Figure 1.
Effects of CCG-1423 on the in vitro interaction between MRTF-A and importin α/β1.
Mixtures of in-translated HA-importin α1, importin β1, and Flag-MRTF-A proteins (A) or β-actin R62D (G-actin) and Flag-MRTF-A proteins (B) were immunoprecipitated with a control gel, anti-HA-affinity matrix, or anti-Flag M2 affinity gel in the presence of either CCG-1423 (+) or vehicle (DMSO; −), and the resulting immunoprecipitates were analyzed by immunoblotting (IB) with the indicated antibodies. Positions of molecular weight markers are indicated on the side of IB panels in kilodaltons (left columns). Control experiments with the control gel (cntl) showed no significant signals on IB (lanes IPC). The respective immunoprecipitation (IP)/IB signal intensities were quantified as described in Materials and Methods (right columns). The percentage values indicate the relative levels of MRTF-A binding to importin α1/β1 (A) or G-actin binding to MRTF-A (B) normalized by the binding of MRTF-A or G-actin in the absence of CCG-1423, which were set at 100% (mean ± s.e.m of the results from three independent experiments). (C) Inhibitory effect of CCG-1423 on the interaction between MRTF-A and importin α/β1 in cultured cells. NIH3T3 cells were transfected with the expression plasmids as described in Materials and Methods. Whole cell extracts from the cells re-stimulated with serum were subjected to IP/IB analysis as described earlier. Representative data are shown (n = 3).
Figure 2.
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.
Figure 3.
Direct binding of CCG-1423 to MRTF-A mediated by NB.
(A and B) Examination of the binding of MRTF-A to CCG-1423 Sepharose. Coomassie brilliant blue (CBB) staining of purified proteins used in these assays are as follows: Flag-MRTF-As [wild-type (wt) and NBmut (m)] (A and B, left columns). (C) Investigations of the binding of Flag-MRTF-A to CCG-1423 Sepharose in the presence of G-actin and the binding of G-actin to CCG-1423 Sepharose. Pull-down assays with CCG-1423 Sepharose were performed using purified Flag-MRTF-A and/or HA-β-actin R62D (G-actin) proteins as inputs. Detailed procedures are described in Materials and Methods. The proteins bound to CCG-Sepharose (CCG) or control Sepharose (cntl) were analyzed by IB with the indicated antibodies (A and B, middle columns and C). The respective pull-down/IB signal intensities were quantified (A and B, right graphs and C, the percentage values on the top of pull-down column). The percentage values indicate the relative levels of MRTF-A binding to CCG-1423 Sepharose or control Sepharose normalized by the binding of MRTF-A in the absence of free CCG-1423 (A), the binding of wild-type MRTF-A to CCG-1423 Sepharose (B), or the binding of MRTF-A in the absence of G-actin (C), which was set at 100% (mean ± s.e.m of the results from three independent experiments).
Figure 4.
Effects of actin dynamics on MRTF-A binding to CCG-1423 Sepharose.
(A) Imaging of F-actin in NIH3T3 cells by staining with phalloidin conjugated to Alexa Fluor 568. NIH3T3 cells were cultured under serum-stimulated conditions. For the final 16 h, they were cultured under either serum-starved (serum−) or serum-stimulated (serum+) conditions. The cells were further incubated with either 50 nM of Jasp (Jasp+) or 2 µM LatB (LatB+) for 10 min. Bars = 20 µm. (B) Whole cell extracts (WE1 and WE2) were prepared from NIH3T3 cells expressing Flag-MRTF-A under either serum-stimulated or serum-starved conditions. Brief explanations of the respective whole cell extracts are given in the upper panel; WE1 from serum-stimulated cells contains Jasp (Jasp+) and WE2 from serum-starved cells contains LatB (LatB+). The details of whole cell extract preparation are described in Materials and Methods. These whole cell extracts were subjected to pull-down assay using CCG-1423 Sepharose in the absence or presence of free CCG-1423 (10 µM). The proteins bound to CCG-Sepharose (CCG) or control Sepharose (cntl) were analyzed by IB with anti-Flag antibody.
Figure 5.
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).
Figure 6.
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).
Figure 7.
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.