Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5

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 (Ser, Ser, Ser, and Thr), of which Ser 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.

Protein–protein interactions in AQP regulation – biophysical characterization of AQP0–CaM and AQP2–LIP5 complex formation

“…9 The affinity observed here from uorescence anisotropy measurements, where we use human LIP5, is thus within error limits the same as that for mouse LIP5. In the earlier study, we used mouse LIP5 that had been uorescently labelled with an amine reactive dye (NT-647-NHS).…”

“…Since AQP2 is shown to be phosphorylated in Pichia pastoris, 9 we dephosphorylated it for our experiments by treating with Alkaline Phosphatase (AP) in 5 mM Tris-HCl, pH 7.9, 10 mM NaCl, 1 mM MgCl 2 and 0.1 mM DTT and incubated at 30 C for 2 hours. The detailed procedure of phosphorylation analysis and dephosphorylation is described in Kinoshita et al 20 with slight modication as mentioned in Ampah-Korsah et al 21 Cloning, expression and purication of human LIP5…”

“…1C), which is involved in targeting AQP2 to multivesicular bodies for subsequent degradation or release as exosomes. 9 The cytoplasmic C-terminal helix is a common structural feature amongst mammalian AQPs, although its relative location in respect to the tetramer differs between human AQP2 and other mammalian AQPs (Fig. 1A).…”

“…9 To our knowledge this is the only study that has investigated AQP proteinprotein interactions using isolated full-length proteins in a quantitative manner. The nding that AQP2 phosphorylation at sites outside the LIP5 binding site affects the interaction highlights the importance of using full-length proteins to fully understand AQP protein-protein interactions and its role in AQP regulation.…”

Thermophoresis for characterizing biomolecular interaction

“…MST and fluorescence correlation spectroscopy (FCS) techniques were used to measure binding constant where the dissociation constants that determined by MST and F K d = 236 nM and K d = 282 nM, respectively. Different applications [43][44][45][46][47][48][49][50] of MST technique in the characterization of protein-protein interactions are summarized in Table 1.…”