The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of a cAMP-dependent protein kinase (PKA) cascade, which ultimately results in the insertion of gastric H,KATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser-66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which ezrin operates in gastric acid secretion. Here we show that phosphorylation of Ser-66 induces a conformational change of ezrin that enables its association with syntaxin 3 (Stx3) and provides a spatial cue for H,K-ATPase trafficking. This conformation-dependent association is specific for Stx3, and the binding interface is mapped to the N-terminal region. Biochemical analyses show that inhibition of ezrin phosphorylation at Ser-66 prevents ezrin-Stx3 association and insertion of H,K-ATPase into the apical plasma membrane of parietal cells. Using atomic force microscopic analyses, our study revealed that phosphorylation of Ser-66 induces unfolding of ezrin molecule to allow Stx3 binding to its N terminus. Given the essential role of Stx3 in polarized secretion, our study presents the first evidence in which phosphorylation-induced conformational rearrangement of the ezrin molecule provides a spatial cue for polarized membrane trafficking in epithelial cells.
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