Crystallographic, kinetic, and calorimetric investigation of PKA interactions with L-type calcium channels and Rad GTPase

Abstract

Abstractβ-adrenergic signalling leads to activation of cAMP-dependent protein kinase (PKA), which can regulate the activity of L-type voltage-gated calcium channels (CaVs) in multiple tissues. In CaV1.2, various sites have been proposed to be involved, including Ser1981 in the C-terminal tail. Its phosphorylation is linked to diabetes progression, synaptic plasticity, and the augmentation of Ca2+currents in smooth muscle. Its role in augmenting cardiac Ca2+currents has been heavily scrutinized, with alternative models including the sites Ser1718 and Ser1535. Recently, the GTPase Rad has been identified as a critical PKA target that mediates the augmentation of cardiac CaV1.2 currents upon its phosphorylation. However, it is unclear which of the four potential sites (Ser25, Ser38, Ser272, and Ser300) are favored by PKA. Using quantitative binding experiments and enzyme kinetics, we show that there are two Tiers of target sites, with CaV1.2 residue Ser1981 and Rad residues Ser25 and Ser272 forming Tier 1 substrates for PKA. The other sites form a second Tier, with PKA only showing minimal detectable activity. The Tier 1 substrates share a common feature with two arginine residues that anchor the peptide into the active site of PKA. We report crystal structures of the PKA catalytic subunit (PKAc) with and without a CaV1.2 substrate that represent different successive conformations prior to product turnover. Different target sites utilize different anchoring residues, highlighting the plasticity of PKAc to recognize substrates.SummaryStress signals can alter the electrical properties of excitable cells. cAMP-dependent protein kinase A (PKA) is a key enzyme that is activated upon β-adrenergic stimulation and can alter the function of L-type voltage-gated calcium channels (CaVs) in various tissues. There is a lot of controversy surrounding the exact recognition and specificity of PKA towards CaV1.2, a key calcium channel located in neuronal, cardiac, and smooth muscle tissue, among others. Using a quantitative and unbiased approach, we determined the substrate specificities of PKA towards various sites in CaV1.2 and Rad, an inhibitory protein. Our work highlights two Tiers of substrates, suggesting a potential graded response. Using X-ray crystallography, we determined a high-resolution structure of PKA bound to its strongest target site in CaV1.2, showing how PKA undergoes multiple structural transitions towards binding and how it makes use of a unique anchoring residue.