mTORC1/S6K1 signaling promotes sustained oncogenic translation through modulating CRL3IBTK-mediated ubiquitination of eIF4A1 in cancer cells
- State Key Laboratory of Genetic Engineering, Shanghai Stomatological Hospital & School of Stomatology, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, School of Life Sciences, Fudan University, China
- Department of Clinical Laboratory, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, China
- Shanghai Key Laboratory of Maternal Fetal Medicine, Shanghai Institute of Maternal-Fetal Medicine and Gynecologic Oncology, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, China
Figures
Figure 1 with 1 supplement
Inhibitor of Bruton's tyrosine kinase (IBTK) interacts with eIF4A1 in cells.
(A) Western blot (WB) analysis of the indicated proteins in the whole cell lysates (WCLs) from FLAG-BirA*-IBTK Tet-on-inducible Flp-In T-REx 293 cells treated with doxycycline (DOX) (10 ng/ml) for 12 hr. (B) Affinity purification of IBTK-containing protein complexes using FLAG-M2 beads was conducted in FLAG-BirA*-IBTK Tet-on-inducible Flp-In T-REx 293 cells. The associated proteins were separated by SDS-PAGE and visualized by Coomassie Blue (CB) staining. (C) The eukaryotic initiation factor (eIF) interactome of IBTK identified by affinity purification coupled with mass spectrometry (AP-MS) analysis in (B). The full list of IBTK-binding partners was shown in Supplementary file 1a. (D–F) WB analysis of the indicated proteins in the WCLs and co-immunoprecipitation (co-IP) samples of anti-FLAG antibody obtained from 293T cells transfected with the indicated plasmids. (G) FLAG-BirA*-IBTK Tet-on-inducible Flp-In T-REx 293 cells were treated with/without DOX (10 ng/ml) for 12 hr, then collected and subjected to co-IP with anti-FLAG antibody. The WCLs and co-IP samples were prepared for WB analysis with the indicated antibodies. (H–K) Co-IP using anti-eIF4A1 (H), eIF4A2 (I), eIF4A3 (J), or IBTK (K) antibody in the WCLs prepared from 293T cells, followed by WB analysis with the indicated antibodies.
-
Figure 1—source data 1
Original file for the western blot analysis in Figure 1.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig1-data1-v1.zip
-
Figure 1—source data 2
Labeled file for the western blot analysis in Figure 1.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig1-data2-v1.zip
Figure 1—figure supplement 1
Inhibitor of Bruton's tyrosine kinase (IBTK) interacts with eIF4A1 in cells.
(A) A workflow for purification of the IBTK protein complexes and identification of potential IBTK interactors. The pcDNA5/FRT/TO vectors were modified to add a FLAG-tag and BirA* CDS. IBTK CDS was inserted to the MCS of this vector. The expression vector was then transfected into Flp-In T-REx 293 cells to establish inducible expressing isogenic cell lines. Affinity purification coupled with mass spectrometry (AP-MS) and biotinylation identification (BioID) methods were applied to purify the IBTK protein complexes, respectively. The purified complexes were then subjected to mass-spectrometric sequencing. (B) The eukaryotic initiation factor (eIF) interactome of IBTK identified by BioID analysis. The full list of IBTK-binding partners was shown in Supplementary file 1b.
Figure 2 with 1 supplement
Inhibitor of Bruton's tyrosine kinase (IBTK) promotes non-degradative ubiquitination of eIF4A1.
(A) Western blot (WB) analysis of the indicated proteins in the whole cell lysates (WCLs) from FLAG-BirA*-IBTK Tet-on-inducible T-REx 293 cells treated with doxycycline (DOX) (10 ng/ml) for the indicated times. (B) WB analysis of the indicated proteins in the WCLs from parental/IBTK-KO SiHa, 293T, and H1299 cells. (C) WB analysis of the indicated proteins in the WCLs from SiHa and HeLa cells infected with lentivirus expressing IBTK-specific shRNA (#1, #2) or negative control (NC). (D) WB analysis of the products of in vivo ubiquitination assays from 293T cells transfected with the indicated plasmids. (E) Parental or IBTK-KO 293T cells were transfected with HA-Ub for 24 hr, then the WCLs were prepared for co-immunoprecipitation (co-IP) with IgG, anti-eIF4A1, eIF4A2, or eIF4A3 antibody. The polyubiquitinated forms of eIF4A1/2/3 were detected by WB with anti-HA antibody. (F) WB analysis of the products of in vitro ubiquitination assays with anti-eIF4A1 antibody. (G) WB analysis of the products of in vivo ubiquitination assays from 293T cells transfected with the indicated plasmids.
-
Figure 2—source data 1
Original file for the western blot analysis in Figure 2.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig2-data1-v1.zip
-
Figure 2—source data 2
Labeled file for the western blot analysis in Figure 2.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig2-data2-v1.zip
Figure 2—figure supplement 1
Inhibitor of Bruton's tyrosine kinase (IBTK) promotes non-degradative ubiquitination of eIF4A1.
(A) Schematic of CRISPR/Cas9-mediated IBTK knockout (KO) in SiHa cells and Sanger sequencing confirming that the IBTK gene was edited by sgRNA #1 or sgRNA #2 in SiHa cells. (B) Schematic of CRISPR/Cas9-mediated IBTK KO in 293T cells and Sanger sequencing confirming that the IBTK gene was edited by sgRNA #1 or sgRNA #2 in 293T cells. (C) Schematic of CRISPR/Cas9-mediated IBTK KO in H1299 cells and Sanger sequencing confirming that the IBTK gene was edited by sgRNA #1 or sgRNA #2 in H1299 cells. (D, E) Western blot (WB) analysis of the indicated proteins in the whole cell lysates (WCLs) from parental and IBTK-KO SiHa cells treated with cycloheximide (CHX,100 μg/ml) and harvested at different time points (D). At each time point, the intensity of eIF4A1, eIF4A2, and eIF4A3 was normalized to the intensity of Actin and then to the value at 0 hr (E). (F) Mass spectrometry (MS) analysis of the immunoprecipitated eIF4A1-Ub conjugates reveals that eIF4A1 ubiquitination occurs at 12 lysine residues. (G) WB analysis of the products of in vivo ubiquitination assays from 293T cells transfected with the indicated plasmids. Twelve ubiquitination sites in eIF4A1 were simultaneously mutated from lysine to arginine (eIF4A1-KR).
-
Figure 2—figure supplement 1—source data 1
Original file for the western blot analysis in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig2-figsupp1-data1-v1.zip
-
Figure 2—figure supplement 1—source data 2
Labeled file for the western blot analysis in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig2-figsupp1-data2-v1.zip
Figure 3 with 1 supplement
Inhibitor of Bruton's tyrosine kinase (IBTK) promotes nascent protein synthesis and cap-dependent translation initiation.
(A) Western blot (WB) analysis of the indicated proteins in the whole cell lysates (WCLs) from parental and IBTK-KO SiHa cells treated with silvestrol (100 nM, 24 hr) and puromycin (1.5 μM, 10 min). (B) WB analysis of the indicated proteins in the WCLs from parental and IBTK-KD HeLa cells treated with silvestrol (100 nM, 24 hr) and puromycin (1.5 μM, 10 min). (C) Representative IF images of parental and IBTK-KO SiHa cells treated with arsenite (AS) (100 μM, 2 hr) and then stained with anti-eIF4A1 (red) and DAPI (blue). Scale bar, 20 μm. (D) The number of eIF4A1 puncta per cell was used to quantify stress granules in (C). Data are presented as means ± SD (n≥10). (E) Representative IF images of HeLa cells transfected with the indicated plasmids, treated with DMSO or AS (100 μM, 2 hr), and then stained with anti-FLAG (green), anti-eIF4A1 (red), and DAPI (blue). The cells successfully transfected with FLAG-IBTK are marked with white dashed lines. Scale bar, 20 μm. (F) Stress granules (SGs) in (E) were quantified by count of eIF4A1 puncta per cell (IBTK transfected vs non-transfected). Data are presented as means ± SD (n≥10). (G) Parental or IBTK-KO SiHa cells were transfected with the reporter pRΔDE·HCVF (pH) or pRΔDE·EMCVF (pE) for 24 hr and the luciferase activities were measured. The counts of Firefly and Renilla were measured and shown in the graph. Data are presented as means ± SD (n=3). (H) Co-immunoprecipitation (Co-IP) assays using IgG or anti-eIF4A1 antibody in the WCLs prepared from parental and IBTK-KO SiHa cells followed by WB analysis with the indicated antibodies. (I) The WCLs of parental and IBTK-KO SiHa cells were incubated with m7GTP-Sepharose beads, and the pull-downed proteins were subjected to WB analysis with the indicated antibodies. p Values are calculated using one-way analysis of variance (ANOVA) test in (D, F, G). ****p<0.0001, n.s. non-significant.
-
Figure 3—source data 1
Original file for the western blot analysis in Figure 3.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig3-data1-v1.zip
-
Figure 3—source data 2
Labeled file for the western blot analysis in Figure 3.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig3-data2-v1.zip
Figure 3—figure supplement 1
Inhibitor of Bruton's tyrosine kinase (IBTK) promotes nascent protein synthesis and cap-dependent translation initiation.
(A) Western blot (WB) analysis of the indicated proteins in the whole cell lysates (WCLs) from parental and IBTK-KO SiHa cells transfected with FLAG-IBTK and treated with puromycin (1.5 μM, 10 min) as indicated. (B) WB analysis of the indicated proteins in the WCLs from parental and IBTK-KO SiHa cells transfected with FLAG-IBTK, treated with silvestrol (100 nM, 24 hr) and puromycin (1.5 μM, 10 min). (C) WB analysis of the indicated proteins in the WCLs from parental and IBTK-KO SiHa cells transfected with Myc-eIF4A1 and treated with puromycin (1.5 μM, 10 min). (D) Representative IF images of SiHa cells transfected with the indicated plasmids, treated with DMSO or sodium arsenite (AS) (100 μM, 2 hr), and then stained with anti-FLAG (green), Caprin1 (red), and DAPI (blue). The cells successfully transfected with FLAG-tag plasmids are marked with white dashed lines. Scale bar, 20 μm. (E) Representative IF images of HeLa cells transfected with FLAG-IBTK, stained with anti-FLAG (green), EDC4 (red), and DAPI (blue). (F) P-bodies in (E) were quantified by counts of EDC4 puncta per cell (IBTK transfected vs non-transfected). Data are presented as means ± SD (n=20). p Values are calculated using unpaired Student’s t-test in (C). n.s. non-significant.
-
Figure 3—figure supplement 1—source data 1
Original file for the western blot analysis in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig3-figsupp1-data1-v1.zip
-
Figure 3—figure supplement 1—source data 2
Labeled file for the western blot analysis in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig3-figsupp1-data2-v1.zip
Figure 4 with 3 supplements
Inhibitor of Bruton's tyrosine kinase (IBTK) deficiency reduces eIF4A1-dependent oncoprotein expression and neoplastic phenotypes in cancer cells.
(A) Western blot (WB) analysis of the indicated proteins in the whole cell lysates (WCLs) from parental and IBTK-KO SiHa cells (left panel) and the WCLs from SiHa cells treated with silvestrol (100 nM) for the indicated times (right panel). (B) Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assessment of the mRNA expression of eIF4A1 target genes in parental and IBTK-KO SiHa cells. The mRNA levels of Actin were used for normalization. Data are shown as means ± SD (n=3). (C) WB analysis of the indicated proteins in the WCLs from FLAG-BirA*-IBTK Tet-on-inducible Flp-In T-REx 293 cells treated with DOX (10 ng/ml) for 12 hr. (D) Cell Counting Kit-8 (CCK-8) cell proliferation analysis of parental and IBTK-KO SiHa cells. Data are shown as means ± SD (n=3). (E) Colony formation analysis of parental and IBTK-KO SiHa cells, and the quantitative data is shown (below). Data are shown as means ± SD (n=3). (F) Cell migration and invasion analysis of parental and IBTK-KO SiHa cells, and the quantitative data is shown (right). Data are shown as means ± SD (n=3). Scale bar, 50 μm. (G) Sphere-formation analysis of parental and IBTK-KO SiHa cells. Representative pictures of SiHa cells after 2 weeks in three-dimensional culture are shown. Average size per sphere and number of spheres per 1000 cells were calculated by ImageJ. The quantitative data is shown (right). Data are shown as means ± SD (n=3). Scale bar, 100 μm. (H) Parental and IBTK-KD HeLa cells were injected subcutaneously (s.c.) into the right flank of BALB/c mice. The tumors in each group at day 20 were harvested and photographed, the quantitative data of tumor weights is shown (below). Data are shown as means ± SD (n=9). (I) WB analysis of the indicated proteins in the WCLs from parental and IBTK-KO SiHa cells treated with silvestrol (100 nM) for the indicated times. (J) Parental and IBTK-KO SiHa cells were treated with silvestrol (100 nM) for the indicated times. Then, annexin-V-FITC/PI dyes were used to stain the harvested cells, after which flow cytometry analysis was performed. Data are shown as means ± SD (n=3). p Values are calculated using unpaired Student’s t-test in (H), one-way analysis of variance (ANOVA) test in (E, F, G), two-way ANOVA test in (B, D, J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. non-significant.
-
Figure 4—source data 1
Original file for the western blot analysis in Figure 4.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig4-data1-v1.zip
-
Figure 4—source data 2
Labeled file for the western blot analysis in Figure 4.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig4-data2-v1.zip
Figure 4—figure supplement 1
Inhibitor of Bruton's tyrosine kinase (IBTK) is indispensable for eIF4A1-related oncogene expression.
(A) Western blot (WB) analysis of the indicated proteins in the whole cell lysates (WCLs) from parental and IBTK-KO H1299 cells (left) and the WCLs from H1299 cells treated with silvestrol (100 nM) for the indicated times (right). (B) Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assessment of the mRNA expression of eIF4A1 targets in parental and IBTK-KO H1299 cells. The mRNA levels of Actin were used for normalization. Data are shown as means ± SD (n=3). (C) WB analysis of the indicated proteins in the WCLs from parental and IBTK-KD CT26 cells (left) and the WCLs from CT26 cells treated with silvestrol (100 nM) for the indicated times (right). (D) RT-qPCR assessment of the mRNA expression of eIF4A1 targets in parental and IBTK-KD CT26 cells. The mRNA levels of Actin were used for normalization. Data are shown as means ± SD (n=3). (E) WB analysis of the indicated proteins in the WCLs from parental and IBTK-KO SiHa cells transfected with EV or Myc-eIF4A1. p Values are calculated using unpaired two-way analysis of variance (ANOVA) test in (B, D). *p<0.05, **p<0.01, n.s. non-significant.
-
Figure 4—figure supplement 1—source data 1
Original file for the western blot analysis in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig4-figsupp1-data1-v1.zip
-
Figure 4—figure supplement 1—source data 2
Labeled file for the western blot analysis in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig4-figsupp1-data2-v1.zip
Figure 4—figure supplement 2
Inhibitor of Bruton's tyrosine kinase (IBTK) deficiency reduces neoplastic phenotypes in cancer cells.
(A) Western blot (WB) analysis of the indicated proteins in the whole cell lysates (WCLs) from parental and IBTK-KO SiHa cells treated with rocaglamide A (200 nM) for the indicated times. (B) Parental and IBTK-KO SiHa cells were treated with rocaglamide A (200 nM) for the indicated times. Then, annexin-V-FITC/PI assays were used to stain the harvested cells, of which later flow cytometry analysis was performed. Data are shown as means ± SD (n=3). (C) Cell Counting Kit-8 (CCK-8) cell proliferation analysis of parental and IBTK-KD HeLa cells. Data are shown as means ± SD (n=3). (D) Cell migration and invasion analysis of parental and IBTK-KD HeLa cells, and the quantitative analysis is shown on the right panel. Data are shown as means ± SD (n=3). Scale bar, 50 μm. (E) Colony formation analysis of parental and IBTK-KD HeLa cells, and the quantitative data are shown as indicated. Data are shown as means ± SD (n=3). (F) Sphere-formation analysis of parental and IBTK-KD HeLa cells. Representative pictures of HeLa cells after 2 weeks in three-dimensional culture are shown. Average size per sphere and number of spheres per 1000 cells were measured by ImageJ and result data are shown as means ± SD (n=3). Scale bar, 100 μm. (G) WB analysis of the indicated proteins in the WCL from parental and IBTK-KD HeLa cells treated with silvestrol (100 nM) for the indicated times. (H) Parental and IBTK-KD HeLa cells were treated with silvestrol (100 nM) for the indicated times. Then, annexin-V-FITC/PI assays were used to stain the harvested cells, of which later flow cytometry analysis was performed. Data are shown as means ± SD (n=3). p Values are calculated using one-way analysis of variance (ANOVA) test in (D, E, F), two-way ANOVA test in (B, C, H). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. non-significant.
-
Figure 4—figure supplement 2—source data 1
Original file for the western blot analysis in Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig4-figsupp2-data1-v1.zip
-
Figure 4—figure supplement 2—source data 2
Labeled file for the western blot analysis in Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig4-figsupp2-data2-v1.zip
Figure 4—figure supplement 3
Inhibitor of Bruton's tyrosine kinase (IBTK) is required for IFN-γ-induced PD-L1 expression.
(A) Western blot (WB) analysis of the indicated proteins in the whole cell lysates (WCLs) from parental and IBTK-KO H1299 cells treated with IFN-γ (200 ng/ml) for the indicated times. (B, C) PD-L1 mean fluorescence intensity (MFI) measurement of parental and IBTK-KO H1299 cells treated with DMSO or IFN-γ (200 ng/ml) for 24 hr. Representative profiles (B) and MFI (C) are shown. Data are shown as means ± SD (n=3). (D) WB analysis of the indicated proteins in the WCLs from parental and IBTK-KD CT26 cells treated with IFN-γ (200 ng/ml) for the indicated times. (E, F) PD-L1 MFI measurement of parental or IBTK-KD CT26 cells treated with DMSO or IFN-γ (200 ng/ml) for 24 hr. Representative profiles (E) and MFI (F) are shown. Data are shown as means ± SD (n=3). (G–I) Parental and IBTK-KD CT26 cells were injected subcutaneously (s.c.) into the right flank of BALB/c mice. The tumors in each group at day 20 were harvested and photographed (G). The quantitative data of tumor weights (H) and tumor volumes (I) are shown. Data are shown as means ± SD (n=8). (J, K) Immunostaining of CD8 and granzyme B in CT26 tumor mass. Scale bar, 200 μm. The number of CD8+ T cells and the intensity of granzyme B were quantified using ImageJ and shown in (K). Data are shown as means ± SD (n=8). p Values are calculated using unpaired Student’s t-test in (H, K), two-way analysis of variance (ANOVA) test in (C, I, F). *p<0.05, ***p<0.001, ****p<0.0001, n.s. non-significant.
-
Figure 4—figure supplement 3—source data 1
Original file for the western blot analysis in Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig4-figsupp3-data1-v1.zip
-
Figure 4—figure supplement 3—source data 2
Labeled file for the western blot analysis in Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig4-figsupp3-data2-v1.zip
Figure 5 with 1 supplement
Inhibitor of Bruton's tyrosine kinase (IBTK)-mediated eIF4A1 ubiquitination is regulated by the mTORC1/S6K1 signaling.
(A) The potential mTOR-regulated phosphorylation sites of IBTK which were sensitive to rapamycin or Torin1 treatment, according to three quantitative phosphoproteomic studies. (B–D) Western blot (WB) analysis of the indicated proteins in the whole cell lysates (WCLs) and co-immunoprecipitation (co-IP) samples of anti-FLAG antibody obtained from 293T cells transfected with the indicated plasmids. (E) Co-IP using anti-IBTK antibody in the WCLs prepared from 293T cells, followed by WB analysis with the indicated antibodies. (F) WB analysis of the indicated proteins in the WCLs from 293T cells transfected with FLAG-IBTK900-1150aa and starved of amino acids (AA) or treated with rapamycin (500 nM) for the indicated times. The phosphorylated forms of IBTK900-1150aa were detected by WB using phos-tag gels. The arrow indicates non-phosphorylated IBTK900-1150aa. (G) WB analysis of the indicated proteins in the WCLs from 293T cells treated with lambda phosphatase (70 U/μl) at 30°C for 1 hr. The phosphorylated form of IBTK900-1150aa was detected by WB using phos-tag gels. (H) WB analysis of the indicated proteins in the WCLs from parental or Raptor-KO 293T cells transfected with FLAG-IBTK900-1150aa. The phosphorylated form of IBTK900-1150aa was detected by WB using phos-tag gels. (I) Recombinant GST-IBTK900-1150aa proteins were subjected to phosphorylation by recombinant mTOR/mLST8, or S6K1, as detected using in vitro kinase assays. The reaction products were separated by SDS-PAGE and visualized by Coomassie Blue (CB) staining. The catalytic domain of human mTOR (1362–2549) was used in this assay. (J) mTOR/mLST8 or S6K1-mediated IBTK in vitro phosphorylation sites identified by mass spectrometry (MS) analysis. (K) WB analysis of the indicated proteins in the WCLs from 293T cells transfected with FLAG-IBTK900-1150aa (WT or 7S/TA mutant) and starved of amino acids or treated with rapamycin (500 nM) for 2 hr. (L) Co-IP using anti-IBTK antibody in the WCLs prepared from 293T cells starved of amino acids (AA-) or treated with rapamycin (500 nM) for 2 hr, followed by WB analysis with the indicated antibodies. (M) WB analysis of the products of in vivo ubiquitination assays from 293T cells transfected with the indicated plasmids and starved of amino acids or treated with rapamycin (500 nM) for 2 hr. (N) WB analysis of the products of in vivo ubiquitination assays from parental or Raptor-KO 293T cells transfected with the indicated plasmids. (O) WB analysis of the products of in vivo ubiquitination assays from 293T cells transfected with the indicated plasmids.
-
Figure 5—source data 1
Original file for the western blot analysis in Figure 5.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig5-data1-v1.zip
-
Figure 5—source data 2
Labeled file for the western blot analysis in Figure 5.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig5-data2-v1.zip
Figure 5—figure supplement 1
mTOR and S6K1 phosphorylates inhibitor of Bruton's tyrosine kinase (IBTK) in vitro.
(A) The mass spectrometry (MS) spectra correspond to phosphorylated IBTK peptides in an in vitro mTOR/mLST8 phosphorylation assay. (B) The MS spectra correspond to phosphorylated IBTK peptides in an in vitro S6K1 phosphorylation assay.
Figure 6
Deficiency in mTORC1/S6K1-mediated eIF4A1 phosphorylation reduces eIF4A1-dependent oncoprotein expression and neoplastic phenotypes in cancer cells.
(A) Western blot (WB) analysis of the indicated proteins in the whole cell lysates (WCLs) from parental and IBTK-KO SiHa cells reconstituted with EV, IBTK-WT, or -7S/TA mutant and treated with puromycin (1.5 μM, 10 min). (B) WB analysis of the indicated proteins in the WCLs from parental and IBTK-KO SiHa cells reconstituted with EV, IBTK-WT, or -7S/TA mutant. (C) Co-immunoprecipitation (Co-IP) assays using anti-eIF4A1 antibody or IgG in the WCLs prepared from parental and IBTK-KO SiHa cells followed by WB analysis with the indicated antibodies. (D) The WCLs of parental and IBTK-KO SiHa cells reconstituted with EV, IBTK-WT, or -7S/TA mutant were incubated with m7GTP-Sepharose beads, and the pull-down proteins were subjected to WB analysis with the indicated antibodies. (E) Cell Counting Kit-8 (CCK-8) cell proliferation analysis of parental and IBTK-KO SiHa cells reconstituted with EV, IBTK-WT, or -7S/TA mutant. Data are shown as means ± SD (n=3). (F, G) Colony formation analysis of parental and IBTK-KO SiHa cells reconstituted with EV, IBTK-WT or, -7S/TA mutant, and the quantitative data is shown in (G). Data are shown as means ± SD (n=3). (H, I) Cell migration analysis of parental and IBTK-KO SiHa cells reconstituted with EV, IBTK-WT or, -7S/TA mutant, and the quantitative analysis is shown in (I). Data are shown as means ± SD (n=3). Scale bar, 100 μm. (J) Parental and IBTK-KO SiHa cells reconstituted with EV, IBTK-WT or, -7S/TA mutant were treated with silvestrol (100 nM) for 24 hr. Then, annexin-V-FITC/PI dyes were used to stain the harvested cells, after which flow cytometry analysis was performed. Data are shown as means ± SD (n=3). p Values are calculated using one-way analysis of variance (ANOVA) test in (G, I), two-way ANOVA test in (E, J). **p<0.01, ***p<0.001, ****p<0.0001, n.s. non-significant.
-
Figure 6—source data 1
Original file for the western blot analysis in Figure 6.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig6-data1-v1.zip
-
Figure 6—source data 2
Labeled file for the western blot analysis in Figure 6.
- https://cdn.elifesciences.org/articles/92236/elife-92236-fig6-data2-v1.zip
Figure 7 with 1 supplement
Overexpression of inhibitor of Bruton's tyrosine kinase (IBTK) correlates with poor survival in cervical cancer.
(A) Kaplan-Meier survival plots of overall survival (OS) were analyzed according to IBTK mRNA expression in cervical squamous cell carcinoma (CESC) specimens from The Cancer Genome Atlas (TCGA) cohort (n=295). (B, C) Immunohistochemistry (IHC) analysis of IBTK protein expression in CESC tissues (n=35) and adjacent normal cervical tissues (n=35). Representative staining results were shown. Scale bar, 30 μm. The relative intensity of IBTK was measured using ImageJ and the quantitative data is shown in (C). (D) IBTK expression in samples with different pathological grade. (E) Kaplan-Meier survival curves of OS comparing high and low expression of IBTK in the TMA (n=117). (F) A proposed model depicting that the mTORC1/S6K1 signaling promotes translation initiation by enhancing IBTK-mediated eIF4A1 ubiquitination. p Values in (A, E) are calculated using log-rank test while p Values in (C, D) are calculated using unpaired Student’s t-test. *p<0.05, ****p<0.0001.
Figure 7—figure supplement 1
Representative images of inhibitor of Bruton's tyrosine kinase (IBTK) immunohistochemistry (IHC) staining in parental and IBTK-KO SiHa cells.
Scale bar,100 μm.
Author response image 1
Author response image 2
Additional files
-
Supplementary file 1
MS data and the information of sequences and reagents.
(a) High-confidence inhibitor of Bruton's tyrosine kinase (IBTK)-interacting protein identified with affinity purification coupled with mass spectrometry (AP-MS) method. (b) High-confidence IBTK-interacting protein identified with biotinylation identification (BioID) method. (c) Ubiquitinated peptide sequences of eIF4A1. (d) Phosphorylated peptide sequences of IBTK by mTOR. (e) Phosphorylated peptide sequences of IBTK by S6K1. (f) Sequence information. (g) Antibody information. (h) Cell lines, cell cultures, chemicals, and kits.
- https://cdn.elifesciences.org/articles/92236/elife-92236-supp1-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/92236/elife-92236-mdarchecklist1-v1.pdf
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
mTORC1/S6K1 signaling promotes sustained oncogenic translation through modulating CRL3IBTK-mediated ubiquitination of eIF4A1 in cancer cells
eLife 12:RP92236.
https://doi.org/10.7554/eLife.92236.3
