β-Adrenergic signaling activates cAMP-dependent PKA, which regulates the activity of L-type voltage-gated calcium channels such as CaV1.2. Several PKA target sites in the C-terminal tail of CaV1.2 have been identified, and their phosphorylation has been suggested to increase currents in specific tissues or heterologous expression systems. However, augmentation of CaV1.2 currents in the heart is instead mediated by phosphorylation of Rad, a small GTPase that can inhibit CaV1.2. It is unclear how each of the proposed target sites in CaV1.2 and Rad rank toward their recognition by PKA, which could reveal a preferential phosphorylation. Here, we used quantitative assays on three CaV1.2 and four Rad sites. Isothermal titration calorimetry and enzyme kinetics show that there are two Tiers of targets, with CaV1.2 residue Ser1981 and Rad residues Ser25 and Ser272 forming tier one substrates for PKA. These share a common feature with two Arginine residues at specific positions that can anchor the peptide into the substrate binding cleft of PKA. In contrast, PKA shows minimal activity for the other, tier two substrates, characterized by low kcat values and undetectable binding via isothermal titration calorimetry. The existence of two tiers suggests that PKA regulation of the CaV1.2 complex may occur in a graded fashion. We report crystal structures of the PKA catalytic subunit with and without a CaV1.2 and test the importance of several anchoring residues via mutagenesis. Different target sites utilize different anchors, highlighting the plasticity of PKAc to recognize substrates.

中文翻译：

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PKA 与 L 型钙通道和 Rad GTP 酶相互作用的晶体学、动力学和量热研究


β-肾上腺素能信号转导激活 cAMP 依赖性 PKA，从而调节 L 型电压门控钙通道（如 CaV1.2）的活性。已经确定了 CaV1.2 的 C 端尾部的几个 PKA 靶位点，并且它们的磷酸化被认为会增加特定组织或异源表达系统中的电流。然而，心脏中 CaV1.2 电流的增强是由 Rad 的磷酸化介导的，Rad 是一种可以抑制 CaV1.2 的小 GTP 酶。目前尚不清楚 CaV1.2 和 Rad 中每个拟议的靶位点如何被 PKA 识别，这可能揭示优先磷酸化。在这里，我们对 3 个 CaV1.2 和 4 个 Rad 位点进行了定量测定。等温滴定量热法和酶动力学表明，有两层靶标，CaV1.2 残基 Ser1981 和 Rad 残基 Ser25 和 Ser272 形成 PKA 的一级底物。它们与特定位置的两个精氨酸残基具有共同特征，可以将肽锚定到 PKA 的底物结合裂隙中。相比之下，PKA 对其他第二层底物的活性最小，其特征是低 kcat 值和通过等温滴定量热法检测不到的结合。两层的存在表明 CaV1.2 复合物的 PKA 调节可能以分级方式发生。我们报道了有和没有 CaV1.2 的 PKA 催化亚基的晶体结构，并通过诱变测试了几种锚定残基的重要性。不同的靶位点利用不同的锚定，突出了 PKAc 识别底物的可塑性。