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The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-β signaling

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Science Signaling  06 Jan 2015:
Vol. 8, Issue 358, pp. ra1
DOI: 10.1126/scisignal.2005379
  • Fig. 1 BUB1 mediates TGF-β ligand–dependent SMAD2/3 phosphorylation, as well as MAPK and AKT activation.

    (A and B) Plot of the induction of a TGFBR1 reporter (BTR) in A549 cells (A) and MDA-231-1833–BTR (B) cells transfected with siRNAs targeting human kinases. Red or blue triangles and circles indicate high-stringency (triangles) or low-stringency (circles) hits either in plate- and experiment-wise analyses (red) or in experiment-wise analysis only (blue). Data are representative of three independent experiments. (C) Heat map of low-stringency hits in A549-BTR and MDA-231-1833–BTR cells from triplicate experiments. Gene name in italics indicates common hits. (D) Western blot analysis of TGF-β effector molecules in A549, NCI-H358, and MDA-231-1833 cells transfected with siRNA against either BUB1 (siBUB1) or TGFBRI (siTGFBRI) or a scrambled control siRNA [nonsilencing siRNA (NSS)] in the presence of TGF-β ligand for 1 hour. Blots are representative of three independent experiments.

  • Fig. 2 BUB1 promotes the recruitment of SMAD3 to TGFBRI, SMAD2/3-SMAD4 complex formation, and transcriptional response.

    (A and B) Immunoprecipitation (IP) for the TGFBRI antibody and then immunoblotting (IB) for the Flag or His tags in HEK293T (A) and A549 (B) cells transfected with control siRNA (NSS), TGFBRI siRNA, or BUB1 siRNA along with Flag-tagged SMAD3 (FL-SMAD3) and 6XHis-tagged TGFBRI (His-TGFBRI) and treated with TGF-β (1 hour). (C) IP for SMAD2/3 followed by blotting for SMAD4 in lysates from A549 cells transfected with control siRNA, TGFBR1 siRNA, or BUB1 siRNA and treated with TGF-β (1 hour). (D) Relative firefly luciferase activity (normalized to Gaussia luciferase) after addition of TGF-β (10 ng/ml) and transfection with mock (NSS) or BUB1 siRNA for 24 to 72 hours in A549 cells transiently transfected with an SBE4-Luc reporter and GLuc plasmids. (E) Immunoblotting for luciferase and BUB1 in lysates from A549 cells transfected and treated as in (D) and harvested 24 hours after TGF-β treatment. (F to H) Relative luciferase activity in NCI-H358 (F), MDA-231-1833 (G), and HeLa (H) transfected as in (D) and treated with TGF-β (10 ng/ml) for 24 hours. Blots are representative of three independent experiments. Data are means ± SEM of three independent experiments. **P < 0.001, two-sided Student’s t test.

  • Fig. 3 BUB1 inhibitor 2OH-BNPP1 abrogates TGF-β signaling in a dose-dependent manner.

    (A) Immunoblotting for total and phosphorylated (p) proteins as indicated in lysates from A549 cells transiently transfected with control siRNA (NSS) or BUB1 siRNA along with wild-type BUB1 (Myc-BUB1 WT) or a kinase-deficient mutant (Myc-BUB1 KD), serum-starved, and then treated with TGF-β (10 ng/ml, 1 hour). (B) Relative luciferase activity after addition of TGF-β in A549 cell cultures described in (A) expressing the SBE4-Luc and GLuc plasmids. Data are means ± SEM of three independent experiments. (C to E) Immunoblots of lysates from A549 (C), NCI-H358 (D), and MDA-231-1833 (E) cells treated with vehicle, vehicle and TGF-β (10 ng/ml), or TGF-β and the indicated concentration of 2OH-BNPP1 for 1 hour. (F and G) IP for SMAD2/3 and then blotting for SMAD4 in lysates from A549 (F) and NCI-H358 (G) cells treated with vehicle, vehicle and TGF-β, or TGF-β and either SB431542 or 2OH-BNPP1 (10 μM) for 1 hour. (H and I) Relative luciferase activity in A549 (H) and NCI-H358 (I) cells treated as in (F) and (G) for 24 hours. Data are means ± SEM of three independent experiments. **P < 0.001, two-sided Student’s t test. Blots are representative of three independent experiments; blots from (C) to (E) are quantified in fig. S7.

  • Fig. 4 BUB1 colocalizes with TGFBRI, coimmunoprecipitates with TGFBRI and TGFBRII, and promotes heteromeric TGFBRI/II complex formation.

    (A) TIRF analysis of BUB1 and TGFBRI colocalization in A549 cells treated with TGF-β (10 ng/ml) for 72 hours. Scale bar, 10 μm. (B) Line scan across BUB1 and TGFBRI particles within inset of (A). (C) Extent of colocalization normalized to the mock sample. Data are means ± SEM of three independent experiments (1000 particles, >20 cells). (D) IP for TGFBR1 and then blotting for Myc in lysates from A549 cells transfected with Myc-BUB1 and/or His-TGFBRI, and treated with TGF-β. 1 hour: 10 ng/ml for 1 hour. (E) IP for His and then blotting for Myc in lysates from A549 cells transfected with Myc-BUB1 and His-tagged cytosolic domain of TGFBRI. (F) IP for TGFBRI and then blotting for TGFBRII and SARA in lysates from A549 cells transfected with control or BUB1 siRNA, serum-starved, and treated with TGF-β. (G) IP for TGFBRI and then blotting for His and TGFBRII in lysates from A549 cells transfected with His-TGFBRI, serum-starved, and treated with SB431542 or 2OH-BNPP1 (1 hour) and then TGF-β. (H) IP for TGFBRI and then blotting for Myc and TGFBRII in lysates from A549 cells transfected with His-TGFBRI and Myc-BUB1, serum-starved, and treated with SB431542 or 2OH-BNPP1 (1 hour) and then TGF-β. (I) IP for Myc and then blotting for TGFBRII in lysates from HEK293T cells transfected with Myc-BUB1 and His-TGFBRI, serum-starved, and treated with 2OH-BNPP1 or SB431542 (10 μM, 1 hour) and then TGF-β. Blots are representative of two (I) or at least three (D to H) independent experiments.

  • Fig. 5 BUB1 inhibitor blocks TGF-β signaling in vivo.

    (A) Representative immunohistochemistry staining for phosphorylated SMAD2 in A549 xenografts harvested from SCID mice 4 hours after treatment with 2OH-BNPP1 (50 mg/kg), SB431542 (10 mg/kg), or vehicle [dimethyl sulfoxide (DMSO)]. Scale bar, 200 μm. (B) Number of cells staining positive for nuclear phosphorylated SMAD2 in control (n = 4), SB431542-treated (n = 2), or 2OH-BNPP1–treated (n = 5) mice bearing tumors. Data are means ± SEM, cell counts from three random fields for each tumor. **P < 0.001, two-sided Student’s t test.

  • Fig. 6 A model for a role of BUB1 in TGF-β signaling.

    On the basis of our findings, we propose that BUB1 forms a ternary complex with TGFBRI and TGFBRII. Our data suggest that the interaction of BUB1 with TGF-β receptors is enhanced upon TGF-β stimulation, requires the kinase activity of BUB1, and promotes stabilization of the heteromeric complex between TGFBRI-TGFBRII, R-SMAD recruitment, and subsequent canonical and noncanonical TGF-β signaling cascades.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/358/ra1/DC1

    Fig. S1. siRNA screening and pathway analysis.

    Fig. S2. BUB1 mediates TGF-β–dependent SMAD2/3 phosphorylation.

    Fig. S3. Depletion of BUB1 leads to reduced SMAD3 recruitment to TGFBRI and TGF-β–dependent accumulation of SMAD2 in the nucleus.

    Fig. S4. Depletion of BUB1 attenuates TGF-β–dependent SMAD2/3-SMAD4 complex formation in A549 cells.

    Fig. S5. Depletion of BUB1 inhibits TGF-β–mediated EMT in A549 and NCI-H358 cells.

    Fig. S6. Depletion of BUB1 inhibits TGF-β–mediated migration and invasion of A549 cells.

    Fig. S7. BUB1 kinase activity mediates TGF-β–dependent phosphorylation and nuclear activity of R-SMAD.

    Fig. S8. TGFBRI and BUB1 colocalization by TIRF microscopy at 1 and 24 hours and coimmunoprecipitation of His-TGFBRI and Myc-BUB1.

    Fig. S9. BUB1 kinase activity mediates TGFBRI-TGFBRII interaction.

    Fig. S10. TGFBRI is not a direct substrate of BUB1 kinase activity.

    Fig. S11. Wild-type Myc-BUB1 interacts with FL-SMAD2 in HEK293T cells.

    Fig. S12. SMAD3 is not a direct substrate of BUB1 kinase activity.

    Table S1. Human kinome siRNA screen in A549-BTR and MDA-231-1833–BTR cells.

    Table S2. Hits obtained in A549-BTR and MDA-231-1833–BTR human kinome screen.

    Table S3. Pathway impact analysis based on the fold induction of the BTR reporter.

  • Supplementary Materials for:

    The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-β signaling

    Shyam Nyati, Katrina Schinske-Sebolt, Sethuramasundaram Pitchiaya, Katerina Chekhovskiy, Areeb Chator, Nauman Chaudhry, Joseph Dosch, Marcian E. Van Dort, Sooryanarayana Varambally, Chandan Kumar-Sinha, Mukesh Kumar Nyati, Dipankar Ray, Nils G. Walter, Hongtao Yu, Brian Dale Ross, Alnawaz Rehemtulla*

    *Corresponding author. E-mail: alnawaz{at}med.umich.edu

    This PDF file includes:

    • Fig. S1. siRNA screening and pathway analysis.
    • Fig. S2. BUB1 mediates TGF-β–dependent SMAD2/3 phosphorylation.
    • Fig. S3. Depletion of BUB1 leads to reduced SMAD3 recruitment to TGFBRI and TGF-β–dependent accumulation of SMAD2 in the nucleus.
    • Fig. S4. Depletion of BUB1 attenuates TGF-β–dependent SMAD2/3-SMAD4 complex formation in A549 cells.
    • Fig. S5. Depletion of BUB1 inhibits TGF-β–mediated EMT in A549 and NCI-H358 cells.
    • Fig. S6. Depletion of BUB1 inhibits TGF-β–mediated migration and invasion of A549 cells.
    • Fig. S7. BUB1 kinase activity mediates TGF-β–dependent phosphorylation and nuclear activity of R-SMAD.
    • Fig. S8. TGFBRI and BUB1 colocalization by TIRF microscopy at 1 and 24 hours and coimmunoprecipitation of His-TGFBRI and Myc-BUB1.
    • Fig. S9. BUB1 kinase activity mediates TGFBRI-TGFBRII interaction.
    • Fig. S10. TGFBRI is not a direct substrate of BUB1 kinase activity.
    • Fig. S11. Wild-type Myc-BUB1 interacts with FL-SMAD2 in HEK293T cells.
    • Fig. S12. SMAD3 is not a direct substrate of BUB1 kinase activity.
    • Legends for tables S1 and S2
    • Table S3. Pathway impact analysis based on the fold induction of the BTR reporter.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 982 KB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Human kinome siRNA screen in A549-BTR and MDA-231-1833–BTR cells.
    • Table S2 (Microsoft Excel format). Hits obtained in A549-BTR and MDA-231-1833–BTR human kinome screen.

    [Download Tables S1 and S2]


    Citation: S. Nyati, K. Schinske-Sebolt, S. Pitchiaya, K. Chekhovskiy, A. Chator, N. Chaudhry, J. Dosch, M. E. Van Dort, S. Varambally, C. Kumar-Sinha, M. K. Nyati, D. Ray, N. G. Walter, H. Yu, B. D. Ross, A. Rehemtulla, The kinase activity of the Ser/Thr kinase BUB1 promotes TGF-β signaling. Sci. Signal. 8, ra1 (2015).

    © 2014 American Association for the Advancement of Science

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