(837.5) 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

Poster Board Number: B64

Omar Hamed (Cumming School of Medicine - University of Calgary), Mark Giembycz (Cumming School of Medicine - University of Calgary)

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 gt;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

CIHR, ALA