Characterization of the putative phosphorylation sites of the AQP2 C terminus and their role in AQP2 trafficking in LLC-PK1 cells.
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.
Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Julian
J
Arthur
Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Jianmin
J
Huang
Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Naohiro
N
Nomura
Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
William W
WW
Jin
Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Wei
W
Li
Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Xiang
X
Cheng
Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
Dennis
D
Brown
Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts Halu@partners.org.
Hua Jenny
HJ
Lu
D000595:Amino Acid Sequence; D000818:Animals; D051399:Aquaporin 2 / Q000235:genetics / Q000378:metabolism*; D003080:Cold Temperature; D005576:Colforsin / Q000494:pharmacology; D004705:Endocytosis; D018374:LLC-PK1 Cells; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D013552:Swine; D014665:Vasodilator Agents / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
D051399:Aquaporin 2; D014665:Vasodilator Agents; D014667:Vasopressins; D005576:Colforsin
LLC-PK1; aquaporin-2; endocytosis; phosphorylation; vasopressin