Identification of protein kinase A signalling molecules in renal collecting ducts.

Body water homeostasis is maintained by the correct balance between water intake and water loss through urine, faeces, sweat and breath. It is known that elevated circulating levels of the antidiuretic hormone vasopressin decrease urine volume to prevent excessive water loss from the body. Vasopressin/cyclic adenosine monophosphate/protein kinase A (PKA) signalling is the canonical pathway in renal collecting ducts for phosphorylating aquaporin-2 (AQP2) water channels, which further leads to the reabsorption of water from urine via AQP2. Although recent omics data have verified various downstream targets of PKA, critical regulators that mediate PKA-induced AQP2 phosphorylation remain unknown mainly because of the fact that vasopressin is usually used to activate PKA as a positive control. Vasopressin is extremely potent and non-specifically phosphorylates various PKA substrates, making the narrowing down of candidate mediators responsible for AQP2 phosphorylation difficult. The intracellular localisation of PKA is tightly regulated by its scaffold proteins, also known as A-kinase anchoring proteins (AKAPs). Further, each AKAP has a target domain that determines its intracellular localisation, enabling to create a local PKA signalling network. Although vasopressin activates most PKAs independently of their intracellular localisation, some chemical compounds preferentially act on PKAs localised on AQP2-containing vesicles while simultaneously phosphorylating AQP2 and its surrounding PKA substrates. Immunoprecipitation with antibodies against phosphorylated PKA substrates followed by mass spectrometry analysis revealed that the PKA substrate in proximity to AQP2 was lipopolysaccharide-responsive and beige-like anchor (LRBA). Further, Lrba knock-out studies revealed that LRBA was required for vasopressin-induced AQP2 phosphorylation. Abstract figure legend Each AKAP separates PKA into specific compartments, such as mitochondria, nucleus or intracellular vesicles, facilitating spatially restricted cAMP/PKA signalling to increase the specificity of the PKA substrate. Although vasopressin activates various PKA signalling pathways, some compounds specifically activate PKA at vesicles. The combined use of compounds and pPKA substrate antibody helps to extract PKA signalling molecules whose phosphorylation levels correlate well with AQP2. This article is protected by copyright. All rights reserved.