Characterization of the putative phosphorylation sites of the AQP2 C terminus and their role in AQP2 trafficking in LLC-PK1 cells

Abstract

Vasopressin (VP) stimulates a signaling cascade that results in phosphorylation and apical membrane accumulation of aquaporin-2 (AQP2), leading to water reabsorption by kidney collecting ducts. However, the roles of most C-terminal phosphorylation events in stimulated and constitutive AQP2 recycling are incompletely understood. Here, we generated LLC-PK1 cells containing point mutations of all potential phosphorylation sites in the AQP2 C terminus: S226, S229, T244, S256, S261, S264, and S269, to determine their impact on AQP2 trafficking. We produced an All Null AQP2 construct in which these serine (S) or threonine (T) residues were mutated to alanine (A) or glycine (G), and we then reintroduced the phosphorylation mimic aspartic acid (D) individually to each site in the All Null mutant. As expected, the All Null mutant does not accumulate at the plasma membrane in response to VP but still undergoes constitutive recycling, as shown by its membrane accumulation when endocytosis is blocked by methyl-β-cyclodextrin (MβCD), and accumulation in a perinuclear patch at low temperature (20°C). Single phosphorylation mimics S226D, S229D, T244D, S261D, S264D, and S269D were insufficient to cause membrane accumulation of AQP2 alone or after VP treatment. However, AQP2 S256 reintroduced into the All Null mutant maintains its trafficking response to VP. We conclude that 1) constitutive recycling of AQP2 does not require phosphorylation at any C-terminal sites; 2) forced "phosphorylation" of sites in the AQP2 C terminus is insufficient to stimulate membrane accumulation in the absence of S256 phosphorylation; and 3) phosphorylation of S256 alone is necessary and sufficient to cause membrane accumulation of AQP2.

Keywords: LLC-PK1; aquaporin-2; endocytosis; phosphorylation; vasopressin.

Fig. 1.

Summary of primers used to generate aquaporin-2 (AQP2) C-terminal phosphorylation mutants and sequence of generated AQP2 mutants. A: list of primers used to generate wild-type AQP2 and AQP2 all A/G mutant. B: comparison of the amino acid sequence of AQP2 C terminus between human and rodents. C: list of primer pairs used for site-directed mutagenesis to generate phosphorylation mimics.

Fig. 2.

Expression of AQP2 C-terminal phosphorylation mutants in LLC-PK₁ cells. A: summary of the amino acid sequence of AQP2 C terminus of wild-type and phosphorylation mutants that were generated. B: immunoblotting of lysates from untransfected and AQP2-transfected LLC-PK₁ cells to confirm AQP2 expression.

Fig. 3.

Regulated trafficking and constitutive recycling of AQP2 in LLC-PK₁ cells transfected with AQP2 phosphorylation mutants. Cells grown on coverslips were treated with vehicle (Cont; A), 10 nM vasopressin (VP; B), 10 μM forskolin (FK; C), or 10 mM methyl-β-cyclodextrin (MβCD; D) for 30 min. Immunofluorescence staining of AQP2 was performed using anti-c-myc antibody. Similar experiments were performed at least 3 times. Scale bar = 10 μm.

Fig. 4.

Assessment of endocytosis in LLC-PK₁ cells expressing AQP2 C-terminal mutant by 20°C cold block. A: AQP2 distribution in cells after cold block by immunofluorescence staining. Cells were incubated at 20°C for 30, 60, and 120 min to block AQP2 release from the trans-Golgi network, forming a “perinuclear patch.” Scale bar = 10 μm. B: quantification of AQP2 perinuclear patch formation after 20°C cold block. The accumulation of intracellular AQP2 fluorescence signal in the perinuclear patches over time was measured by quantifying mean patch fluorescence intensity using Velocity software as was described previously (20). Values are means ± SE; n = ≥13 for each data point. Similar experiments were repeated at least 3 times.