IFN-γ-response mediator GBP-1 represses human cell proliferation by inhibiting the Hippo signaling transcription factor TEAD

Abstract

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that exerts important functions in inflammation, infectious diseases, and cancer. The large GTPase human guanylate-binding protein 1 (GBP-1) is among the most strongly IFN-γ-induced cellular proteins. Previously, it has been shown that GBP-1 mediates manifold cellular responses to IFN-γ including the inhibition of proliferation, spreading, migration, and invasion and through this exerts anti-tumorigenic activity. However, the mechanisms of GBP-1 anti-tumorigenic activities remain poorly understood. Here, we elucidated the molecular mechanism of the human GBP-1-mediated suppression of proliferation by demonstrating for the first time a cross-talk between the anti-tumorigenic IFN-γ and Hippo pathways. The α9-helix of GBP-1 was found to be sufficient to inhibit proliferation. Protein-binding and molecular modeling studies revealed that the α9-helix binds to the DNA-binding domain of the Hippo signaling transcription factor TEA domain protein (TEAD) mediated by the ³⁷⁶VDHLFQK³⁸² sequence at the N-terminus of the GBP-1-α9-helix. Mutation of this sequence resulted in abrogation of both TEAD interaction and suppression of proliferation. Further on, the interaction caused inhibition of TEAD transcriptional activity associated with the down-regulation of TEAD-target genes. In agreement with these results, IFN-γ treatment of the cells also impaired TEAD activity, and this effect was abrogated by siRNA-mediated inhibition of GBP-1 expression. Altogether, this demonstrated that the α9-helix is the proliferation inhibitory domain of GBP-1, which acts independent of the GTPase activity through the inhibition of the Hippo transcription factor TEAD in mediating the anti-proliferative cell response to IFN-γ.

Keywords: cell proliferation; colorectal cancer; guanylate-binding proteins; interferons.

Figure 1.. The α9-helix of GBP-1 is sufficient to inhibit cell proliferation of colorectal carcinoma cells.

(A–H) DLD1 cells were transiently transfected with the indicated Flag-tagged expression plasmids, the plain vector (Ctrl.) or were left untransfected (UT). (A) Proliferation was determined by cell counting after 4 days (n = 3). (B–H) Proliferation of transfected cells was analyzed using EdU. Scale bar represents 80 µm. (B) Detection of total cells (DAPI, blue), transfected cells (Flag, green) and proliferating cells (EdU, red). Representatives of nuclei segmentation (top, right) and signal thresholds (middle, lower right) as applied in the automatic signal detection procedure are shown for cells expressing α12–13. Cells exhibiting fluorescence intensity below the threshold are indicated by asterisks (left). (C) Distribution of green fluorescence intensities. A fluorescence threshold (red vertical line) was chosen that discriminates between non-transfected and transfected cells (above the threshold) and included high numbers of transfected cells for the subsequent quantitative evaluation of the proliferation rates. (D) Numbers of cells analyzed (total (Dapi) and transfected (Flag)) are given. (E) Representative results of the automatic cell detection (transfected cells (green), EdU-positive cells (red), transfected proliferating cells (yellow, arrows)). (F) Proliferation rates of exclusively transfected cells are shown for all conditions except for Ctrl., where the proliferation rate of all cells was calculated (n = 3). Green bars indicate transfections where the α9-helix was present. (G) Flag-tagged GBP-1-α9 (DLD1-α9 #1, #2, #3) or GBP-1 full-length protein (DLD1-GBP-1 #1, #2, #3) expressed in three independent single-cell-derived lines were detected by Flag staining (green). Cell nuclei are stained with Dapi. Cells stably transfected with the control vector (DLD1–CV) or untransfected cells (DLD1) were used as negative controls. Scale bar represents 500 µm. (H) Proliferation of stably transfected DLD1 cells was determined by cell counting after 7 days (n = 3). (I) Spreading of transfected cell was analyzed by determining the cell surface area 20 min after seeding. Percentages of cells possessing an area below 120 µm² and above 120 µm² are shown (n = 6). Control cells (DLD1/CV) included DLD1–CV and untransfected DLD1 cells.

Figure 2.. GBP-1 interacts with and inhibits TEAD.

(A and B) DLD1 or HeLa cells were either left untransfected (UT) or transiently transfected with Flag-tagged GBP-1 (F-GBP-1), respective fragments (F-α7–11, F-α12–13, F-Glo), GFP (F-GFP) or Ctrl. and subjected to co-immunoprecipitation (top). Expression of transfected Flag-tagged proteins and their binding to the beads were detected by Flag immunoprecipitation and subsequent western blot (bottom). Immunoglobulin heavy and light chains are indicated by asterisks. All proteins showed a clear signal at the corresponding sizes. (A) After α-Flag co-precipitation, TEAD was detected by western blot using a pan-antibody. (B) After α-Flag co-precipitation, TEAD1, TEAD2 and TEAD3 were detected by western blot using specific antibodies (upper). (C) HeLa cells were either stimulated with 50 U/ml IFN-γ or were not treated (NT) and subjected to co-immunoprecipitation. After co-precipitation, TEAD was detected by western blot using a pan-antibody (top). Expression of endogenous GBP-1 and its binding to the beads was detected by α-GBP-1 immunoprecipitation and subsequent western blot (bottom). No immunoglobulin chains appeared as the antibody was covalently linked to the beads. (D) Immunofluorescent double staining of F-GBP-1 (green, anti-Flag) and TEAD (red, anti-pan TEAD) in DLD1 cells stably transfected by either control vector (control) or human GBP-1 (F-GBP-1). Nuclei were counterstained by DAPI (blue). Staining was recorded by a confocal microscope. Size bar indicates 25 µm. Cytoplasmic localization of TEAD is indicated by white arrows. (E) Cells from (D) were fractionated and analyzed by western blotting using anti-Flag and anti-pan TEAD antibodies. Lamin A/C and GAPDH were used as controls for the nuclear and cytoplasmic fractions, respectively. The ratio of TEAD relative to GAPDH was quantified using ImageJ and normalized to the control cells. (F) Activity of a TEAD-dependent luciferase reporter was measured in DLD1-GBP-1 (n = 6) cells and compared with the control cells (DLD1/CV) showing the mean of six experiments with control vector-transfected (n = 3) or untransfected DLD1 cells (n = 3). In HeLa cells (right), the luciferase activity was determined in IFN-γ-treated (10 U/ml) and non-treated (NT) cells (n = 2). SiRNA against GBP-1 (GBP-1-siRNA) and control siRNA (Ctrl.-siRNA) were used as indicated. Data shown were normalized to Renilla luciferase activity. (G) mRNA expression of FOXM1 (left), CTGF (middle) and C-MYC (right) were measured by TaqMan qPCR in GBP-1 or control vector (CV)-stably transfected DLD1 cells and IFN-γ- (100 U/ml) treated or non-treated (NT) HeLa cells (n = 6).

Figure 3.. GBP-1 inhibits proliferation by interacting with TEAD.

(A) Amino acid sequences of indicated GBP-1 fragments and mutants are shown. Alanine substitutions within the α9-helix (green) are highlighted in blue/purple. The A1-7/α mutants begin at the α7 and end after the α13 lacking the CAAX motif and contain an N-terminal Flag tag. (B) Indicated proteins were detected in whole-cell lysates of transiently transfected HeLa cells via the Flag epitope. (C) Lysates shown in (B) were subjected to α-Flag co-precipitation. TEAD was detected by western blot using a pan-antibody (top). Expression of transfected Flag-tagged proteins and their binding to the beads was detected by Flag immunoprecipitation and subsequent western blot (lower). (D) Quantification of three independent co-immunoprecipitation experiments (n = 3). TEAD signal was divided by Flag-per-IgG signal followed by normalization to positive control (pos.) (pos. shows the mean of three experiments with GBP-1 (n = 2) or wild-type α7–13 (n = 1)). (E and F) Mode of TEAD GBP-1 interaction derived from docking simulations. GBP-1 is shown in space-filled presentation (gray) with residues 376–382 highlighted in yellow and the remaining parts of the α9-helix in cyan. The TEAD1 docking models are shown in ribbon presentation and colored individually per model. (E) Distribution of the 10 top scoring TEAD1 docking models (m1–m10). Residues 27–105 containing the DNA-binding domain are shown. (F) Detailed view of TEAD1 atoms that are closer than 10 Å to the GBP-1 residues 376–382 are shown in ribbon presentation (m1, blue; m3, red; m4, green; m6, orange; m9, magenta; m10, brown). The view is rotated by ∼70° around the horizontal axis compared with e. (G–J) Proliferation of transiently transfected DLD1 cells was analyzed using EdU (n = 9). (G) Distribution of green fluorescence intensities and applied threshold (red line). (H) Numbers of cells analyzed (total (Dapi), transfected (Flag)). (I) Proliferation rates represent the ratio between transfected proliferating and total transfected cells. Experiment was performed independently three times, each in triplicates. (J) Representative results of the automatic cell detection (transfected cells (green), EdU-positive cells (red), and transfected proliferating cells (yellow, arrows)). Scale bar represents 80 µm.