We demonstrated that the active Ezrin (phosphorylated Ezrin on Thr-567) was essential for forming the flat shape of MDCK cells with cell-cell contacts. The importance of Ezrin in shaping the cells was evidenced by the fact that depletion of Ezrin or dephosphorylation of Ezrin resulted in the columnar cell shape change of MDCK cells. Ezrin has been reported to be involved in the determination of cell shape and polarity (7
). Although the active Ezrin is known to change the cell shape of MDCK cells (21
), the upstream signal activating Ezrin in the presence of cell-cell contacts has remained unclear. In this study, we focused on the signaling that causes dephosphorylation of Thr-567 in the MDCK cells stimulated with ephrinA1, because we previously found that ephrinA1 induces compaction of MDCK cells.
The active Ezrin inhibited the compaction, whereas depletion of Ezrin in the cells resulted in the compaction with polarity. When the active Ezrin is overexpressed in the mouse egg, the compaction that is usually found at the 8- or 16-cell stage is inhibited (33
). In addition, the active Ezrin induces the formation of abnormal membrane protrusions (33
). These morphological changes are also observed in MDCK cells expressing Ezrin T567D (32
). Ezrin knock-out mice show the loss of villous morphogenesis (34
), suggesting that active Ezrin might have the membrane extension activity. Consistently, we found that MDCK cells expressing active Ezrin exhibited a flat shape by horizontally extending the membrane even though cell-cell contacts were preserved.
For Ezrin, cycling between inactivation and activation might be important for shaping the cells to form the organs and tissues. In Ezrin knock-out mice, disorganized intestinal epithelial cells without polarization are found (34
), suggesting the important role for Ezrin in the formation of a multicellular epithelium. Activation or inactivation of Ezrin parallels phosphorylation or dephosphorylation of Thr-567. Extracellular stimuli, including epidermal growth factor, platelet-derived growth factor, and hepatocyte growth factor, can induce the phosphorylation of Thr-567 through Ser/Thr kinases including protein kinase Cα, Rho kinase, and NF-κB-inducing kinase (14
). Activation of Fas, T-cell receptor and B-cell receptor leads to dephosphorylates ERM protein (35
). We here reported that ephrinA1/EphA2 induced dephosphorylation of Thr-567 via inactivation of RhoA-Rho kinase through p190RhoGAP-A. These data indicate that the formation of cell-cell contact negatively regulates Ezrin and vice versa, because Ezrin decreases the height of lateral domain at the cell-cell contacts. Therefore, activation and inactivation of Ezrin appear to be essential for organogenesis that requires the cell-cell contacts.
Besides the activation of Ezrin indicated by phosphorylation of Thr-567, PIP2
was required for activated Ezrin-dependent morphological change (). It is controversial whether ephrinA/EphA signal activates phosphatidylinositol 3-kinase. EphrinA1 stimulation results in the recruitment of Src homology 2-containing inositol-5′-phosphatase and subsequently inhibiting the conversion from PIP2
to phosphatidylinositol 3,4,5-triphosphate (39
), suggesting the possibility of an increase in PIP2
. In contrast, there is a contrasting report that EphA2 activation leads to the activation of phosphatidylinositol 3-kinase in endothelial cells (40
), suggesting the possibility of a decrease in PIP2
. PIP5 kinase is an effector of Arf6 (30
). We previously demonstrated that ephrinA1/EphA2 signal induces compaction of MDCK cells via inactivation of Arf6 and found that PIP2
was essential for maintaining the flat morphology of MDCK cells in this study (). We thus tried to explore whether ephrinA1/EphA2 signal affected the localization of Ezrin in a manner dependent on PIP2
. Depletion of PIP5K did not result in any morphological changes, excluding the possibility that Arf6-PIP5K-mediated PIP2
regulation in ephrinA1/EphA2 signal induces compaction. In addition, Arf6 depletion did not affect dephosphorylation of Ezrin (A
). These results indicate that ephrinA1/EphA2 signal mainly regulates the inactivation of Ezrin by inhibiting RhoA-Rho kinase signal in a manner independent on Arf6, although it is not clear whether EphA2 phosphorylates p190RhoGAP-A directly or indirectly via other tyrosine kinases.
The changes of cell shape are accompanied with the changes in the domains of plasma membrane, pointing to the polarity. Throughout the experiments, the localization of Ezrin was restricted to the apical domain even in flat or columnar states, suggesting that the activation or inactivation is not dependent on the localization of Ezrin but dependent on the degree of phosphorylation of Thr-567. At present, we have not yet clarified the downstream signaling that controls cell morphology by Ezrin at the apical domain, although we here demonstrated the regulation of phosphorylation by ephrinA1/EphA2 signaling initiated by cell-cell contacts. Ezrin at the border between apical domain and lateral domain is known to bind to actin filament and to Bitesize to control actin organization at the cell-cell junction (42
). It is required to explore the molecular mechanism by which active Ezrin maintains the flat morphology. In conclusion, we delineated the signal that regulates ephrinA1/EphA2-induced compaction of MDCK cells (): the engagement ephrinA1 with EphA2 results in the phosphorylation of p190RhoGAP-A and subsequent inactivation of RhoA-Rho kinase signaling, thereby leading to the dephosphorylation of Thr-567 of Ezrin that is essential for maintaining the flat morphology of MDCK cells.