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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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Preparation of CCG-1423 Sepharose.(A) Synthesis of the photoaffinity linker, 2-(2-{2-[4-(3-trifluoromethyl-3H-diazirin-3-yl)benzamido]ethoxy}ethoxy)ethylammonium trifluoroacetate. The photoaffinity linker was synthesized by the indicated three-step reactions. The steps were based on the following methods: step 1 [24] and steps 2 and 3 [25]. Reagents needed for the respective reactions are indicated by their abbreviations (above the arrows) and are as follows: (Boc)2O, di-tert-butyl dicarbonate; EDCI, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, DMAP, 4-(dimethylamino)pyridine; TFA, trifluoroacetic acid. Solvents and reaction time for the respective reactions are indicated (below the arrows). The structures of the respective synthesized products were characterized by NMR. (B) Crosslinking of CCG-1423 to Sepharose with the photoaffinity linker. This step was performed according to the method published by McIntyre et al. [26]. Activated CH Sepharose 4B beads were coupled with the photoaffinity linker, and the beads were treated with 1 M ethanolamine (pH 11) to block the remaining reactive groups. The Sepharose beads with photoaffinity linker were agitated with 50 mM Tris-HCl (pH 7.4) buffer containing 0.1 mM CCG-1423, and then were exposed to UV light for 1 h. CCG-1423 was randomly coupled with the photoaffinity linkers on Sepharose by UV irradiation. The CCG-1423 Sepharose was washed with methanol and dried.
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pone-0089016-g002: Preparation of CCG-1423 Sepharose.(A) Synthesis of the photoaffinity linker, 2-(2-{2-[4-(3-trifluoromethyl-3H-diazirin-3-yl)benzamido]ethoxy}ethoxy)ethylammonium trifluoroacetate. The photoaffinity linker was synthesized by the indicated three-step reactions. The steps were based on the following methods: step 1 [24] and steps 2 and 3 [25]. Reagents needed for the respective reactions are indicated by their abbreviations (above the arrows) and are as follows: (Boc)2O, di-tert-butyl dicarbonate; EDCI, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, DMAP, 4-(dimethylamino)pyridine; TFA, trifluoroacetic acid. Solvents and reaction time for the respective reactions are indicated (below the arrows). The structures of the respective synthesized products were characterized by NMR. (B) Crosslinking of CCG-1423 to Sepharose with the photoaffinity linker. This step was performed according to the method published by McIntyre et al. [26]. Activated CH Sepharose 4B beads were coupled with the photoaffinity linker, and the beads were treated with 1 M ethanolamine (pH 11) to block the remaining reactive groups. The Sepharose beads with photoaffinity linker were agitated with 50 mM Tris-HCl (pH 7.4) buffer containing 0.1 mM CCG-1423, and then were exposed to UV light for 1 h. CCG-1423 was randomly coupled with the photoaffinity linkers on Sepharose by UV irradiation. The CCG-1423 Sepharose was washed with methanol and dried.

Mentions: We covalently coupled Sepharose with CCG-1423 (CCG-1423 Sepharose) using a photo-crosslinking agent (Figure 2) and performed a pull-down assay using the CCG-1423 Sepharose to examine MRTF-A binding to CCG-1423 (Figure 3A, B). Because MRTF-A is associated with various proteins including G-actin, SRF, Smad, and other protein factors in cells, these protein factors may affect the interaction between MRTF-A and CCG-1423 Sepharose. In these assays, to rule out this possibility, in vitro-translated Flag-tagged proteins were purified using an anti-Flag M2 affinity gel and were used as inputs (Figure 3A, B, left columns). Wild-type MRTF-A protein clearly bound to CCG-1423 Sepharose, but such binding was severely reduced by free CCG-1423 (20.6±6.3% of the binding level in the absence of free CCG-1423) (Figure 3A, middle and right columns). However, this protein did not bind to a control Sepharose without CCG-1423 coupling (Figure 3B, middle and right columns). An MRTF-A protein with mutation in NB (MRTF-A NBmut), in which the NB sequence KLKRAR was mutated to ALAAAR, exhibited a low binding level (11.2±8.5% of the wild-type protein level) (Figure 3B, middle and right columns). These results strongly suggest that CCG-1423 binds specifically and directly to MRTF-A under mediation by NB. The basic amino acids in the NB sequence (Figure 3B) play a critical role in CCG-1423 binding to NB.


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)

Preparation of CCG-1423 Sepharose.(A) Synthesis of the photoaffinity linker, 2-(2-{2-[4-(3-trifluoromethyl-3H-diazirin-3-yl)benzamido]ethoxy}ethoxy)ethylammonium trifluoroacetate. The photoaffinity linker was synthesized by the indicated three-step reactions. The steps were based on the following methods: step 1 [24] and steps 2 and 3 [25]. Reagents needed for the respective reactions are indicated by their abbreviations (above the arrows) and are as follows: (Boc)2O, di-tert-butyl dicarbonate; EDCI, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, DMAP, 4-(dimethylamino)pyridine; TFA, trifluoroacetic acid. Solvents and reaction time for the respective reactions are indicated (below the arrows). The structures of the respective synthesized products were characterized by NMR. (B) Crosslinking of CCG-1423 to Sepharose with the photoaffinity linker. This step was performed according to the method published by McIntyre et al. [26]. Activated CH Sepharose 4B beads were coupled with the photoaffinity linker, and the beads were treated with 1 M ethanolamine (pH 11) to block the remaining reactive groups. The Sepharose beads with photoaffinity linker were agitated with 50 mM Tris-HCl (pH 7.4) buffer containing 0.1 mM CCG-1423, and then were exposed to UV light for 1 h. CCG-1423 was randomly coupled with the photoaffinity linkers on Sepharose by UV irradiation. The CCG-1423 Sepharose was washed with methanol and dried.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089016-g002: Preparation of CCG-1423 Sepharose.(A) Synthesis of the photoaffinity linker, 2-(2-{2-[4-(3-trifluoromethyl-3H-diazirin-3-yl)benzamido]ethoxy}ethoxy)ethylammonium trifluoroacetate. The photoaffinity linker was synthesized by the indicated three-step reactions. The steps were based on the following methods: step 1 [24] and steps 2 and 3 [25]. Reagents needed for the respective reactions are indicated by their abbreviations (above the arrows) and are as follows: (Boc)2O, di-tert-butyl dicarbonate; EDCI, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, DMAP, 4-(dimethylamino)pyridine; TFA, trifluoroacetic acid. Solvents and reaction time for the respective reactions are indicated (below the arrows). The structures of the respective synthesized products were characterized by NMR. (B) Crosslinking of CCG-1423 to Sepharose with the photoaffinity linker. This step was performed according to the method published by McIntyre et al. [26]. Activated CH Sepharose 4B beads were coupled with the photoaffinity linker, and the beads were treated with 1 M ethanolamine (pH 11) to block the remaining reactive groups. The Sepharose beads with photoaffinity linker were agitated with 50 mM Tris-HCl (pH 7.4) buffer containing 0.1 mM CCG-1423, and then were exposed to UV light for 1 h. CCG-1423 was randomly coupled with the photoaffinity linkers on Sepharose by UV irradiation. The CCG-1423 Sepharose was washed with methanol and dried.
Mentions: We covalently coupled Sepharose with CCG-1423 (CCG-1423 Sepharose) using a photo-crosslinking agent (Figure 2) and performed a pull-down assay using the CCG-1423 Sepharose to examine MRTF-A binding to CCG-1423 (Figure 3A, B). Because MRTF-A is associated with various proteins including G-actin, SRF, Smad, and other protein factors in cells, these protein factors may affect the interaction between MRTF-A and CCG-1423 Sepharose. In these assays, to rule out this possibility, in vitro-translated Flag-tagged proteins were purified using an anti-Flag M2 affinity gel and were used as inputs (Figure 3A, B, left columns). Wild-type MRTF-A protein clearly bound to CCG-1423 Sepharose, but such binding was severely reduced by free CCG-1423 (20.6±6.3% of the binding level in the absence of free CCG-1423) (Figure 3A, middle and right columns). However, this protein did not bind to a control Sepharose without CCG-1423 coupling (Figure 3B, middle and right columns). An MRTF-A protein with mutation in NB (MRTF-A NBmut), in which the NB sequence KLKRAR was mutated to ALAAAR, exhibited a low binding level (11.2±8.5% of the wild-type protein level) (Figure 3B, middle and right columns). These results strongly suggest that CCG-1423 binds specifically and directly to MRTF-A under mediation by NB. The basic amino acids in the NB sequence (Figure 3B) play a critical role in CCG-1423 binding to NB.

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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Related in: MedlinePlus