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. 2016 Oct 1;473(19):3159-75.
doi: 10.1042/BCJ20160648. Epub 2016 Jul 21.

cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry

Dominic P Byrne  1 , Matthias Vonderach  1 , Samantha Ferries  1 , Philip J Brownridge  1 , Claire E Eyers  1 , Patrick A Eyers  1
Affiliations

cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry

Dominic P Byrne et al. Biochem J. .

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.

Keywords: complex; inhibitor; ion mobility; mass spectrometry; protein kinase A; protein structure.

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Figures

Figure 1.
Figure 1.. Analysis of purified recombinant proteins that make up the PKA signaling complex.
(A) Coomassie blue staining of purified recombinant PKA proteins: 1.5 μg of PKA catalytic (WT, K72H or R133A proteins) and regulatory (RIIα subunits and 0.6 μg of PKI protein were analyzed by SDS–PAGE and proteins stained with Coomassie Blue). Coomassie staining of PKI is weak due to its small size and amino acid composition. (B) CD spectra demonstrating similar secondary structures of WT and K72H PKAc in the absence or presence of 1 mM ATP and 10 mM Mg2+ ions. The mean average of three replicate spectra of 0.6 mg/ml WT (red) and K72H (blue) PKAc in 10 mM sodium phosphate (pH 7.4), and 25 mM NaF, recorded in a 0.1 cm cell are shown. (C) ESI mass spectra of the 39+ charge state of intact recombinant PKAc WT (PKA) under denaturing conditions, in the absence (top) or presence of Mn2+-λ protein phosphatase (λPP), and for PKAc K72H and PKAc R133A. Peaks are annotated with the number of phosphate groups.
Figure 2.
Figure 2.. IM-MS of PKAc variants reveals conformationally distinct forms.
(A) ESI mass spectrum of PKAc obtained under nondenaturing ‘native’ conditions. (B) TWCCSN2→He for the [M+13H]13+ form of untreated WT PKAc (PKA), PKAc following treatment with Mn2+-λ protein phosphatase (PKA λPP), and the K72H and R133A variants. Two overlapping conformations of PKA are indicated, the more extended of which (green) matches the CCSD of the other PKA species. (C; top to bottom) CIU profiles of PKAc WT, λPP-treated, K72H and R133A.
Figure 3.
Figure 3.. PKA binding of nucleotides is detectable by DSF.
(A) TSA of PKA WT, K72H and R133A (5 μM) in the presence of 10 mM MgCl2 and 1 mM (blue), 2 mM (green) or 4 mM (orange) ATP; buffer control is in red. (B) ΔTm for WT, K72H and R133A PKA upon nucleotide binding, as measured by DSF. Mean ΔTm values ± SD (n = 2) were calculated by subtracting the control Tm value (buffer, no nucleotide) from the measured Tm value.
Figure 4.
Figure 4.. PKI protein binds stably to PKAc WT, but not K72H or R133A protein.
(A) Native ESI mass spectra of PKAc WT, K72H and R133A, in the absence or presence of equimolar PKI. (B) TSA of WT, K72H and R133A PKAc proteins measured in the presence of the indicated concentration of ATP and 10 mM MgCl2 ± 10 μM PKI. Mean ΔTm values ± SD (n = 2) are shown. (C) TWCCSN2→He for [M+13H]13+ and [M+14H]14+ forms of WT PKAc in the absence (top) or presence (bottom) of PKI. CCS distribution of non-PKI-bound form of PKAc is presented [PKA(PKI)]. (D and E) CIU profiles of PKAc upon the addition of PKI: (D) PKI-bound PKA (PKA/PKI) and (E) non-PKI-bound PKAc.
Figure 5.
Figure 5.. PKI disrupts non-covalent complexes between PKAc and RII.
Native ESI mass spectra of WT PKAc (C, blue dots), PKA regulatory subunit RIIα (R, green squares) and equimolar ratios of the two (C + R) in the absence or presence of PKI proteins (red triangles).
Figure 6.
Figure 6.. Binding of small-molecule kinase inhibitors to PKAc as determined by DSF.
Thermal stability of WT (A), K72H (B) or R133A (C) PKAc (5 μM) was assessed as a function of inhibitor binding at the indicated concentrations. Mean ΔTm values ± SD (n = 2) are shown.
Figure 7.
Figure 7.. Binding of small-molecule inhibitors to PKAc and effect on disruption of PKA holoenzyme by PKI.
Native ESI mass spectra (A) and TWCCSN2→He distributions (B) acquired in the presence of DMSO vehicle or with 10-fold molar excess of staurosporine (STS), H89 or AT13148. CCSD are presented for [M+11H]11+ (red dotted line), [M+12H]12+ (blue line) and [M+13H]13+ (black line). (C) Equimolar ratios of WT PKAc (C, blue dots) and PKA RIIα (R, green squares) were preincubated with vehicle control, STS, K252a or AT13148, prior to the addition of equipmolar PKI (red triangles). Native ESI spectra are annotated with identified complexes.
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