RPEL proteins are the molecular targets for CCG-1423, an inhibitor of Rho signaling.
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 accu...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3928398?pdf=render |
| _version_ | 1818013485044334592 |
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| author | Ken'ichiro Hayashi Bunta Watanabe Yoshiaki Nakagawa Saki Minami Tsuyoshi Morita |
| author_facet | Ken'ichiro Hayashi Bunta Watanabe Yoshiaki Nakagawa Saki Minami Tsuyoshi Morita |
| author_sort | Ken'ichiro Hayashi |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-14T06:33:45Z |
| format | Article |
| id | doaj.art-8a8b937a9b454b97a5def99bbb6c4c45 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-14T06:33:45Z |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-8a8b937a9b454b97a5def99bbb6c4c452022-12-22T02:07:32ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-0192e8901610.1371/journal.pone.0089016RPEL proteins are the molecular targets for CCG-1423, an inhibitor of Rho signaling.Ken'ichiro HayashiBunta WatanabeYoshiaki NakagawaSaki MinamiTsuyoshi MoritaEpithelial-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.http://europepmc.org/articles/PMC3928398?pdf=render |
| spellingShingle | Ken'ichiro Hayashi Bunta Watanabe Yoshiaki Nakagawa Saki Minami Tsuyoshi Morita RPEL proteins are the molecular targets for CCG-1423, an inhibitor of Rho signaling. PLoS ONE |
| title | RPEL proteins are the molecular targets for CCG-1423, an inhibitor of Rho signaling. |
| title_full | RPEL proteins are the molecular targets for CCG-1423, an inhibitor of Rho signaling. |
| title_fullStr | RPEL proteins are the molecular targets for CCG-1423, an inhibitor of Rho signaling. |
| title_full_unstemmed | RPEL proteins are the molecular targets for CCG-1423, an inhibitor of Rho signaling. |
| title_short | RPEL proteins are the molecular targets for CCG-1423, an inhibitor of Rho signaling. |
| title_sort | rpel proteins are the molecular targets for ccg 1423 an inhibitor of rho signaling |
| url | http://europepmc.org/articles/PMC3928398?pdf=render |
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