Abstract
The interaction between the renal water channel aquaporin-2 (AQP2) and the lysosomal trafficking regulator-interacting protein LIP5 targets AQP2 to multivesicular bodies and facilitates lysosomal degradation. This interaction is part of a process that controls AQP2 apical membrane abundance in a vasopressin-dependent manner, allowing for urine volume adjustment. Vasopressin regulates phosphorylation at four sites within the AQP2 C terminus (Ser256, Ser261, Ser264, and Thr269), of which Ser256 is crucial and sufficient for AQP2 translocation from storage vesicles to the apical membrane. However, whether AQP2 phosphorylation modulates AQP2-LIP5 complex affinity is unknown. Here we used far-Western blot analysis and microscale thermophoresis to show that the AQP2 binds LIP5 in a phosphorylation-dependent manner. We constructed five phospho-mimicking mutants (S256E, S261E, S264E, T269E, and S256E/T269E) and a C-terminal truncation mutant (ΔP242) that lacked all phosphorylation sites but retained a previously suggested LIP5-binding site. CD spectroscopy indicated that wild-type AQP2 and the phospho-mimicking mutants had similar overall structure but displayed differences in melting temperatures possibly arising from C-terminal conformational changes. Non-phosphorylated AQP2 bound LIP5 with the highest affinity, whereas AQP2-ΔP242 had 20-fold lower affinity as determined by microscale thermophoresis. AQP2-S256E, S261E, T269E, and S256E/T269E all had reduced affinity. This effect was most prominent for AQP2-S256E, which fits well with its role in apical membrane targeting. AQP2-S264E had affinity similar to non-phosphorylated AQP2, possibly indicating a role in exosome excretion. Our data suggest that AQP2 phosphorylation allosterically controls its interaction with LIP5, illustrating how altered affinities to interacting proteins form the basis for regulation of AQP2 trafficking by post-translational modifications.
Highlights
The interaction between the renal water channel aquaporin-2 (AQP2) and the lysosomal trafficking regulator-interacting protein LIP5 targets AQP2 to multivesicular bodies and facilitates lysosomal degradation
Wild-type human AQP2, AQP2 phospho-mimicking mutants (S256E, S261E, S264E, and T269E), and AQP2 truncated after Pro242 (AQP2-⌬P242) were cloned into yeast expression vectors, overproduced in the yeast P. pastoris, and purified
Because P. pastoris is known to be able to phosphorylate recombinant proteins, including aquaporins [28], we decided to investigate whether this holds true for human AQP2
Summary
The interaction between the renal water channel aquaporin-2 (AQP2) and the lysosomal trafficking regulator-interacting protein LIP5 targets AQP2 to multivesicular bodies and facilitates lysosomal degradation. Phosphorylation of Thr269 is able to override the ubiquitin-internalization signal [8], and phosphorylation of Ser261 has been shown to increase ubiquitinated AQP2 levels [13] This suggests that there is an intricate cross-talk between the post-translational modifications that determine AQP2 apical membrane abundance. We further show that the distal part of the C terminus that contains the phosphorylation sites was not critical for the AQP2-LIP5 interaction, removal of this region resulted in 20-fold lower affinity This suggests that the distal AQP2 C terminus and its phosphorylation status plays an important role in modulating the affinity to LIP5, thereby fine-tuning its sorting into MVBs
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