γ-phosphate to an appropriate side chain in a substrate [1]. Both exhibit CIU profiles more typical of the less stable, more open conformer of λPP-treated WT PKAc. That both PKA variants exhibit similar CCS values of lower conformational stability suggests that these structural effects are not primarily dependent on the extent of phosphorylation.
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