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

Hayashi K, Watanabe B, Nakagawa Y, Minami S, Morita T - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience (D13), Osaka University Graduate School of Medicine, Osaka, Japan.

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.

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Binding specificity of CCG-1423.(A) The sequences of Nrf2 NLSs are aligned. (B) In vitro interaction between Nrf2 and importin α/β1. Mixtures of in vitro-translated HA-importin α1, importin β1, and Flag-Nrf2 proteins were immunoprecipitated with a control gel (cntl) or anti-HA-affinity matrix, and the resulting immunoprecipitates were analyzed by IB with the indicated antibodies. Positions of molecular weight markers are indicated on the side of IB panels in kilodalton. (C) Examination of the binding of purified Flag-Nrf2 to CCG-1423 Sepharose. Procedures for the pull-down assays are similar to those described in the legend for Figure 3. Representative data are shown (n  = 3). (D) Examination of the binding of importin α/β1 to CCG-1423 Sepharose. A mixture of in vitro-translated HA-importin α1 and importin β1 proteins was incubated for 1 h on ice to form a heterodimeric complex. The complex thus obtained was purified using anti-HA-affinity matrix and was used as input. The pull-down assays were performed as described in the legend for Figure 3. Representative data are shown (n  = 3).
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pone-0089016-g005: Binding specificity of CCG-1423.(A) The sequences of Nrf2 NLSs are aligned. (B) In vitro interaction between Nrf2 and importin α/β1. Mixtures of in vitro-translated HA-importin α1, importin β1, and Flag-Nrf2 proteins were immunoprecipitated with a control gel (cntl) or anti-HA-affinity matrix, and the resulting immunoprecipitates were analyzed by IB with the indicated antibodies. Positions of molecular weight markers are indicated on the side of IB panels in kilodalton. (C) Examination of the binding of purified Flag-Nrf2 to CCG-1423 Sepharose. Procedures for the pull-down assays are similar to those described in the legend for Figure 3. Representative data are shown (n  = 3). (D) Examination of the binding of importin α/β1 to CCG-1423 Sepharose. A mixture of in vitro-translated HA-importin α1 and importin β1 proteins was incubated for 1 h on ice to form a heterodimeric complex. The complex thus obtained was purified using anti-HA-affinity matrix and was used as input. The pull-down assays were performed as described in the legend for Figure 3. Representative data are shown (n  = 3).

Mentions: We then investigated whether CCG-1423 binds specifically to NLS of MRTF-A. It has been reported that the nuclear import of Nrf2, a transcription factor essential for antioxidant response element-mediated gene expression, is mediated by three distinct basic amino acid-rich NLSs (Figure 5A) and importin α/β1 [28]. We confirmed that Nrf2 forms a complex with importin α/β1 (Figure 5B). Although the sequences of Nrf2 NLSs are rich in basic amino acids (Figure 5A), significant binding of Nrf2 to CCG-1423 Sepharose was not observed (Figure 5C). Furthermore, the pull-down assay showed that importin α/β1 did not bind to CCG-1423 Sepharose (Figure 5D). These results suggest that CCG-1423 does not bind to any protein with a basic amino acid-rich NLS.


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

Hayashi K, Watanabe B, Nakagawa Y, Minami S, Morita T - PLoS ONE (2014)

Binding specificity of CCG-1423.(A) The sequences of Nrf2 NLSs are aligned. (B) In vitro interaction between Nrf2 and importin α/β1. Mixtures of in vitro-translated HA-importin α1, importin β1, and Flag-Nrf2 proteins were immunoprecipitated with a control gel (cntl) or anti-HA-affinity matrix, and the resulting immunoprecipitates were analyzed by IB with the indicated antibodies. Positions of molecular weight markers are indicated on the side of IB panels in kilodalton. (C) Examination of the binding of purified Flag-Nrf2 to CCG-1423 Sepharose. Procedures for the pull-down assays are similar to those described in the legend for Figure 3. Representative data are shown (n  = 3). (D) Examination of the binding of importin α/β1 to CCG-1423 Sepharose. A mixture of in vitro-translated HA-importin α1 and importin β1 proteins was incubated for 1 h on ice to form a heterodimeric complex. The complex thus obtained was purified using anti-HA-affinity matrix and was used as input. The pull-down assays were performed as described in the legend for Figure 3. Representative data are shown (n  = 3).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928398&req=5

pone-0089016-g005: Binding specificity of CCG-1423.(A) The sequences of Nrf2 NLSs are aligned. (B) In vitro interaction between Nrf2 and importin α/β1. Mixtures of in vitro-translated HA-importin α1, importin β1, and Flag-Nrf2 proteins were immunoprecipitated with a control gel (cntl) or anti-HA-affinity matrix, and the resulting immunoprecipitates were analyzed by IB with the indicated antibodies. Positions of molecular weight markers are indicated on the side of IB panels in kilodalton. (C) Examination of the binding of purified Flag-Nrf2 to CCG-1423 Sepharose. Procedures for the pull-down assays are similar to those described in the legend for Figure 3. Representative data are shown (n  = 3). (D) Examination of the binding of importin α/β1 to CCG-1423 Sepharose. A mixture of in vitro-translated HA-importin α1 and importin β1 proteins was incubated for 1 h on ice to form a heterodimeric complex. The complex thus obtained was purified using anti-HA-affinity matrix and was used as input. The pull-down assays were performed as described in the legend for Figure 3. Representative data are shown (n  = 3).
Mentions: We then investigated whether CCG-1423 binds specifically to NLS of MRTF-A. It has been reported that the nuclear import of Nrf2, a transcription factor essential for antioxidant response element-mediated gene expression, is mediated by three distinct basic amino acid-rich NLSs (Figure 5A) and importin α/β1 [28]. We confirmed that Nrf2 forms a complex with importin α/β1 (Figure 5B). Although the sequences of Nrf2 NLSs are rich in basic amino acids (Figure 5A), significant binding of Nrf2 to CCG-1423 Sepharose was not observed (Figure 5C). Furthermore, the pull-down assay showed that importin α/β1 did not bind to CCG-1423 Sepharose (Figure 5D). These results suggest that CCG-1423 does not bind to any protein with a basic amino acid-rich NLS.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience (D13), Osaka University Graduate School of Medicine, Osaka, Japan.

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.

Show MeSH
Related in: MedlinePlus