L. stimulation activates PKA, which phosphorylates MST4 at Thr-178 and then promotes MST4 kinase activity. Interestingly, activated MST4 then phosphorylates ezrin prephosphorylated by PKA. Importantly, MST4 is important for acid secretion in parietal cells because either suppression of MST4 or overexpression of non-phosphorylatable MST4 prevents the apical membrane reorganization and proton pump translocation elicited by histamine stimulation. In addition, overexpressing MST4 phosphorylation-deficient ezrin results in an inhibition of gastric acid secretion. Taken together, these results define a novel molecular mechanism linking the PKA-MST4-ezrin signaling cascade to polarized epithelial secretion in gastric parietal cells. (5) and preferentially bound to the -actin isoform (6). It has been postulated that ezrin couples the activation of protein kinase A (PKA) to the apical membrane remodeling associated with parietal cell secretion (3, 7). In fact, we have mapped the PKA phosphorylation site on ezrin and demonstrated its functional importance in histamine-elicited gastric acid secretion (3). Using mouse genetics, Tamura (8) have demonstrated that knocking down ezrin in stomachs to <5% of the wild-type levels results in severe achlorhydria. In these parietal cells, H,K-ATPase-containing tubulovesicles failed to fuse with the apical membrane, suggesting an essential role of ezrin in tubulovesicle docking. A recent study has shown that the levels of ezrin phosphorylation on Thr-567 are low in resting parietal cells and that histamine stimulation results MT-4 in a slight Rabbit polyclonal to AARSD1 increase of ezrin phosphorylation at Thr-567 (9). However, it was unclear how ezrin phosphorylation of Thr-567 is orchestrated and whether it links to remodeling of the apical membrane and cytoskeleton during parietal cell activation. Our studies demonstrate the functional significance of the vesicle trafficking machinery Stx3 (10), VAMP2 (11), and SNAP25 (12) in parietal cell secretion. Using atomic force microscopic analyses, we show that phosphorylation of Ser-66 unfolds MT-4 the three compact lobes of the FERM (protein 4.1, ezrin, radixin, moesin) domain and that this conformational change enables association of Stx3 with ezrin (13). Our study provides novel insights into the spatial control of H,K-ATPase docking by phosphorylation-coupled ezrin-Stx3 interaction in parietal cells. Mammalian MST4 kinase is a conserved element of the STE20 signaling cascade underlying cell polarity control (14). A recent study has shown that MST4 phosphorylates ezrin at Thr-567 at the apical membrane of intestinal cells, which induces brush borders (15). Here we show that MST4 is downstream MT-4 from MT-4 histamine-stimulated PKA activation and that activation of MST4 is important for parietal cell acid secretion by phosphorylation of Ser-66-phosphorylated ezrin. Therefore, our study provides novel insights into the PKA-MST4-ezrin signaling axis in polarized secretion in epithelial cells. Materials and Methods Isolation MT-4 of Gastric Glands and Aminopyrine Uptake Assay Gastric glands were isolated from New Zealand White rabbits as modified by Yao (5). Briefly, the rabbit stomach was perfused under high pressure with PBS (2.25 mm K2HPO4, 6 mm Na2HPO4, 1.75 mm NaH2PO4, and 136 mm NaCl (pH7.4)) containing 1 mm CaCl2 and 1 mm MgSO4. The gastric mucosa was scraped from the smooth muscle layer, minced, and then washed twice with minimal essential medium buffered with 20 mm HEPES (pH7.4) (HEPES-minimal essential medium). The minced mucosa was then digested with 15 mg of collagenase (Sigma). Intact gastric glands were collected from the digestion mixture for 20C25 min and then washed three times in HEPES-minimal essential medium. In all subsequent gland experiments (AP8 uptake assay), glands were resuspended at 5% cytocrit (v/v) in the appropriate buffer containing histamine receptor 2 blockers (cimetidine or famotidine, 5 m) for the final assay. Stimulation of intact and Streptolysin O (SLO)-permeabilized rabbit gastric glands was quantified using the AP uptake assay as described by Ammar (16). Briefly, intact glands in HEPES-minimal essential medium were washed twice by settling at 4 C in ice-cold K buffer (10 mm Tris base, 20 mm HEPES acid, 100 mm KCl, 20 NaCl, 1.2 mm MgSO4, 1 mm NaH2PO4, and 40 mm mannitol (pH7.4)). SLO was added to a.