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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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Summary of the inhibitory effect of CCG-1423 on the importin α/β1-mediated nuclear import of MRTF-A.When the G-actin pool is depleted by extracellular stimuli, MRTF-A is imported into the nucleus under mediation by importin α/β1. However, in the presence of CCG-1423, this process is competitively inhibited because CCG-1423 binds to NB and masks the binding site for importin α/β1. The basic amino acids indicated with red letters play a critical role in CCG-1423 binding to NB (upper panel). In contrast, in serum-starved cells, MRTF-A forms a complex with G-actin under mediation by RPEL motifs. Thus, both CCG-1423 and importin α/β1 are not accessible to NB because the binding affinities of CCG-1423 and importin α/β1 to MRTF-A are weaker than those of G-actin to MRTF-A (lower panel). Because MRTF-A associated with G-actin exhibits a low binding affinity to CCG-1423, G-actin-free MRTF-A is a more suitable CCG-1423 target protein. The abbreviations used are as follows: NB, N-terminal basic domain; CB, central basic domain; SAP, SAP domain; CC, coiled-coil domain.
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pone-0089016-g007: Summary of the inhibitory effect of CCG-1423 on the importin α/β1-mediated nuclear import of MRTF-A.When the G-actin pool is depleted by extracellular stimuli, MRTF-A is imported into the nucleus under mediation by importin α/β1. However, in the presence of CCG-1423, this process is competitively inhibited because CCG-1423 binds to NB and masks the binding site for importin α/β1. The basic amino acids indicated with red letters play a critical role in CCG-1423 binding to NB (upper panel). In contrast, in serum-starved cells, MRTF-A forms a complex with G-actin under mediation by RPEL motifs. Thus, both CCG-1423 and importin α/β1 are not accessible to NB because the binding affinities of CCG-1423 and importin α/β1 to MRTF-A are weaker than those of G-actin to MRTF-A (lower panel). Because MRTF-A associated with G-actin exhibits a low binding affinity to CCG-1423, G-actin-free MRTF-A is a more suitable CCG-1423 target protein. The abbreviations used are as follows: NB, N-terminal basic domain; CB, central basic domain; SAP, SAP domain; CC, coiled-coil domain.

Mentions: Because NB is in close proximity to the G-actin-binding RPEL motifs (Figure 7), we predicted that CCG-1423 binding to NB competes with G-actin binding to RPEL motifs. We addressed this possibility using purified MRTF-A and β-actin R62D (G-actin) proteins. In the presence of G-actin, MRTF-A binding to CCG-1423 Sepharose reduced, but G-actin was clearly detected in the bound fraction (Figure 3C, left column). G-actin did not bind solely to CCG-1423 Sepharose (Figure 3C, right column). Taken together with the data shown in Figure 1B, CCG-1423 does not inhibit G-actin binding to MRTF-A, suggesting that in the presence of G-actin, MRTF-A preferentially forms a complex with G-actin because of its high binding affinity for G-actin and results in inhibition of CCG-1423 binding to MRTF-A. Thus, G-actin-free MRTF-A rather than MRTF-A associated with G-actin is the more likely CCG-1423 target protein.


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)

Summary of the inhibitory effect of CCG-1423 on the importin α/β1-mediated nuclear import of MRTF-A.When the G-actin pool is depleted by extracellular stimuli, MRTF-A is imported into the nucleus under mediation by importin α/β1. However, in the presence of CCG-1423, this process is competitively inhibited because CCG-1423 binds to NB and masks the binding site for importin α/β1. The basic amino acids indicated with red letters play a critical role in CCG-1423 binding to NB (upper panel). In contrast, in serum-starved cells, MRTF-A forms a complex with G-actin under mediation by RPEL motifs. Thus, both CCG-1423 and importin α/β1 are not accessible to NB because the binding affinities of CCG-1423 and importin α/β1 to MRTF-A are weaker than those of G-actin to MRTF-A (lower panel). Because MRTF-A associated with G-actin exhibits a low binding affinity to CCG-1423, G-actin-free MRTF-A is a more suitable CCG-1423 target protein. The abbreviations used are as follows: NB, N-terminal basic domain; CB, central basic domain; SAP, SAP domain; CC, coiled-coil domain.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089016-g007: Summary of the inhibitory effect of CCG-1423 on the importin α/β1-mediated nuclear import of MRTF-A.When the G-actin pool is depleted by extracellular stimuli, MRTF-A is imported into the nucleus under mediation by importin α/β1. However, in the presence of CCG-1423, this process is competitively inhibited because CCG-1423 binds to NB and masks the binding site for importin α/β1. The basic amino acids indicated with red letters play a critical role in CCG-1423 binding to NB (upper panel). In contrast, in serum-starved cells, MRTF-A forms a complex with G-actin under mediation by RPEL motifs. Thus, both CCG-1423 and importin α/β1 are not accessible to NB because the binding affinities of CCG-1423 and importin α/β1 to MRTF-A are weaker than those of G-actin to MRTF-A (lower panel). Because MRTF-A associated with G-actin exhibits a low binding affinity to CCG-1423, G-actin-free MRTF-A is a more suitable CCG-1423 target protein. The abbreviations used are as follows: NB, N-terminal basic domain; CB, central basic domain; SAP, SAP domain; CC, coiled-coil domain.
Mentions: Because NB is in close proximity to the G-actin-binding RPEL motifs (Figure 7), we predicted that CCG-1423 binding to NB competes with G-actin binding to RPEL motifs. We addressed this possibility using purified MRTF-A and β-actin R62D (G-actin) proteins. In the presence of G-actin, MRTF-A binding to CCG-1423 Sepharose reduced, but G-actin was clearly detected in the bound fraction (Figure 3C, left column). G-actin did not bind solely to CCG-1423 Sepharose (Figure 3C, right column). Taken together with the data shown in Figure 1B, CCG-1423 does not inhibit G-actin binding to MRTF-A, suggesting that in the presence of G-actin, MRTF-A preferentially forms a complex with G-actin because of its high binding affinity for G-actin and results in inhibition of CCG-1423 binding to MRTF-A. Thus, G-actin-free MRTF-A rather than MRTF-A associated with G-actin is the more likely CCG-1423 target protein.

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