CRISPR/Cas9 screening reveals key role of STK11/LKB1 in vasopressin-mediated regulation of Aqp2 transcription

ABSTRACT: Identification of signaling networks is an essential goal in systems biology. Here, we use CRISPR knockout screening (employing a whole kinome sgRNA library) to identify functionally critical protein kinases in a well-studied Gs-dependent G-protein coupled receptor (GPCR)-signaling model, namely the vasopressin V2 receptor (V2R). Screening was done using a specially-designed fluorescence-based reporter cell line with green-fluorescent protein (GFP) co-transcribed with Aqp2, a gene whose transcription is dependent on vasopressin-mediated activation of protein kinase A (PKA). The reporter line endogenously expresses V2R. Positive regulators (n=16) included PKA-catalytic subunit α (but not β). Mark2 (Par1b) and Dyrk1a (minibrain homolog). Negative regulators (n=13) included PKA-regulatory subunit type Iα, Stk11 (liver kinase B1 [LKB1]), and three TGF-β receptor subunits (Tgfbr1, Tgfbr2, Tgfbr3) (see https://esbl.nhlbi.nih.gov/Databases/Kinome-CRISPR-screen/ for full list). Dyrk1a and Stk11/LKB1 knockout cell lines were created for further study. Dyrk1a knockout cell lines failed to express AQP2 protein and exhibited a profound decrease in AQP2 mRNA. RNA-sequencing demonstrated widespread increases in cell-cycle transcripts, with a general defect in cell differentiation, accounting for AQP2 loss. Stk11/LKB1 knockout lines displayed marked increases in AQP2 protein and mRNA. RNA-sequencing and phospho-proteomic findings point to a signaling model in which Stk11/LKB1- and PKA-mediated phosphorylation events exert counteracting effects on the activities of cAMP-responsive transcriptional coactivator (CRTC) proteins. Finally, additional studies confirmed that TGF-β exposure to un-transformed cells results in a profound decrease in AQP2 mRNA abundance along with most recognized differentiation markers, consistent with our prior conclusion that TGF-β represses Aqp2 gene expression by inducing epithelial-mesenchymal transition.

ORGANISM(S): Mus musculus

PROVIDER: GSE326336 | GEO | 2026/03/30

REPOSITORIES: GEO