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

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 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.