Phosphorylation of UT-A1 urea transporter at serines 486 and 499 is important for vasopressin-regulated activity and membrane accumulation

Abstract

The UT-A1 urea transporter plays an important role in the urine concentrating mechanism. Vasopressin (or cAMP) increases urea permeability in perfused terminal inner medullary collecting ducts and increases the abundance of phosphorylated UT-A1, suggesting regulation by phosphorylation. We performed a phosphopeptide analysis that strongly suggested that a PKA consensus site(s) in the central loop region of UT-A1 was/were phosphorylated. Serine 486 was most strongly identified, with other potential sites at serine 499 and threonine 524. Phosphomutation constructs of each residue were made and transiently transfected into LLC-PK1 cells to assay for UT-A1 phosphorylation. The basal level of UT-A1 phosphorylation was unaltered by mutation of these sites. We injected oocytes, assayed [14C]urea flux, and determined that mutation of these sites did not alter basal urea transport activity. Next, we tested the effect of stimulating cAMP production with forskolin. Forskolin increased wild-type UT-A1 and T524A phosphorylation in LLC-PK1 cells and increased urea flux in oocytes. In contrast, the S486A and S499A mutants demonstrated loss of forskolin-stimulated UT-A1 phosphorylation and reduced urea flux. In LLC-PK1 cells, we assessed biotinylated UT-A1. Wild-type UT-A1, S486A, and S499A accumulated in the membrane in response to forskolin. However, in the S486A/S499A double mutant, forskolin-stimulated UT-A1 membrane accumulation and urea flux were totally blocked. We conclude that the phosphorylation of UT-A1 on both serines 486 and 499 is important for activity and that this phosphorylation may be involved in UT-A1 membrane accumulation.

Fig. 1.

Tandem mass spectrometry (MS/MS) detects three protein kinase A (PKA) consensus sites in forskolin-stimulated UT-A1. UT-A1 was isolated from rat inner medulla treated with forskolin (10 μM) (methods). Potential vasopressin (AVP)-stimulated phosphorylation sites between residues 482 and 558 (highlighted in gray) were revealed. PKA consensus sites: S486, S499, and T524 (indicated by *) in the intracellular loop of UT-A1.

Fig. 2.

S486 and S499 are targets for PKA phosphorylation. Shown are a representative autoradiogram (A) and a Western blot (B) of [³²P]orthophosphate-labeled wild-type UT-A1 or UT-A1 phosphomutants immunoprecipitated from transiently transfected LLC-PK₁ cells. Cells were treated with 10 μM forskolin (+) or vehicle (−), and untransfected LLC-PK₁ cells were used as a negative control (ctrl). Densitometry was performed on Western blots and film with Licor and Image J, respectively, and phosphate-to-protein ratios were determined (C). Graph represents n = 6 independent transfection experiments. Data are means ± SE. *P < 0.05.

Fig. 3.

Phosphorylation of both S486 and S499 is required for UT-A1 trafficking to the membrane. LLC-PK₁ cells that were transiently transfected with UT-A1 and mutant constructs were treated with either vehicle control (−) or 10 μM forskolin (+) for 30 min before biotinylation was performed. Shown is a representative Western blot of biotinylated samples (A) and whole cell lysates (B). Densitometry was performed with Licor to determine the biotin-to-protein ratio (C). Graph represents n = 4 independent transfection experiments. Data are means ± SE. *P < 0.05.

Fig. 4.

PKA phosphorylation at sites S486 and S499 is needed for urea transport activity in Xenopus oocytes. Oocytes were injected with indicated cRNA or a mixture of cRNAs as described in methods. A: before urea flux was measured, oocytes were incubated in the absence (open bars) or presence (filled bars) of 500 μM 8-(4-chlorophenylthio)-adenosine-3′,5′-cyclic monophosphate (CPT-cAMP), 500 μM 3-isobutyl-1-methylxanthine (IBMX), and 50 μM forskolin for 2 h. Each bar shows the mean ± SE obtained from 6 individual oocytes from 3 different experiments. *P < 0.05. B: representative Western blot analysis shows equal expression of wild-type UT-A1 and each of the UT-A1 mutant proteins generated by oocytes.