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.

Local protein kinase A action proceeds through intact holoenzymes

“…Native mass spectrometry (MS) allows the measurement of molecular mass and is used to determine the stoichiometry of intact protein complexes in the gas phase (7–9). In the presence of cAMP, which copurifies with RII, we observed higher-order complexes, including the intact 2RII:2C PKA holoenzyme and the pentameric AKAP79:2RII:2C assembly (Fig.…”

Covalent Aurora A regulation by the metabolic integrator coenzyme A

“…1E). We also evaluated effects of CoA and dpCoA on the gas phase conformation of Aurora A using Ion Mobility Mass Spectrometry (IM-MS), which is a new approach for probing kinase conformational dynamics (Byrne et al, 2016). The calculated cross-sectional area of intact Aurora A (nm 2 ) in both 14 + and 15 + charge states increased significantly after pre-incubation with both CoA or dpCoA, consistent with their ability to bind to Aurora A and induce a conformational change.…”

“…IM-MS analysis was performed on a Waters Synapt G2-Si instrument. Aurora A was buffer exchanged into 50 mM NH4OAc (LC grade, Sigma) as previously described (Byrne et al, 2016). Typically, 1-3 µl of 2-5 µM sample was analyzed using borosilicate emitters (Thermo ES 387).…”

“…The exact hard sphere scattering (EHSS) model implemented in the Mobcal software was used to calculate CCS values on the basis of X-ray structures, as described previously (Byrne et al, 2016).…”

Characterization of aminoacyl-tRNA synthetase stability and substrate interaction by differential scanning fluorimetry

“…Many examples of DSF have been reported for proteins such as Carbonic Anhydrase-II [16], cytoplasmic sulfotransferase 1C1 [17], cAMP-dependent protein kinase (PKA) [18], MAPK13 [19], and pregnane xenobiotic receptor (PXR) [20] to name a few [21]. A particular advantage of the DSF method is that the experimental set up can be implemented in a high-throughput mode by use of a 96-well microtiter plate [22].…”