Investigation of PKA interactions with cardiac voltage gated calcium channel and Rad GTPase

The β-adrenergic signaling pathway leads to PKA-mediated activation of L-type voltage-gated calcium channels (CaVs) in multiple tissues. Various sites have been proposed to be involved In CaV1.2. Notably, PKA-mediated phosphorylation of Ser1981 is reported in diabetes progression, synaptic plasticity, and the increase of cardiac calcium currents during the fight-or-flight response. However, the role of Ser1981 phosphorylation is heavily scrutinized, with alternative sites Ser1718 and Ser1535 proposed to be important during β-adrenergic activation of CaV1.2. Recently, a constitutive inhibitor of CaV1.2 called Rad was also identified as a target of PKA during cardiac excitation. However, it is unclear which of the four potential sites (Ser25, Ser38, Ser272, and Ser300) are preferentially phosphorylated by PKA. Using quantitative binding and kinetic experiments we measured significant binding and activity towards Ser1981 on CaV1.2 and Ser25 and Ser272 on Rad, with no measurable PKA binding or kinetic activity found for the other sites. We also captured multiple PKAc high resolution structures using X-ray crystallography. Our PKAc:AMP-PNP/Mg2+:CaV peptide ternary complex reveal a novel mode of PKAc substrate recognition, involving a phenylalanine at the P+1 position anchored into a hydrophobic pocket of the enzyme's active site. Our work thus provides valuable mechanistic insight into PKA substrate recognition and suggest the likely physiological target sites for PKA on CaV1.2 and Rad.