IBTK interacts with eIF4A1 in cells.

(A) 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 h.

(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 CB staining. (C)The eIF interactome of IBTK identified by AP-MS analysis in (B). The full list of IBTK binding partners was showed in Supplementary Table 1.

(D-F) WB analysis of the indicated proteins in the WCL and 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 h, then collected and subjected to co-IP with anti-FLAG antibody. The WCL 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.

IBTK promotes non-degradative ubiquitination of eIF4A1.

(A) WB analysis of the indicated proteins in the WCLs from FLAG-BirA*-IBTK Tet-on-inducible T-REx 293 cells treated with 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 h, then the WCLs were prepared for 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.

IBTK promotes nascent protein synthesis and cap-dependent translation initiation.

(A) WB analysis of the indicated proteins in the WCLs from parental and IBTK-KO SiHa cells treated with silvestrol (100 nM, 24 h) 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 h) and puromycin (1.5 μM, 10 min).

(C) Representative IF images of parental and IBTK-KO SiHa cells treated with AS (100 μM, 2 h) 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 ± S.D. (n ≥10).

(E) Representative IF images of HeLa cells transfected with the indicated plasmids, treated with DMSO or AS (100 μM, 2 h), 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) SGs in (E) were quantified by count of eIF4A1 puncta per cell (IBTK transfected vs non-transfected). Data are presented as means ± S.D. (n ≥10).

(G) Parental or IBTK-KO SiHa cells were transfected with the reporter pRΔDE·HCVF (pH) or pRΔDE·EMCVF (pE) for 24 h and the luciferase activities were measured. The counts of Firefly and Renilla were measured and shown in the graph. Data are presented as means ± S.D. (n = 3).

(H) 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-down proteins were subjected to WB analysis with the indicated antibodies.

P values are calculated using One-way ANOVA test in (D, F, G). ****p<0.0001, n.s. non-significant.

IBTK deficiency reduces eIF4A1-dependent oncoprotein expression and neoplastic phenotypes in cancer cells.

(A) WB analysis of the indicated proteins in the 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) 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 ± S.D. (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 h.

(D) CCK-8 cell proliferation analysis of parental and IBTK-KO SiHa cells. Data are shown as means ± S.D. (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 ± S.D. (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 ± S.D. (n=3). Scale bar, 50 μm.

(G) Sphere-formation analysis of parental and IBTK-KO SiHa cells. Representative pictures of SiHa cells after two 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 ± S.D. (n=3). Scale bar, 100 μm.

(H) Parental and IBTK-KD HeLa cells were injected 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 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.

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) WB analysis of the indicated proteins in the WCLs and co-IP samples of anti-FLAG antibody obtained from 293T cells transfected with the indicated plasmids.

(E) Co-IP using anti-IBTK antibodies 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℃ for 1 h. 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 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 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 h.

(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 h, 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 h.

(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.

Deficiency in mTORC1/S6K1-mediated eIF4A1 phosphorylation reduces eIF4A1-dependent oncoprotein expression and neoplastic phenotypes in cancer cells.

(A) 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 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-IP assays using anti-eIF4A1 antibody in the WCLs prepared from parental and IBTK-KO SiHa cells followed by WB analysis with the indicated antibodies.

(D) 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) 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 ± S.D. (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 ± S.D. (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 on in (I). Data are shown as means ± S.D. (n=3). Scale bar, 100 μm.

(J) Parental and IBTK-KO SiHa cells were treated with silvestrol (100 nM) for 24 h. 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 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.

Overexpression of 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 CESC specimens from TCGA cohort (n=295).

(B, C) IHC analysis of IBTK protein expression in 35 CESC tissues and 35 adjacent normal cervical tissues. 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 overall survival 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.