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First published online 6 September 2005
doi: 10.1242/jcs.02559

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Phosphorylation of ezrin on threonine 567 produces a change in secretory phenotype and repolarizes the gastric parietal cell

¹ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
² School of Life Science, University of Science and Technology of China, Hefei 230027, Peoples Republic of China

View larger version (26K): [in a new window]   Fig. 1. Western blots of parietal cell cultures probed for ezrin, CFP-ezrin construct expression, and for H,K-ATPase. Conditions for cell culture included control cells (C, no treatment), control virus infected cells (CV), and cells infected with rAD including CFP-constructs as wild-type ezrin (WT), T567A mutant ezrin (TA), and T567D mutant ezrin (TD). (A) Blot probed with an antibody against ezrin that recognizes 80 kDa native ezrin, the CFP-constructs of ezrin (106 kDa) and an N-terminal 55 kDa breakdown product of ezrin. (B) Blot probed with antibody against GFP which also recognizes CFP. For blots A and B, protein bands are marked by arrowheads indicating lanes for ezrin (ezrin), the CFP-tagged ezrin constructs (CFP-ez), the approximately 55 kDa ezrin breakdown product (55 kD), and CFP alone (CFP) in the cells infected with the control rAd construct. (C) Blot probed with antibody against the ß-subunit of H,K-ATPase (HKß) to show the approximate equivalency of parietal cells in the assay. The antibody against the ß-subunit of H,K-ATPase ordinarily migrates as a very broad band, 60-80 kDa, because of the high degree of glycosylation.

View larger version (138K): [in a new window]   Fig. 2. Expression of CFP-ezrin constructs in live parietal. (A) Examples of cells infected with rAD-CFP-ezrin WT. (B) Cells infected with rAD-CFP-ezrin T567A. (C) Cells infected with rAD-CFP-ezrin T567D. For all cases images are shown for parietal cells maintained in the resting, nonsecreting, state (rest) and 20-30 minutes after cells had been stimulated to secrete acid (stim) by histamine plus IBMX. For WT and the T567A mutant, CFP-ezrin was mainly targeted to relatively large vacuoles that are derived from the apical plasma membrane when the cells are cultured, thus we refer to them as apical membrane vacuoles. There are lesser amounts of CFP-ezrin WT and T567A displayed on the basolateral membrane (surrounding plasma membrane) and in the cytoplasm. Cells expressing both CFP-ezrin WT and T567A respond well to stimulation with a large expansion of apical membrane vacuoles that fill with secreted acid. Note: the rest/stim images at the lower left are the same cells expressing CFP-ezrin WT at rest and after 20 minutes stimulation. The targeting of CFP-ezrin T567D mutant was totally different. The CFP signal was detected primarily at the basolateral membrane, often in the form of elaborate surface projections, and the cells did not display the normal secretory response to stimulation. Bars, 10 µm.

View larger version (110K): [in a new window]   Fig. 3. Parietal cell cultures double stained for expression of H,K-ATPase (HK) and ezrin (or CFP-ezrin). Resting cells infected with control virus displayed native ezrin primarily on apical membrane vacuoles and basolateral membrane to some extent, and HK staining was distributed throughout the cytoplasm, presumably on tubulovesicles. After stimulation ezrin and HK were localized to some extent on expanded apical secretory vacuoles. Cells infected with rAD-CFP-ezrin WT, CFP-ezrin T567A mutant and CFP-ezrin T567D mutant were stained for HK and CFP (using a GFP antibody). In resting cells expressing CFP-ezrin WT and T567A mutant where it colocalized with a subset of CFP staining. This was highly unusual because the resting cells displayed none of the morphological characteristics of the secretory phenotype (which promptly occurred after the cells were stimulated with histamine plus IBMX). The targeting of CFP-ezrin T567D mutant was similar to that described in Fig. 2, where the CFP signal was detected primarily at the basolateral membrane. Not only was the normal secretory response absent in the T567D-expressing cells, the HK was completely `repolaraized' to the basolateral membrane, there colocalizing with CFP-ezrin T567D.

View larger version (120K): [in a new window]   Fig. 4. Time course of stimulatory response for cells infected with rAD-CFP-ezrin T5687D mutant. Cells were placed in an incubating chamber (37°C) and viewed by spinning disc confocal microcopy using the CFP channel. Histamine plus IBMX was added at zero time and the cells were periodically examined with images being selected at the times (in minutes) shown. For the two parietal cells expressing CFP-ezrin T567D in the main panel the CFP signal was at the basolateral surface in the form of long surface projections; there was no significant morphological response to stimulation. The rectangular inset at the lower left has digitally amplified an extremely faint image (virtually autofluorescence) of that very region and the signal to show that `hidden' parietal cells on the same slide, but not expressing T567D, respond normally to stimulation. Bar, 20 µm for all images.

View larger version (17K): [in a new window]   Fig. 5. Acid secretory response of parietal cells infected with control rAd and with CFP-ezrin constructs expressing WT and the T567A and T567D mutants. The aminopyrine (AP) uptake was measured for the various cultures of parietal cells in the presence of the presence of the H2-receptor blocker cimetidine (Cim), maximally stimulated by histamine plus IBMX (His+IBMX), and stimulated in the presence of the H,K-ATPase blocking drug SCH-28080 (His+IBMX+SCH). The data were collected from assays on six separate culture preparations, and normalized within each experimental run by setting the AP uptake of His+IBMX for control virus at 100% and shown as mean±s.e.m. All preparations showed highly significant AP uptakes in response to stimulation by His+IBMX. Statistical analysis (t-test) of the maximally stimulated AP uptake revealed that P>0.05 between control virus and ezrin-WT; P=0.05 between ezrin-WT and T567A; and P=0.004 between ezrin-WT and T567D.

View larger version (92K): [in a new window]   Fig. 6. Confocal microscopy of parietal cell cultures double stained for expression of H,K-ATPase (HK, red) and CFP-ezrin T567D mutant (green). Resting cells displayed T567D ezrin primarily on the surface basolateral membrane often appearing as extensive surface projections. HK staining was distributed (relocated) to the basolateral membrane colocalized as a subset of T567D staining. After treating the cultures with histamine plus IBMX, parietal cells prominently expressing T567D ezrin did not respond to the stimulation, but neighboring cells expressing little or no T567D ezrin assumed the typical secretory phenotype. Bars, 10 µm.

View larger version (129K): [in a new window]   Fig. 7. Scanning electron microscopy of parietal cell cultures in control conditions and those infected with rAd-CFP-ezrin constructs expressing WT and the T567A and T567D mutants. The cells were cultured on silicon wafers, exposed to virus (or not) for 48 hours, then fixed, dried, coated with carbon and examined in a Hitachi scanning microscope as described in Materials and Methods. For most of the cells/treatments the plasma membrane (basolateral membrane) is studded with short projections and folds, with occasional lamellipodia and longer microvillar projections. In the case of T567D the basolateral membrane was often found to be replete with extensive long microvillar projections. Bars, 5 µm.