Vasopressin-Dependent β-Catenin Phosphorylation at Ser552 and Branching Structure of Mouse Collecting Duct System

BackgroundPhosphoproteomics studies in both cultured and native collecting duct (CD) cells showed that vasopressin strongly increases PKA-dependent phosphorylation of {beta}-catenin at Ser552. Relatively little is known about the role of Ser552 phosphorylation. MethodsTo address the role of {beta}-catenin Ser552 phosphorylation in the mature renal CD, we have inserted a Ser552Ala mutation in mice using CRISPR-Cas9. ResultsThe mutation did not affect the renal abundance of the vasopressin-regulated water channel aquaporin-2 (AQP2) or urinary osmolality. However, the structure of the CD system was altered. Specifically, the cortical branching ratio (the number of nephrons that merge to form one cortical CD) was reduced from 6.18 {+/-} 0.66 in control mice to 3.33 {+/-} 0.82 in Ser552Ala mutant mice. This was associated with a greater number of cortical and medullary CDs with smaller average diameter. The total number of nephrons (glomerular counts) was not different between control and Ser552Ala mutant mice (both [~]13,500 per kidney). RNA-seq in microdissected cortical CDs of the mice revealed a highly significant enrichment of genes involved in regulation of mitosis and the cell cycle, along with decreases in mRNAs coding for two CDK inhibitor proteins, Cdkn1b and Cdkn1c. At the same time, there were no changes in abundances of major transporter mRNAs, indicative of sustained CD differentiation. A subset of cortical CD cells showed an increase in DNA content, consistent with G2/M cell-cycle arrest. ConclusionsThe observed structural changes in the collecting duct system of adult mice point to a role of vasopressin-mediated post-translational modification of {beta}-catenin at Ser552 in collecting duct development, presumably PKA-mediated Ser552 phosphorylation. We speculate that vasopressin may act to slow or halt branching morphogenesis perinatally and may affect the collecting duct elongation process that normally produces the unbranched region of the CD system in the cortex and outer medulla. Key pointsO_LIVasopressin regulates collecting duct (CD) transport by triggering phosphorylation of multiple proteins including {beta}-catenin at Ser552. C_LIO_LIMutating Ser552 to a non-phosphorylable amino acid in mice resulted in altered CD branching without loss of differentiation in adult CDs. C_LIO_LIThe findings point to a role for {beta}-catenin Ser552 phosphorylation in CD branching and sub-segmental CD elongation. C_LI