The RhoA-binding protein, rhophilin-2, regulates actin cytoskeleton organization

J Biol Chem. 2002 Nov 15;277(46):43924-32. doi: 10.1074/jbc.M203569200. Epub 2002 Sep 6.


The Rho GTPases regulate the actin cytoskeleton through interactions with various downstream effector molecules. Here we have identified a ubiquitously expressed human RhoA-binding protein, designated Rhophilin-2. Rhophilin-2 shows 40% amino acid similarity to human Rhophilin-1 and contains an N-terminal Rho-binding, a central Bro1-like, and a C-terminal PDZ domain. Glutathione S-transferase-capture experiments revealed that Rhophilin-1 and Rhophilin-2 interacted with both GDP- and GTP-bound RhoA in vitro. Despite the ability of Rhophilin-1 and Rhophilin-2 to interact with RhoA in a nucleotide-independent fashion, Rho-induced serum response element transcriptional activity was not altered by expression of either of these molecules. Although Rhophilin-2-expressing HeLa cells showed a loss of actin stress fibers, Rhophilin-1 expression had no noticeable effect on the actin cytoskeleton. Coexpression of Rhophilin-2 with a constitutively active Rho mutant reversed the disassembly phenotype, in which the coexpressing cells were more spread and less contracted than Rho alone-expressing cells. Expression of various Rhophilin-2 deletion and point mutants containing the N-terminal RhoA-binding domain but lacking other regions suggested that the disassembly of F-actin stress fibers was not simply caused by Rho sequestration. In addition, the Bro1 and PDZ domains of Rhophilin-2 were required for disassembly. RhoA activity assays also revealed that Rhophilin-2-expressing cells showed increased levels of RhoA-GTP suggesting that the Rhophilin-2-induced disassembly of stress fibers was not mediated by decreased RhoA activity. Based on the biochemical and biological activity, Rhophilin-2 may function normally in a Rho pathway to limit stress fiber formation and/or increase the turnover of F-actin structures in the absence of high levels of RhoA activity.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Actins / metabolism*
  • Adaptor Proteins, Signal Transducing*
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blotting, Northern
  • Carrier Proteins / chemistry*
  • Carrier Proteins / metabolism
  • Carrier Proteins / physiology*
  • Cloning, Molecular
  • Cytoskeleton / metabolism*
  • DNA, Complementary / metabolism
  • Gene Library
  • Genes, Reporter
  • Genetic Vectors
  • Glutathione Transferase / metabolism
  • HeLa Cells
  • Humans
  • Luciferases / metabolism
  • Mice
  • Molecular Sequence Data
  • Protein Binding
  • Protein Isoforms
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / metabolism*
  • Protein-Serine-Threonine Kinases / physiology*
  • Recombinant Fusion Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Signal Transduction
  • Tissue Distribution
  • Transfection
  • rhoA GTP-Binding Protein / metabolism


  • Actins
  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • DNA, Complementary
  • Protein Isoforms
  • RHPN2 protein, human
  • Recombinant Fusion Proteins
  • rhophilin
  • Luciferases
  • Glutathione Transferase
  • Protein-Serine-Threonine Kinases
  • rhoA GTP-Binding Protein