Ezrin function is required for ROCK-mediated fibroblast transformation by the Net and Dbl oncogenes

The small G protein RhoA and its GDP/GTP exchange factors (GEFs) Net and Db l can transform NM 3T3 fibroblasts, dependent on the activity of the RhoA e ffector kinase ROCK. We investigated the role of the cytoskeletal linker pr otein ezrin in this process. RhoA effector loop mutants which can bind ROCK induce relocalization of ezrin to dorsal actin-containing cell surface pro trusions, as do Net and Dbl. Both processes are inhibited by the ROCK inhib itor Y27632, which also inhibits association of ezrin with the cytoskeleton , and phosphorylation of T567, conserved between ezrin and its relatives ra dixin and moesin. ROCK can phosphorylate the ezrin C-terminus in vitro. The ezrin mutant T567A cannot be relocalized by activated RhoA, Net or Dbl or by ROCK itself, and also inhibits RhoA-mediated contractility and focal adh esion formation. Moreover, ezrin T567A, but not wild-type ezrin, restores c ontact inhibition to Net- and Dbl-transformed cells, and inhibits the activ ity of Net and Ras in focus formation assays. These results implicate ROCK- mediated ezrin C-terminal phosphorylation in transformation by RhoGEFs.