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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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Direct binding of CCG-1423 to other Mycd family members and Phactr1.(A) The sequences of NLSs of Mycd family members and Phactr1 are aligned. Amino acids indicated with red letters play a critical role as an NLS of each RPEL-containing protein. The sequence of NLS of all members of the Mycd family is completely conserved. Phactr1 N-terminal NLS abuts the first RPEL motif, and Phactr1 C-terminal NLS is located between the third and fourth RPEL motifs. (B) Examination of the binding of purified Flag-MRTF-B, Flag-Mycd, or Flag-Phactr1 to CCG-1423 Sepharose. The pull-down assays were performed as described in the legend for Figure 3. (C) Effects of CCG-1423 on the subcellular localization of Phactr1. NIH3T3 cells were transfected with Flag-Phactr1 expression plasmid for 4 h. For further 20 h, the cells were cultured under serum-starved conditions (serum−) in the presence of either 10 µM CCG-1423 (+) or vehicle (DMSO) and were then re-stimulated with 10% serum for 15 min (serum+). The cells were stained with anti-DYKDDDDK (Flag) antibody and Hoechst 33258 (upper panel). Bar  = 20 µm. The images were quantified as described in Materials and Methods: nuclear-specific localization (N), diffuse distribution in the nucleus and the cytoplasm (NC), and cytoplasmic localization (C) (lower panel). Asterisks indicate differences from the values under serum re-stimulated conditions without CCG-1423 in the respective localization categories (*P  = 0.0002 and **P  = 0.0002).
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pone-0089016-g006: Direct binding of CCG-1423 to other Mycd family members and Phactr1.(A) The sequences of NLSs of Mycd family members and Phactr1 are aligned. Amino acids indicated with red letters play a critical role as an NLS of each RPEL-containing protein. The sequence of NLS of all members of the Mycd family is completely conserved. Phactr1 N-terminal NLS abuts the first RPEL motif, and Phactr1 C-terminal NLS is located between the third and fourth RPEL motifs. (B) Examination of the binding of purified Flag-MRTF-B, Flag-Mycd, or Flag-Phactr1 to CCG-1423 Sepharose. The pull-down assays were performed as described in the legend for Figure 3. (C) Effects of CCG-1423 on the subcellular localization of Phactr1. NIH3T3 cells were transfected with Flag-Phactr1 expression plasmid for 4 h. For further 20 h, the cells were cultured under serum-starved conditions (serum−) in the presence of either 10 µM CCG-1423 (+) or vehicle (DMSO) and were then re-stimulated with 10% serum for 15 min (serum+). The cells were stained with anti-DYKDDDDK (Flag) antibody and Hoechst 33258 (upper panel). Bar  = 20 µm. The images were quantified as described in Materials and Methods: nuclear-specific localization (N), diffuse distribution in the nucleus and the cytoplasm (NC), and cytoplasmic localization (C) (lower panel). Asterisks indicate differences from the values under serum re-stimulated conditions without CCG-1423 in the respective localization categories (*P  = 0.0002 and **P  = 0.0002).

Mentions: We addressed the binding specificity of CCG-1423 to other Mycd family members (MRTF-B and Mycd) and one of other RPEL containing proteins (Phactr1). Figure 6A shows the sequences of NLSs of Mycd family members and Phactr1. The sequence of NLS of the Mycd family (NB) is conserved among all members from different species and is located between the second and third RPEL motifs [17]. The conserved amino acids of NLS across Mycd family members and Phactr1 were highlighted. Similarly, Phactr1 C-terminal NLS is located between the third and fourth RPEL motifs [20]. We performed a pull-down assay using CCG-1423 Sepharose to examine the binding of respective RPEL-containing proteins to CCG-1423 (Figure 6B). In these assays, in vitro- translated Flag-tagged proteins were purified using an anti-Flag M2 affinity gel and were used as inputs. These analyses revealed that MRTF-B, Mycd, and Phactr1 bound to CCG-1423 Sepharose. Bindings of Flag-MRTF-B and Phactr1 to CCG-1423 Sepharose were also observed in the binding assay using whole cell extracts (Figure S2). The binding of mutant MRTF-B protein with mutation in NB (MRTF-B NBmut) to CCG-1423 Sepharose severely reduced, suggesting that CCG-1423 also binds to MRTF-B under mediation by NB (Figure S3A). We then examined the effect of CCG-1423 on the subcellular localization of exogenously expressed Flag-MRTF-B (Figure S3B) and Flag-Phactr1 (Figure 6C) in NIH3T3 cells under serum-starved and serum-stimulated conditions. In almost all of the cells expressing Flag-MRTF-B under serum-starved conditions, the protein was primarily observed in the cytoplasm. In contrast, in a large proportion (51.7±1.0%) of serum-stimulated cells, Flag-MRTF-B protein accumulated primarily in the nucleus. CCG-1423 treatment significantly reduced (25.1±0.1%) the proportion of cells showing the nuclear accumulation of the protein and increased (48.3±4.0%) the proportion of cells showing the cytoplasmic localization of the protein. (Figure S3B). Similarly, in almost all of the cells expressing Flag-Phactr1 under serum-starved conditions, the protein was located entirely in the cytoplasm. However, in most of the cells under serum-stimulated conditions, the protein was evenly distributed in the cytoplasm and nucleus. CCG-1423 treatment reduced (66.2±2.0%) the proportion of such cells and increased (33.8±2.0%) the proportion of cells showing the cytoplasmic localization of the protein. These results suggest that CCG-1423 inhibits the serum-induced nuclear import of MRTF-B and Phactr1. However, CCG-1423 did not affect the subcellular localization of constitutively nuclear Mycd (data not shown).


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)

Direct binding of CCG-1423 to other Mycd family members and Phactr1.(A) The sequences of NLSs of Mycd family members and Phactr1 are aligned. Amino acids indicated with red letters play a critical role as an NLS of each RPEL-containing protein. The sequence of NLS of all members of the Mycd family is completely conserved. Phactr1 N-terminal NLS abuts the first RPEL motif, and Phactr1 C-terminal NLS is located between the third and fourth RPEL motifs. (B) Examination of the binding of purified Flag-MRTF-B, Flag-Mycd, or Flag-Phactr1 to CCG-1423 Sepharose. The pull-down assays were performed as described in the legend for Figure 3. (C) Effects of CCG-1423 on the subcellular localization of Phactr1. NIH3T3 cells were transfected with Flag-Phactr1 expression plasmid for 4 h. For further 20 h, the cells were cultured under serum-starved conditions (serum−) in the presence of either 10 µM CCG-1423 (+) or vehicle (DMSO) and were then re-stimulated with 10% serum for 15 min (serum+). The cells were stained with anti-DYKDDDDK (Flag) antibody and Hoechst 33258 (upper panel). Bar  = 20 µm. The images were quantified as described in Materials and Methods: nuclear-specific localization (N), diffuse distribution in the nucleus and the cytoplasm (NC), and cytoplasmic localization (C) (lower panel). Asterisks indicate differences from the values under serum re-stimulated conditions without CCG-1423 in the respective localization categories (*P  = 0.0002 and **P  = 0.0002).
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Related In: Results  -  Collection

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

pone-0089016-g006: Direct binding of CCG-1423 to other Mycd family members and Phactr1.(A) The sequences of NLSs of Mycd family members and Phactr1 are aligned. Amino acids indicated with red letters play a critical role as an NLS of each RPEL-containing protein. The sequence of NLS of all members of the Mycd family is completely conserved. Phactr1 N-terminal NLS abuts the first RPEL motif, and Phactr1 C-terminal NLS is located between the third and fourth RPEL motifs. (B) Examination of the binding of purified Flag-MRTF-B, Flag-Mycd, or Flag-Phactr1 to CCG-1423 Sepharose. The pull-down assays were performed as described in the legend for Figure 3. (C) Effects of CCG-1423 on the subcellular localization of Phactr1. NIH3T3 cells were transfected with Flag-Phactr1 expression plasmid for 4 h. For further 20 h, the cells were cultured under serum-starved conditions (serum−) in the presence of either 10 µM CCG-1423 (+) or vehicle (DMSO) and were then re-stimulated with 10% serum for 15 min (serum+). The cells were stained with anti-DYKDDDDK (Flag) antibody and Hoechst 33258 (upper panel). Bar  = 20 µm. The images were quantified as described in Materials and Methods: nuclear-specific localization (N), diffuse distribution in the nucleus and the cytoplasm (NC), and cytoplasmic localization (C) (lower panel). Asterisks indicate differences from the values under serum re-stimulated conditions without CCG-1423 in the respective localization categories (*P  = 0.0002 and **P  = 0.0002).
Mentions: We addressed the binding specificity of CCG-1423 to other Mycd family members (MRTF-B and Mycd) and one of other RPEL containing proteins (Phactr1). Figure 6A shows the sequences of NLSs of Mycd family members and Phactr1. The sequence of NLS of the Mycd family (NB) is conserved among all members from different species and is located between the second and third RPEL motifs [17]. The conserved amino acids of NLS across Mycd family members and Phactr1 were highlighted. Similarly, Phactr1 C-terminal NLS is located between the third and fourth RPEL motifs [20]. We performed a pull-down assay using CCG-1423 Sepharose to examine the binding of respective RPEL-containing proteins to CCG-1423 (Figure 6B). In these assays, in vitro- translated Flag-tagged proteins were purified using an anti-Flag M2 affinity gel and were used as inputs. These analyses revealed that MRTF-B, Mycd, and Phactr1 bound to CCG-1423 Sepharose. Bindings of Flag-MRTF-B and Phactr1 to CCG-1423 Sepharose were also observed in the binding assay using whole cell extracts (Figure S2). The binding of mutant MRTF-B protein with mutation in NB (MRTF-B NBmut) to CCG-1423 Sepharose severely reduced, suggesting that CCG-1423 also binds to MRTF-B under mediation by NB (Figure S3A). We then examined the effect of CCG-1423 on the subcellular localization of exogenously expressed Flag-MRTF-B (Figure S3B) and Flag-Phactr1 (Figure 6C) in NIH3T3 cells under serum-starved and serum-stimulated conditions. In almost all of the cells expressing Flag-MRTF-B under serum-starved conditions, the protein was primarily observed in the cytoplasm. In contrast, in a large proportion (51.7±1.0%) of serum-stimulated cells, Flag-MRTF-B protein accumulated primarily in the nucleus. CCG-1423 treatment significantly reduced (25.1±0.1%) the proportion of cells showing the nuclear accumulation of the protein and increased (48.3±4.0%) the proportion of cells showing the cytoplasmic localization of the protein. (Figure S3B). Similarly, in almost all of the cells expressing Flag-Phactr1 under serum-starved conditions, the protein was located entirely in the cytoplasm. However, in most of the cells under serum-stimulated conditions, the protein was evenly distributed in the cytoplasm and nucleus. CCG-1423 treatment reduced (66.2±2.0%) the proportion of such cells and increased (33.8±2.0%) the proportion of cells showing the cytoplasmic localization of the protein. These results suggest that CCG-1423 inhibits the serum-induced nuclear import of MRTF-B and Phactr1. However, CCG-1423 did not affect the subcellular localization of constitutively nuclear Mycd (data not shown).

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