Inhibition of arginyltransferase 1 induces transcriptional activity of myocardin-related transcription factor A (MRTF-A) and promotes directional migration

Abstract

Myocardin-related transcription factor A (MRTF-A/MAL/MKL1/BSAC) regulates the expression of serum-response factor (SRF)-dependent target genes in response to the Rho-actin signaling pathway. Overexpression or activation of MRTF-A affects shape, migration, and invasion of cells and contributes to human malignancies, including cancer. In this study, we report that inhibition of arginyltransferase 1 (ATE1), an enzyme mediating post-transcriptional protein arginylation, is sufficient to increase MRTF-A activity in MCF-7 human breast carcinoma cells independently of external growth factor stimuli. In addition, silencing or inhibiting ATE1 disrupted E-cadherin-mediated cell-cell contacts, enhanced formation of actin-rich protrusions, and increased the number of focal adhesions, subsequently leading to elevated chemotactic migration. Although arginylated actin did not differentially affect MRTF-A, a rapid loss of E-cadherin and F-actin reorganization preceded MRTF-A activation upon ATE1 inhibition. Conversely, ectopic ATE1 expression was sufficient to render MRTF-A inactive, both in resting cells and in cells with exogenously activated RhoA-actin pathways. In this study, we provide a critical link between protein arginylation and MRTF-A activity and place ATE1 upstream of myocardin-related transcription factor.

Keywords: Actin; Adherens Junction; Breast Cancer; Cell Adhesion; Cell Signaling; Myocardin; Rho (Rho GTPase); Transcriptional Co-activator.

FIGURE 1.

Silencing of ATE1 expression or inhibition of ATE1 activity induces translocation of MRTF-A to the nucleus. A, MCF-7 cells were serum-starved and treated with 15 μm tannic acid or 100 μm hemin. siCtrl or siATE1 was added, and cells were stimulated with FCS for 60 min. The localization of MRTF-A and F-actin was detected. Line scans of orthogonal sections along the indicated line demonstrate the distribution of MRTF-A (green line) and F-actin (red line). Green bars indicate the nuclear region in the line scan, and red arrows represent cell-cell borders. The localization of MRTF-A staining was determined by quantifying 100 cells each of four independent experiments. Shown is the mean ± S.E. C, cytoplasmic staining; C/N, cytoplasmic/nuclear staining; N, nuclear staining. B, MCF-7 cells were transfected with siCtrl or siATE1, respectively, and the efficacy of ATE1 knockdown was assessed by real time PCR (RNA level) and immunoblotting (protein level). Shown is the fold-change difference of siATE1 versus siCtrl of four (RNA level) or three (protein level) independent experiments ±95% CI. C, MCF-7 cells were transfected with siCtrl or siATE1, respectively, and protein lysates were immunoblotted (IB) with an anti-ATE1, an anti-β-actin and an anti-tubulin antibody. D, cells were serum-starved and treated with the indicated concentrations of tannic acid or 100 μm hemin. FCS was added for 3 h. Cells were lysed and immunoblotted (IB) with an anti-RGS4 and an anti-tubulin antibody.

FIGURE 2.

MRTF-A-dependent transcription in resting MCF-7 cells is increased in cells lacking ATE1 activity. A, MCF-7 cells were transfected with the firefly and the Renilla luciferase plasmids and were treated with 30 μm tannic acid (Tannic A.) or hemin (100 μm). Data represent four independent experiments (tannic acid, three independent experiments) with mean ± S.E. a.u., arbitrary units. B, cells were transfected as before (A) and were treated with increasing concentrations of tannic acid or hemin. Cells were serum-starved in 0.5% FCS-containing medium for 24 h prior to the assay. Data represent four independent experiments with means ± 95% CI. The 95% CI for +FCS control is indicated (dotted lines). a.u., arbitrary units. C, cells were transfected as before (A), and siRNA was added. Cells were either serum-starved (white bars) or stimulated for 7 h with FCS (black bars). Data represent three independent experiments with mean ± S.E. a.u., arbitrary units. D–F, MCF-7 cells were transfected with FLAG-actin and MRTF-A-HA. Cells were treated with siRNA for 72 h, for 2 h with FCS, for 24 h with indicated concentrations of tannic acid, or for 4 h with hemin, and cell extracts were immunoprecipitated (IP) with an anti-FLAG antibody. Immunoprecipitates and input controls were immunoblotted (IB) with an anti-HA and an anti-FLAG antibody. The semiquantitative analyses of band intensities of three independent immunoprecipitations (D) ± S.E. are shown on the right side (E).

FIGURE 3.

Formation of actin-rich protrusions is increased in resting cells lacking ATE1 activity. A, MCF-7 cells were treated with 30 μm tannic acid and 150 μm hemin. siRNA was added, and FCS was added to a final concentration of 20% for 60 min. The localization of F-actin was detected. Middle panels show magnified portions of each image, as indicated (rectangles). Arrows point toward the formation of actin-rich protrusions, and open arrowheads indicate stress fibers. B, number of cells without actin-rich protrusions, with actin-rich protrusions, or ambiguous morphology was quantified by counting 100 cells of four independent experiments. ns, not significant. Data are shown as mean ± 95% CI with p < 0.0001 (***).

FIGURE 4.

Silencing of gene expression or inhibition of ATE1 leads to an increase in small sized focal adhesions. A, cells were transfected with siCtrl or siATE1, respectively. Cells were serum-starved 24 h prior to the experiment. FCS was added to a final concentration of 20% for 60 min, where indicated. The localization of paxillin and F-actin was detected. Inset panels show magnified portions of each image (paxillin staining), as indicated (rectangles). B, number of cell adhesions per cell was counted for 50 cells in six replicates. ns, not significant. Boxplot whiskers represent 5–95 percentile ranges. C, adhesion sites of siATE-transfected and FCS-stimulated cells were calculated and plotted according to different size classes, as indicated. ns, not significant. Boxplot whiskers represent 5–95 percentile ranges.

FIGURE 5.

E-cadherin-mediated cell-cell adhesion is reduced upon ATE1 inhibition or depletion. A, MCF-7 cells were treated with 30 μm tannic acid or 150 μm hemin. siRNA was transfected, and FCS was added to a final concentration of 20% for 60 min. The localization of MRTF-A and E-cadherin was detected. Inset panels show magnified portions of each image (E-cadherin) as indicated (rectangles). B, in parallel, samples of A were lysed and immunoblotted (IB) with an anti-E-cadherin and an anti-tubulin antibody. The semiquantitative analyses of band intensities of three independent immunoblots are shown on the right side. Data are shown as mean ± S.E. ns, not significant.

FIGURE 6.

ATE1-dependent cytoskeletal rearrangements occur prior to MRTF-A activation. A, MCF-7 cells were treated with 150 μm hemin for increasing time intervals, as indicated. Cells were subsequently immunoblotted (IB) with an anti-E-cadherin, an anti-MRTF-A, and an anti-tubulin antibody. The fold change differences in protein expression compared with t = 0 min are indicated below the respective blots. B and C, MCF-7 cells were serum-starved, and 150 μm hemin was added for the indicated time intervals. F-actin and MRTF-A localization was visualized with phalloidin or an anti-MRTF-A antibody, and the percentage of cells exhibiting actin-rich protrusion (black bars) or MRTF-A nuclear localization (red line), respectively, was counted for 200 cells in two independent experiments (C; ±S.E.). D, MCF-7 cells were transfected with the firefly and the Renilla luciferase plasmids. Cells were serum-starved and treated with 150 μm hemin for indicated time points. Data represent three independent experiments with mean ± S.E. *, p < 0.05; **, p <0.01.

FIGURE 7.

ATE1 activity is sufficient to sequester MRTF-A activity independent of actin arginylation. A, MCF-7 cells were transfected with the firefly and the Renilla luciferase plasmids and were co-transfected with ATE1-eGFP. Cells were serum-starved (white circles) and stimulated for 7 h with FCS (black circles), as indicated. Data represent four independent experiments with mean ± S.E. ns, not significant. B, MCF-7 cells were transfected with the firefly and the Renilla luciferase plasmids and were co-transfected with MRTF-A-HA, Ub-M-actin or Ub-R-actin, as indicated. Cells were serum-starved for 24 h. Data represent three independent replicates with mean ± S.E. ns, not significant.

FIGURE 8.

Silencing of ATE1 expression increases cellular motility. Cells were either transfected with siRNA or transfected with pEF-MRTF-A-HA-ΔN or respective empty plasmid control (pEF-HA control). Wild-type control (WT) remained untreated. Cell migration was quantified using a modified Boyden chamber assay. Data represent the mean number of cells per field of view (five different fields of view) of five independent biological replicates (pEF-HA control plasmid and pEF-MRTF-A-HA-ΔN transfected cells were assayed in duplicate) ±S.E. Representative field of view is shown below the plot.