Downregulation of urea transporter UT-A1 activity by 14-3-3 protein

Abstract

Urea transporter (UT)-A1 in the kidney inner medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the kidney inner medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione- S-transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.

TSS-Seq analysis of low pH-induced gene expression in intercalated cells in the renal collecting duct

“…Josd1 is a deubiquitinating enzyme that is localized to the plasma membrane [ 38 ]. Mdm2 is a ubiquitin ligase that can interact with 14-3-3γ and degrade UT-A1 [ 39 ]. Siah1 degrades homeo-domain interacting protein kinase 2 (HIPK2), a key molecule in the induction of kidney fibrosis and the epithelial-to-mesenchymal transition in various nephropathies such as human immunodeficiency virus (HIV)-associated kidney disease [ 40 ].…”

USP7 (Ubiquitin-Specific Protease 7)

“…Vasopressin stimulates UT-A1 phosphorylation at both S486 and S499 with a similar time course (Hoban et al 2015). 14-3-3g binds to UT-A1, and this binding is stimulated by PKA (Feng et al 2015). 14-3-3g binding decreases urea transport and increases UT-A1 ubiquitination and degradation by interacting with MDM2, an E3 ubiquitin ligase, MDM2 (Feng et al 2015).…”

Urea transport and clinical potential of urearetics

“…14-3-3γ was shown to bind to UT-A1, and activation of PKA enhanced this binding (18). 14-3-3γ decreased urea transport and increased UT-A1 ubiquitination and degradation by interacting with the E3 ubiquitin ligase, MDM2 (18). Thus, PKA activation increases UT-A1 phosphorylation, and the subsequent binding of 14-3-3γ leads to UT-A1 degradation.…”

“…Thus, PKA activation increases UT-A1 phosphorylation, and the subsequent binding of 14-3-3γ leads to UT-A1 degradation. If these two opposing effects are separated temporally, they could provide a negative feedback mechanism to return UT-A1 function to its basal state following stimulation by vasopressin (18) (Figure 3). The data showing these opposite effects of PKA are established, but whether there is a negative feedback loop, or whether only one effect is dominant in vivo, remains to be determined.…”