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.
Highlights
EXPERIMENTAL PROCEDURESReagents—[14C]Aminopyrine and [32P]ATP were obtained from PerkinElmer Life Sciences. Monoclonal antibody (JL-18) against rGFP was purchased from Clontech (Palo Alto, CA), whereas ezrin antibody 4A5 was produced and described by Hanzel et al [28]
Using fluorescence resonance energy transfer monitored by lar membrane associated with stimulation by histamine, fluorescence lifetime imaging microscopy and chemotaxis asindicating that PKA-mediated phosphorylation of ezrin is says [8], it has been shown that protein kinase C-mediated necessary for acid secretion
To test if any of these acidic spots are related to phosphoserine induced by histamine stimulation, we carried out Western blotting using two-dimensional SDS-PAGE of separated ezrin spots
Summary
Reagents—[14C]Aminopyrine and [32P]ATP were obtained from PerkinElmer Life Sciences. Monoclonal antibody (JL-18) against rGFP was purchased from Clontech (Palo Alto, CA), whereas ezrin antibody 4A5 was produced and described by Hanzel et al [28]. To probe for the nature of ezrin phosphorylation, aliquots of gastric glands were treated with cimetidine and histamine plus IBMX for different time intervals and harvested for two-dimensional SDS-PAGE. To analyze the relative level of ezrin associated with cytoskeleton, transfected cells were raised with PHEM buffer (100 mM Pipes, 20 mM Hepes, pH 6.9, 5 mM EGTA, 2 mM MgCl2, and 4 M glycerol) twice followed by incubation of PHEM buffer containing a proteinase inhibitor mixture (pepstatin-A, leupeptin, aprotinin, and chymostatin; final concentration 5 g/ml for each inhibitor) plus 0.1% Triton X-100 for 1 min at room temperature to allow cytosolic proteins to be released into extracellular medium [33]. The blot was stripped with SDS-PAGE sample buffer at 55 °C for 20 min followed by validation of serine phosphorylation on GFP-ezrin using a phosphoserine antibody. The band intensity was quantified using a PhosphorImager (Amersham Biosciences)
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