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

EMBO J. 2000 Sep 1;19(17):4565-76. doi: 10.1093/emboj/19.17.4565.

Abstract

The small G protein RhoA and its GDP/GTP exchange factors (GEFs) Net and Dbl can transform NIH 3T3 fibroblasts, dependent on the activity of the RhoA effector kinase ROCK. We investigated the role of the cytoskeletal linker protein ezrin in this process. RhoA effector loop mutants which can bind ROCK induce relocalization of ezrin to dorsal actin-containing cell surface protrusions, as do Net and Dbl. Both processes are inhibited by the ROCK inhibitor Y27632, which also inhibits association of ezrin with the cytoskeleton, and phosphorylation of T567, conserved between ezrin and its relatives radixin 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 adhesion formation. Moreover, ezrin T567A, but not wild-type ezrin, restores contact inhibition to Net- and Dbl-transformed cells, and inhibits the activity of Net and Ras in focus formation assays. These results implicate ROCK-mediated ezrin C-terminal phosphorylation in transformation by RhoGEFs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Animals
  • Cell Transformation, Neoplastic / genetics*
  • Cytoskeletal Proteins
  • Cytoskeleton / enzymology
  • Guanine Nucleotide Exchange Factors
  • Intracellular Signaling Peptides and Proteins
  • Mice
  • Oncogene Proteins*
  • Oncogenes*
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism
  • Phosphoproteins / physiology*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / physiology*
  • Proto-Oncogene Proteins c-ets
  • Retroviridae Proteins, Oncogenic / genetics
  • Signal Transduction
  • Transcription Factors / genetics
  • rho-Associated Kinases

Substances

  • Cytoskeletal Proteins
  • Elk3 protein, mouse
  • Guanine Nucleotide Exchange Factors
  • Intracellular Signaling Peptides and Proteins
  • Mcf2 protein, mouse
  • Oncogene Proteins
  • Phosphoproteins
  • Proto-Oncogene Proteins c-ets
  • Retroviridae Proteins, Oncogenic
  • Transcription Factors
  • ezrin
  • Protein-Serine-Threonine Kinases
  • rho-Associated Kinases