Divalent Metal Ions Mg²⁺ and Ca²⁺ Have Distinct Effects on Protein Kinase A Activity and Regulation

Abstract

cAMP-dependent protein kinase (PKA) is regulated primarily in response to physiological signals while nucleotides and metals may provide fine-tuning. PKA can use different metal ions for phosphoryl transfer, yet some, like Ca2+, do not support steady-state catalysis. Fluorescence Polarization (FP) and Surface Plasmon Resonance (SPR) were used to study inhibitor and substrate interactions with PKA. The data illustrate how metals can act differentially as a result of their inherent coordination properties. We found that Ca2+, in contrast to Mg2+, does not induce high-affinity binding of PKA to pseudosubstrate inhibitors. However, Ca2+ works in a single turnover mode to allow for phosphoryl-transfer. Using a novel SPR approach, we were able to directly monitor the interaction of PKA with a substrate in the presence of Mg2+ATP. This allows us to depict the entire kinase reaction including complex formation as well as release of the phosphorylated substrate. In contrast to Mg2+, Ca2+ apparently slows down the enzymatic reaction. A focus on individual reaction steps revealed that Ca2+ is not as efficient as Mg2+ in stabilizing the enzyme:substrate complex. The opposite holds true for product dissociation where Mg2+ easily releases the phospho-substrate while Ca2+ traps both reaction products at the active site. This explains the low steady-state activity in the presence of Ca2+. Furthermore, Ca2+ is able to modulate kinase activity as well as inhibitor binding even in the presence of Mg2+. We therefore hypothesize that the physiological metal ions Mg2+ and Ca2+ both play a role in kinase activity and regulation. Since PKA is localized close to calcium channels and may render PKA activity susceptible to Ca2+, our data provide a possible mechanism for novel crosstalk between cAMP and calcium signaling.

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

“…We therefore exploited DSF to investigate the binding of nucleotides to PKAc. As expected [65,66], thermostabilization was readily observed for WT PKAc using a broad range of nucleotides (particularly ATP and ADP) in the presence of divalent cations (Figure 3B), and this was recapitulated with the R133A variant. In contrast with the increase in T m observed for WT PKAc (Δ T m  = 4.24 ± 0.09°C) and R133A (Δ T m  = 3.49 ± 0.16°C) with 1 mM ATP and Mg 2+ , incubation of K72H PKAc (or a less stable K72R PKAc mutant, data not shown) with any combination of nucleotides or cations failed to promote any stabilization under these conditions (Δ T m for K72H = −0.49 ± 0.04°C upon incubation with ATP/Mg 2+ ions), even at very high concentrations (up to 4 mM) of ATP.…”

Activating PRKACB somatic mutation in cortisol-producing adenomas

“…A Biacore T200 SPR instrument (GE Healthcare) was used for kinetic analysis of the interaction of PKA-Cβ WT and S54L with GST-PKA-RIα. For this, an anti-GST (glutathione S-transferase) antibody (Carl Roth) functionalized CM5 sensor chip (GE Healthcare) was used as described previously (35). GST-PKA-RIα was captured on this anti-GST antibody surface and several concentrations of either WT (0-8 nM) or S54L (0-32 nM) PKA-Cβ (isoform 1) were injected in running buffer (20 mM MOPS, pH 7, 150 mM NaCl, 50 μM EDTA, 1 mM ATP, 10 mM MgCl 2 , 0.01% P20) at a flow rate of 30 μl/min at 25°C for 300 seconds.…”

“…Regeneration of the chip surface was performed by injecting 10 mM glycine (pH 1.9) for 30 seconds at 30 μl/min at the end of each measurement cycle. Binding of PKA-Cβ to GST-tagged PKIα was performed as described previously (35). Rate constants were determined by nonlinear curve fitting (global fit analysis) using the software T200 Evaluation 3.0 (GE Healthcare).…”

Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer

“…Binding of wild-type and Y204A PKA kinase catalytic subunit to various nucleotides was ascertained by the changes in the kinase's intrinsic tryptophan fluorescence as described previously (37). The reaction buffer was used as 25 mM Mops, pH 7.0, 100 mM NaCl, 10 mM MgCl 2 , and 2 mM DTT.…”