RHO-KINASE PHOSPHORYLATES COOH-TERMINAL THREONINES OF EZRIN RADIXIN/MOESIN (ERM) PROTEINS AND REGULATES THEIR HEAD-TO-TAIL ASSOCIATION/

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament /plasma membrane interaction that is regulated by Rho. We examined whe ther ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COO H-terminal half radixin were incubated with constitutively active cata lytic domain of Rho-kinase, and similar to 30 and similar to 100% of t hese molecules, respectively, were phosphorylated mainly at the COOH-t erminal threonine (T564). Next, to detect Rho-kinase-dependent phospho rylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb reveale d that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 p hosphorylation of recombinant COOH-terminal half radixin did not affec t its ability to bind to actin filaments in vitro but significantly su ppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase-dependent phosphoryla tion interferes with the intramolecular and/or intermolecular head-to- tail association of ERM proteins, which is an important mechanism of r egulation of their activity as actin filament/plasma membrane cross-li nkers.