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Mass spectrometry-based analysis of LRBA phosphorylation

ABSTRACT: Vasopressin/cAMP/protein kinase A (PKA) signaling phosphorylates AQP2 water channels in renal collecting ducts to reabsorb water from urine for the prevention of further water loss. Lipopolysaccharide-responsive and beige-like anchor protein (LRBA) mediates vasopressin-induced AQP2 phosphorylation; therefore LRBA is essential for urinary concentration. LRBA is identified as the PKA substrates in a mouse cortical collecting duct principal cell line (mpkCCDcl4) whose phosphorylation levels are nearly perfectly correlated with those of AQP2. Although mouse LRBA contains several consensus PKA phosphorylation sites, their phosphorylation status in response to vasopressin remain unknown. Post-translational modification analysis revealed that RRDS1607 and RRIS2189 were phosphorylated by vasopressin.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: YU HARA

LAB HEAD: Shinichi Uchida

PROVIDER: PXD033859 | Pride | 2022-07-23

REPOSITORIES: Pride

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cAMP/PKA signaling phosphorylates LRBA in renal collecting duct

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2022-07-23 | PXD033852 | Pride

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Identification of PKA-dependent signaling network using CRISPR-Cas9 coupled with quantitative transcriptomics, proteomics and phosphoproteomics

Project description:Purpose: PKA plays a crucial role in vasopressin signaling of renal collecting duct cells. To understand regulation of mRNA expression mediated by vasopressin/PKA signaling, mRNA expression was profiled by RNA-Seq in double knockout cells (both PKA catalytic genes) generated from mouse cortical collecting duct mpkCCD cell line versus control lines with intact PKA expression. Methods: PKA double knockout (dKO) cell lines were generated from mouse cortical collecting duct mpkCCDc11 cells by CRISPR/Cas-9 genome editing method. For mRNA profiling using RNA-Seq analysis, three biological replicates of control (not mutated in PKA two catalytic subunits) cell lines and PKA double knockout cell lines were used. The reads uniquely mapped on GENCODE mouse gene set were analyzed with HOMER (v4.8) and edgeR (v3.10.5). Results and conclusion: About 40-50 million sequence reads per sample were sucessfully mapped in the mouse genome (GENCODE, GPCm38.p5). Among total transcripts of the mouse genome, 10,190 transcripts (cutoff: Counts Per Million > 4 by edgeR) were considered as genes expressed in the cell lines. In differential expression analysis by standard edgeR analysis, 354 transcripts were differentially expressed between control cell lines and PKA dKO cell lines (FDR < 0.05). We also identified nine genes that were markedly decreased in PKA dKO cell lines (log2 PKA dKO/Control < -2, FDR < 0.05) including aquaporin-2 (Aqp2) and two genes that were markedly increased in PKA dKO cell lines (log2 PKA dKO/Control > 2, FDR < 0.05). These results suggest PKA signaling is important for regulation of expression of a very limited number of genes in vasopressin-responsive renal collecting duct cells.

2018-01-02 | GSE95008 | GEO

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2020-05-25 | PXD011245 | Pride

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