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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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			Action	DRS
	MSB56659Chym012722.mgf	Mgf
	MSB56659Chym012722.raw	Raw
	MSB56659Chym012722.raw_20220201_Byonic.zip	Raw
	MSB56659Elas012722.mgf	Mgf
	MSB56659Elas012722.raw	Raw

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cAMP/PKA signaling phosphorylates LRBA in renal collecting duct

Project description:Vasopressin/cAMP/protein kinase A (PKA)/aquaporin-2 (AQP2) water channels signaling in the kidneys is a canonical pathway that determines AQP2 activity in response to body fluid balance. AQP2 phosphorylation by PKA increases water reabsorption from urine for the prevention of further water loss. We used several activators of cAMP/PKA signaling as screening tools to generate various phosphorylation patterns of PKA substrates and AQP2 in a mouse cortical collecting duct principal cell line (mpkCCDcl4). We next quantified phosphorylation levels of PKA substrates and AQP2 after western blot analysis using a phospho-PKA substrates antibody. Finally, we screen for PKA substrates whose phosphorylation levels were well correlated with those of AQP2. Immunoprecipitation with a phospho-PKA substrates antibody followed by mass spectrometry analysis revealed that the leading candidate in this assay proved to be a lipopolysaccharide-responsive and beige-like anchor protein (LRBA). Phosphorylation levels of LRBA was nearly perfectly correlated with those of AQP2.

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