FIGURE 3.

Identification of potential Akt phosphorylation sites in NHE3. A, amino acids defining the F1–4 domains of the rabbit NHE3 C terminus. B, purified fusion proteins were phosphorylated by active Akt-Ser⁴⁷³ in vitro and then detected by a Pro-Q® Diamond phosphoprotein gel stain kit. The Akt phosphorylation site is located in the NHE3-F1 domain (aa 475–589), but not in F2 or F3. C, densitometry for F1,2,3 phosphorylation with a Pro-Q diamond phosphorylation stain kit. The results of densitometry were normalized to amount of fusion protein with F1 fusion protein set to 100 for each experiment. The results are means ± S.E. of three identical experiments. D, the illustration presents a cluster of three serines (Ser⁵¹⁵, Ser⁵²², and Ser⁵²⁶) located in the same putative NHE3-F1 domain α-helix (aa 509–527) as the previously demonstrated direct ezrin-binding domain (Lys⁵¹⁶, Arg⁵²⁰, and Arg⁵²⁷) (12). E, illustrates the presence in the same NHE3-F1 domain of an established phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3,4,5-trisphosphate-binding domain (20) and of a putative PDK1-binding site, as well as an Akt consensus phosphorylation sequence. F, sequence alignment of NHE3 putative α-helix region showing amino acids conserved across species, including the serine cluster. G, Akt phosphorylation of NHE3-F1 fusion protein is abolished with NHE3-S515A mutation, significantly reduced with S526A and not significantly altered with S522A mutation. A single experiment is shown. H, densitometric analyses as in G for NHE3-F1 wild type, -S515A, -S522A, and -S526A were normalized to Coomassie Blue signal with NHE3 wild type F1 set to 100% for each experiment. The results are means ± S.E. of at least five separate studies. p values are in comparison to NHE3 wild type F1.