ESBL-Transcriptomic Data-Tranche-3

Identification of signaling networks is an essential goal in systems biology. Here, we use CRISPR/Cas9 knockout screening (employing a whole kinome sgRNA library) to identify functionally critical protein kinases in a well-studied Gas-dependent G-protein coupled receptor (GPCR)-signaling model, namely the vasopressin V2 receptor (V2R) pathway. 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). Positive regulators (n=14) included PKA-catalytic subunit a (Prkaca) and Dyrk1a (minibrain homolog). Negative regulators (n=12) included PKA-regulatory subunit type Ia, Stk11 (catalytic subunit of liver kinase B1 [LKB1] complex), and three TGF-b receptor subunits (Tgfbr1, Tgfbr2, Tgfbr3) (see https://esbl.nhlbi.nih.gov/Databases/Kinome-CRISPR-screen/ for full list). 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. TGF-b exposure to un-transformed cells results in a profound decrease in V2R and AQP2 mRNA abundance along with multiple additional transcriptional targets of V2R-PKA signaling, consistent with prior findings in TGF-b-mediated vasopressin ‘escape’. Stk11/LKB1 knockout lines displayed marked increases in AQP2 protein and mRNA, even in the absence of vasopressin. RNA-sequencing showed a marked similarity between the responses to Stk11/LKB1 deletion and vasopressin exposure in untransformed cells. Phospho-proteomic data point to opposing roles of Stk11/LKB1 and PKA in the regulation of cAMP-responsive transcriptional coactivator (CRTC) proteins in the transcriptional response to V2R-PKA signaling. Data sets are currently publicly available and can be accessed at the following sites: 1. https://esbl.nhlbi.nih.gov/Databases/Kinome-CRISPR-screen/ 2. https://esbl.nhlbi.nih.gov/Databases/Dyrk1a-KO-RNA-seq/ 3. https://esbl.nhlbi.nih.gov/Databases/TGF-beta-response-RNA-seq/ 4. https://esbl.nhlbi.nih.gov/Databases/Vasopressin-RNA-seq-mpkCCD/ 5. https://esbl.nhlbi.nih.gov/Databases/Stk1-KO-no-VP/ 6. https://esbl.nhlbi.nih.gov/Databases/Stk11-KO-RNA-seq/