Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association

J Cell Biol. 1998 Feb 9;140(3):647-57. doi: 10.1083/jcb.140.3.647.

Abstract

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and approximately 30 and approximately 100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase-dependent phosphorylation 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 revealed 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 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase-dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Actins / metabolism
  • Animals
  • Antibodies, Monoclonal
  • Blood Proteins / chemistry
  • Blood Proteins / immunology
  • Blood Proteins / metabolism*
  • Cell Membrane / metabolism
  • Cytoskeletal Proteins*
  • Immunoblotting
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins / chemistry
  • Membrane Proteins / immunology
  • Membrane Proteins / metabolism*
  • Mice
  • Microfilament Proteins*
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Phosphothreonine / metabolism*
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proteins / chemistry
  • Proteins / metabolism*
  • Recombinant Proteins / metabolism
  • rho-Associated Kinases

Substances

  • Actins
  • Antibodies, Monoclonal
  • Blood Proteins
  • Cytoskeletal Proteins
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Microfilament Proteins
  • Phosphoproteins
  • Proteins
  • Recombinant Proteins
  • ezrin
  • Phosphothreonine
  • moesin
  • radixin
  • Protein-Serine-Threonine Kinases
  • rho-Associated Kinases