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
Notice bibliographique
Résumé
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
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,002 | 0,001 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».