Vasopressin/cAMP/protein kinase A (PKA)/aquaporin-2 (AQP2) 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. However, critical mediators in this phosphorylation process remain unknown.
We used several PKA activators as screening tools to generate various phosphorylation patterns of PKA substrates in renal collecting ducts. We extracted the most important PKA substrate (Protein X) whose phosphorylation levels were almost perfectly correlated with those of AQP2. We confirmed the renal physiological role of Protein X by generating Protein X^-/- mice. AQP2 was not phosphorylated even by the administration of exogenous vasopressin in Protein X^-/- mice, resulting in polyuric phenotype.
Protein X unexpectedly had a characteristic of an anchor protein that interacted with PKA (AKAP). We had previously reported that an AKAP–PKA interactions inhibitor, FMP-API-1/27, can directly activate PKA/AQP2 in renal collecting ducts (PMID: 34224008). Notably, not only FMP-API-1/27 but also vasopressin preferentially dissociated the Protein X–PKA interaction rather than other AKAPs–PKA interactions. Therefore, Protein X is a potential novel therapeutic target in the development of anti-aquaretic drugs.