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.