Differential Roles of the α and β Catalytic Subunits of PKA in the Regulation of Formoterol‐modulated Gene Expression Changes in Human Airway Epithelial Cells
Bibliographic record
Abstract
Introduction Protein kinase A (PKA) is the most studied cAMP effector and phosphorylates different downstream proteins to control various cellular functions including gene expression. PKA is a heterotetramer composed of two regulatory (R) subunits and two catalytic (C) subunits of which there are three main variants: Cα, Cβ and Cγ. Whereas PKA‐Cα and Cβ are expressed ubiquitously, Cγ appears to be restricted to the testis. The specificity of PKA to phosphorylate different substrates has been attributed to the C subunits; however, this suggestion remains controversial given that several common features are shared between Cα and Cβ including the approximately 90.5% sequence homology in the catalytic core region. Hypothesis PKA Cα and Cβ subunits differentially regulate gene changes induced by the β2‐ adrenoceptor agonist, formoterol, in the BEAS‐2B human airway epithelial cell line. Summary of Results RNA‐sequencing determined that PRKACA transcripts encoding Cα were abundantly expressed in BEAS‐2B cells at a level that was 9.3‐fold higher than PRKACB, which encodes Cβ. Cα and Cβ were also identified at the protein level by western blotting. siRNA mediated knockdown of C subunits was employed to assess their role in regulating cAMP response element (CRE)‐dependent transcription following β2‐adrenoceptor stimulation. In BEAS‐2B cells depleted of Cα or Cβ, formoterol‐induced CRE binding protein (CREB) phosphorylation was inhibited by 66.1% and 31.4%, respectively and by 89.6% when both catalytic subunits were knocked‐down simultaneously. Similarly, the increase in formoterol‐induced CRE reporter luciferase activity was reduced by 38.5% and 21.5% in Cα‐ and Cβ‐depleted cells, respectively and by 66.5% following knockdown of both PKA catalytic subunits. These data were confirmed by using CRISPR/Cas9‐ mediated deletion of Cα in BEAS‐2B cells wherein formoterol induced CREB phosphorylation was reduced by 81%. However, transfection of Cα‐deficient cells with Cβ siRNAs abolished formoterolinduced CREB phosphorylation indicating that both C subunits regulated CRE‐dependent transcription. Formoterol also promoted the PKA‐dependent phosphorylation of c‐Raf in BEAS‐2B cells at Ser43 and Ser259, which inhibited the transcription of the Elk‐1‐regulated gene, EGR1. However, unlike CREB phosphorylation, formoterol‐induced c‐Raf phosphorylation was unaffected by Cα depletion but, nevertheless, abolished in cell concurrently depleted of Cβ. Such a discrepancy was investigated further by examining the regulation of 14 formoterol‐sensitive genes using qPCR. Of those, the expression of five was attenuated by >50% in Cα knockout cells whereas the remainder was considerably more resistant with NR4A3 (70% inhibition, CREB‐dependent) and EGR1 (13% inhibition, ElK‐1‐dependent) lying at the extremes of this continuum. The expression of all 14 genes was abrogated in Cα‐deficient BEAS‐2B cells depleted of Cβ. Conclusions These data suggest that PKA Cα and Cβ subunits serve distinct, non‐redundant roles in BEAS‐2B cells that cooperatively regulate β2‐adrenoceptor‐mediated gene expression changes in a transcript‐dependent manner that may depend on the substrate (e.g., c‐Raf vs. CREB) targeted by PKA
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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 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
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".