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1 Molecular & Cell Biology, Univ. of California, Berkeley, California, United States
2 Dept. of Molecular and Cell Biology, University of California
* To whom correspondence should be addressed. E-mail: jforte{at}berkeley.edu.
In its dormant state the membrane cytoskeletal linker protein ezrin takes on N-terminal to C-terminal (N-C) binding conformation. In vitro evidence suggests that eliminating the N-C binding conformation by Thr567 phosphorylation leads to ezrin activation. Here we found for resting gastric parietal cells that the levels of ezrin phosphorylation on Thr567 are low, and can be increased to a small extent (~40%) by stimulating secretion via the cAMP pathway. Treatment of the cells with protein phosphatase inhibitors led to a rapid, dramatic, increase in Thr567 phosphorylation by 400% over resting levels, prompting an hypothesis that ezrin activity is regulated by turnover of phosphorylation on Thr567. In vitro and in vivo FRET (fluorescence resonance energy transfer) analysis demonstrated that Thr567 phosphorylation opens N-C interaction. However, even in the closed conformation, ezrin localizes to membranes by an exposed N-terminal binding site. Importantly, the opened phosphorylated form of ezrin more readily co-sediments with F-actin and binds more tightly to membrane than the closed forms. Furthermore, FRAP analysis in live cells showed that the Thr567Asp mutant has longer recovery times than wild-type or the Thr567Ala mutant, indicating the Thr567 phospho-form of ezrin is tightly associated with F-actin and membrane, restricting normal activity. These data demonstrate and emphasize the functional importance of reversible phosphorylation of ezrin on F-actin binding. A novel model is proposed whereby ezrin and closely associated kinase and phosphatase proteins represent a within a complex of proteins to establish a dynamic relationship between the varying membrane surface area and filamentous actin length.
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