title: RPEL proteins are the molecular targets for CCG-1423, an inhibitor of Rho signaling.
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.
authors.affiliation: Department of Neuroscience (D13), Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Ken'ichiro
authors.initials: K
authors.lastname: Hayashi
authors.affiliation: Institute for Chemical Research, Kyoto University, Kyoto, Japan.
authors.forename: Bunta
authors.initials: B
authors.lastname: Watanabe
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Nakagawa
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Saki
authors.initials: S
authors.lastname: Minami
authors.affiliation: Department of Neuroscience (D13), Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Morita
mesh_terms: D000199:Actins / Q000378:metabolism; D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000813:Anilides / Q000378:metabolism*; D001549:Benzamides / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D028884:Karyopherins / Q000378:metabolism; D008954:Models, Biological; D015394:Molecular Structure; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000737:chemistry; D011485:Protein Binding; D012685:Sepharose; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D015534:Trans-Activators; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D000199:Actins; D000813:Anilides; D001549:Benzamides; C523455:CCG 1423; D004268:DNA-Binding Proteins; D028884:Karyopherins; C431575:MRTFA protein, human; D015514:Oncogene Proteins, Fusion; D019920:Photoaffinity Labels; D015534:Trans-Activators; D012685:Sepharose; D020741:rho GTP-Binding Proteins