RPEL proteins are the molecular targets for CCG-1423, an inhibitor of Rho signaling

PLoS One. 2014 Feb 18;9(2):e89016. doi: 10.1371/journal.pone.0089016. eCollection 2014.

Abstract

Epithelial-msenchymal transition (EMT) is closely associated with cancer and tissue fibrosis. The nuclear accumulation of myocardin-related transcription factor A (MRTF-A/MAL/MKL1) plays a vital role in EMT. In various cells treated with CCG-1423, a novel inhibitor of Rho signaling, the nuclear accumulation of MRTF-A is inhibited. However, the molecular target of this inhibitor has not yet been identified. In this study, we investigated the mechanism of this effect of CCG-1423. The interaction between MRTF-A and importin α/β1 was inhibited by CCG-1423, but monomeric G-actin binding to MRTF-A was not inhibited. We coupled Sepharose with CCG-1423 (CCG-1423 Sepharose) to investigate this mechanism. A pull-down assay using CCG-1423 Sepharose revealed the direct binding of CCG-1423 to MRTF-A. Furthermore, we found that the N-terminal basic domain (NB) of MRTF-A, which acts as a functional nuclear localization signal (NLS) of MRTF-A, was the binding site for CCG-1423. G-actin did not bind to CCG-1423 Sepharose, but the interaction between MRTF-A and CCG-1423 Sepharose was reduced in the presence of G-actin. We attribute this result to the high binding affinity of MRTF-A for G-actin and the proximity of NB to G-actin-binding sites (RPEL motifs). Therefore, when MRTF-A forms a complex with G-actin, the binding of CCG-1423 to NB is expected to be blocked. NF-E2 related factor 2, which contains three distinct basic amino acid-rich NLSs, did not bind to CCG-1423 Sepharose, but other RPEL-containing proteins such as MRTF-B, myocardin, and Phactr1 bound to CCG-1423 Sepharose. These results suggest that the specific binding of CCG-1423 to the NLSs of RPEL-containing proteins. Our proposal to explain the inhibitory action of CCG-1423 is as follows: When the G-actin pool is depleted, CCG-1423 binds specifically to the NLS of MRTF-A/B and prevents the interaction between MRTF-A/B and importin α/β1, resulting in inhibition of the nuclear import of MRTF-A/B.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Active Transport, Cell Nucleus / physiology*
  • Anilides / metabolism*
  • Benzamides / metabolism*
  • Cell Nucleus / metabolism*
  • DNA-Binding Proteins / metabolism*
  • Epithelial-Mesenchymal Transition / genetics
  • Epithelial-Mesenchymal Transition / physiology*
  • Humans
  • Immunohistochemistry
  • Karyopherins / metabolism
  • Models, Biological
  • Molecular Structure
  • Oncogene Proteins, Fusion / metabolism*
  • Photoaffinity Labels / chemical synthesis
  • Photoaffinity Labels / chemistry
  • Protein Binding
  • Sepharose
  • Signal Transduction / genetics
  • Signal Transduction / physiology*
  • Trans-Activators
  • rho GTP-Binding Proteins / metabolism

Substances

  • Actins
  • Anilides
  • Benzamides
  • CCG 1423
  • DNA-Binding Proteins
  • Karyopherins
  • MRTFA protein, human
  • Oncogene Proteins, Fusion
  • Photoaffinity Labels
  • Trans-Activators
  • Sepharose
  • rho GTP-Binding Proteins

Grant support

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (23590332 to K. H.) and Project MEET, Osaka University Graduate School of Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.