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E. R. Buck, R. K. Crouch, K. L. Schey; Mutation of N- and C- Terminal Phosphorylation Sites of Aquaporin-0 Affects Plasma Membrane Trafficking. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2097.
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© ARVO (1962-2015); The Authors (2016-present)
AQP0 is the most abundant membrane protein in the lens and is responsible for the regulation of lens fiber cell water permeability. The absence of AQP0 in an animal model and trafficking defects caused by AQP0 mutations in humans and mice lead to cataract formation. Furthermore, because AQP0 is located within fiber cells of the ocular lens, it does not undergo protein turnover and therefore is regulated and modified through post-translational modification, including phosphorylation, as fiber cells age. The purpose of this study is to investigate the effect of N- and C- terminal phosphorylation on plasma membrane trafficking.
HEK-293 cells were grown in 12-well plates, pre-treated with poly-D-lysine and grown to 80% confluency. Cells were transfected with cDNA for WT-AQP0 or mutant-AQPO and expressed for 48 hours. Cells were then harvested, lysed, and proteins analyzed by Western blotting. For confocal studies, cells were grown on glass cover slips pre-treated with poly-D-lysine and then transfected in the same fashion as for Western blotting. Cells then underwent standard immunofluorescence fixing protocols. Site-directed mutagenesis was used to generate alanine and aspartic acid mutations of serine residues in N- and C- terminal regions of AQP0.
Mutation of S229 and S235 to aspartic acid (S229D, S235D) caused an increase in plasma membrane expression of AQP0 compared to wild-type AQP0, while mutation of these sites to alanine (S229A, S235A) inhibited trafficking. This effect was not seen when S231 was mutated to either residue. A newly characterized site in the N-terminal, S6, also appeared to impair trafficking when mutated to S6A. MUT-AQP0 and WT-AQP0 expressed at similar levels from Western blot data.
These results suggest that two phosphorylation sites in the C-terminal region of AQP0 (S229 and S235) as well as a site in the N-terminal region (S6) play a role in proper AQP0 trafficking to the plasma membrane.
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