PKA-mediated protein phosphorylation regulates ezrin-WWOX interaction.
The ezrin-radixin-moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton, and also participate in signal-transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the cAMP-dependent protein kinase activation cascade to the regulated HCl secretion in gastric parietal cells. Our recent studies have mapped the PKA-mediated phosphorylation site to Ser(66) and established its functional role in parietal cell activation [R. Zhou et al., Characterization of protein kinase A-mediated phosphorylation of ezrin in gastric parietal cell activation, J. Biol. Chem. 278 (2003) 35651-35659], but the underlying basis for this regulation is not known. Here, we provide the first evidence that PKA-mediated phosphorylation of Ser(66)regulates the interaction of ezrin with WWOX, a WW domain-containing protein. Our biochemical study reveals that ezrin directly binds to the first WW domain of WWOX via its C-terminal tyrosine-containing polyproline sequence (470)PPPPPPVY(477). Mutational analyses further demonstrate that tyrosine(477) is essential for the ezrin-WWOX interaction. In addition, our study shows that PKA-mediated phosphorylation of ezrin is essential and sufficient for the apical localization of WWOX protein as disruption of ezrin-WWOX interaction eliminated the apical localization of WWOX. Finally, our study demonstrates the essential role of ezrin-WWOX interaction in the apical membrane remodeling associated with H,K-ATPase recruitment. Taken together, these results define a novel molecular mechanism underlying phospho-regulation of ezrin function by PKA in parietal cell activation.
Jin
Changjiang
C
Laboratory of Cellular Dynamics, University of Science and Technology of China, Heifei National Laboratory for Physical Sciences at the Microscale, Hefei 230027, China.
Ge
Ling
L
Ding
Xia
X
Chen
Yong
Y
Zhu
Hongling
H
Ward
Tarsha
T
Wu
Fang
F
Cao
Xinwang
X
Wang
Qichen
Q
Yao
Xuebiao
X
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D025521:Tumor Suppressor Proteins; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D000074584:WW Domain-Containing Oxidoreductase; D000099149:Ezrin
D017868:Cyclic AMP-Dependent Protein Kinases; D003598:Cytoskeletal Proteins; D010088:Oxidoreductases; D025521:Tumor Suppressor Proteins; D014443:Tyrosine; D000074584:WW Domain-Containing Oxidoreductase; D000099149:Ezrin; C486071:WWOX protein, human