Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation.
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.
Zhou
Rihong
R
Laboratory of Cell Dynamics, School of Life Science, University of Science and Technology of China, Hefei 230027, China.
Cao
Xinwang
X
Watson
Charles
C
Miao
Yong
Y
Guo
Zhen
Z
Forte
John G
JG
Yao
Xuebiao
X
D000255:Adenosine Triphosphate / Q000378:metabolism; D000632:Aminopyrine / Q000493:pharmacokinetics; D000818:Animals; D001692:Biological Transport; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D006636:Histamine Release; D010295:Parietal Cells, Gastric / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D000099149:Ezrin
D000255:Adenosine Triphosphate; D000632:Aminopyrine; D017868:Cyclic AMP-Dependent Protein Kinases; D003598:Cytoskeletal Proteins; D010750:Phosphoproteins; D010768:Phosphoserine; D011993:Recombinant Fusion Proteins; D012694:Serine; D000099149:Ezrin