The four and a half LIM-only protein 2 (FHL2) activates transforming growth factor β (TGF-β) signaling by regulating ubiquitination of the E3 ligase Arkadia

J Biol Chem. 2013 Jan 18;288(3):1785-94. doi: 10.1074/jbc.M112.439760. Epub 2012 Dec 4.


Arkadia is a RING-based ubiquitin ligase that positively regulates TGF-β signaling by targeting several pathway components for ubiquitination and degradation. However, little is known about the mechanisms controlling Arkadia activity. Here we show that the LIM-only protein FHL2 binds and synergistically cooperates with Arkadia to activate Smad3/Smad4-dependent transcription. Knockdown of FHL2 by RNA interference decreases Arkadia level and restricts the amplitude of Arkadia-induced TGF-β target gene responses. We found that Arkadia is ubiquitinated via K63- and K27-linked polyubiquitination. A single mutation at the RING domain that abolishes the E3 activity diminishes Arkadia ubiquitination, indicating that this modification partly involves autocatalytic process. Mutation of seven lysines at the C-terminal region of Arkadia severely impairs ubiquitination through the K27 but not the K63 linkage and slows down the turnover of Arkadia, suggesting that K27-linked polyubiquitination might promote proteolysis-dependent regulation of Arkadia. We show that FHL2 increases the half-life of Arkadia through inhibition of ubiquitin chain assembly on the protein, which provides a molecular basis for functional cooperation between Arkadia and FHL2 in enhancing TGF-β signaling. Our study uncovers a novel regulatory mechanism of Arkadia by ubiquitination and identifies FHL2 as important regulator of Arkadia ubiquitination and TGF-β signal transduction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Cell Line, Tumor
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Gene Expression Regulation
  • Genes, Reporter
  • Half-Life
  • Humans
  • LIM-Homeodomain Proteins / genetics*
  • LIM-Homeodomain Proteins / metabolism
  • Luciferases
  • Mice
  • Muscle Proteins / genetics*
  • Muscle Proteins / metabolism
  • Mutation
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Signal Transduction
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Transfection
  • Transforming Growth Factor beta / genetics*
  • Transforming Growth Factor beta / metabolism
  • Ubiquitin / genetics*
  • Ubiquitin / metabolism
  • Ubiquitin-Protein Ligases / genetics*
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination


  • FHL2 protein, human
  • Fhl2 protein, mouse
  • LIM-Homeodomain Proteins
  • Muscle Proteins
  • Nuclear Proteins
  • Transcription Factors
  • Transforming Growth Factor beta
  • Ubiquitin
  • Luciferases
  • RNF111 protein, human
  • Ubiquitin-Protein Ligases