Crystallographic, kinetic, and calorimetric investigation of PKA interactions with L-type calcium channels and Rad GTPase
Bibliographic record
Abstract
Abstract β-adrenergic signalling leads to activation of cAMP-dependent protein kinase (PKA), which can regulate the activity of L-type voltage-gated calcium channels (Ca V s) in multiple tissues. In Ca V 1.2, 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 Ca 2+ currents in smooth muscle. Its role in augmenting cardiac Ca 2+ 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 Ca V 1.2 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 Ca V 1.2 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 Ca V 1.2 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. Summary Stress 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 (Ca V s) in various tissues. There is a lot of controversy surrounding the exact recognition and specificity of PKA towards Ca V 1.2, 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 Ca V 1.2 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 Ca V 1.2, showing how PKA undergoes multiple structural transitions towards binding and how it makes use of a unique anchoring residue.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".