Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates moesin at Thr558 in vitro. Here, using a site- and phosphorylation state-specific antibody, we found that the expression of dominant active RhoA in COS7 cells induced moesin phosphorylation and the formation of microvilli-like structures at apical membranes where the Thr558-phosphorylated moesin accumulated, whereas the expression of dominant negative Rho-kinase inhibited both of these processes. The expression of dominant active Rho-kinase also induced moesin phosphorylation. When COS7 cells expressing moesin or moesin(T558A) (substitution of Thr by Ala) were cultured under serum-depleted conditions, there were few microvilli-like structures, whereas microvilli-like structures remained in the cells expressing moesin(T558D) (substitution of Thr by Asp). The expression of moesin(T558A) inhibited the dominant active RhoA-induced formation of microvilli-like structures. These results indicate that Rho- kinase regulates moesin phosphorylation downstream of Rho in vivo and that the phosphorylation of moesin by Rhokinase plays a crucial role in the formation of microvilli-like structures.
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