Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation
ABSTRACT Gastric ezrin was initially identified as a phosphoprotein associated with parietal cell activation. To explore the nature of ezrin phosphorylation, proteins from resting and secreting gastric glands were subjected to two-dimensional SDS-PAGE. Histamine triggers acid secretion and a series of acidic isoforms of ezrin on two-dimensional SDS-PAGE. Mass spectrometric analysis of these acidic ezrin spots induced by stimulation suggests that Ser66 is phosphorylated. To determine whether Ser66 is a substrate of protein kinase A (PKA), recombinant proteins of ezrin, both wild type and S66A mutant, were incubated with the catalytic subunit of PKA and [32P]ATP. Incorporation of 32P into wild type but not the mutant ezrin verified that Ser66 is a substrate of PKA. In addition, expression of S66A mutant ezrin in cultured parietal cells attenuates the dilation of apical vacuolar membrane associated with stimulation by histamine, indicating that PKA-mediated phosphorylation of ezrin is necessary for acid secretion. In fact, expression of phosphorylation-like S66D mutant in parietal cells mimics histamine-stimulated apical vacuole remodeling. Further examination of H,K-ATPase distribution revealed a blockade of stimulation-induced proton pump mobilization in S66A but not S66D ezrin-expressing parietal cells. These data suggest that PKA-mediated phosphorylation of ezrin plays an important role in mediating the remodeling of the apical membrane cytoskeleton associated with acid secretion in parietal cells.
Cancer Research 12/2004; 64(24):8994-9001. DOI:10.1158/0008-5472.CAN-04-2052 · 9.28 Impact Factor
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ABSTRACT: 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,K-ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser66 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 Ser66 induces a conformational change of ezrin which 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 binding interface is mapped to the N-terminal region. Biochemical analyses show that inhibition of ezrin phosphorylation at Ser66 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 Ser66 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 ezrin molecule for provides a spatial cue for polarized membrane trafficking in epithelial cells.Journal of Biological Chemistry 10/2014; DOI:10.1074/jbc.M114.581280 · 4.60 Impact Factor
CellBio 01/2013; 02(04):179-185. DOI:10.4236/cellbio.2013.24020