Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling.

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: Fig. 1. TGFBR2 is a BUB1 kinase. (A) Radio-phosphoimages of in vitro kinase assay where His-tagged TGFBR1-WT and TGFBR1-cyto domain only (purified using Ni-NTA beads from HEK293T cells) were used as kinases. Varying concentrations of Sf9 baculovirally purified BUB1-WT (200 ng, 500 ng, 1 mg/lane) and BUB1-KD (1 mg/lane) were used as substrates. The reactions were run at 37 C for 3 h with 5–10 mCi 32p-ATP. Laemmli quenched reactions were run on SDS-PAGE gels and imaged on phosphoimager. Arrows point to the proteins as detected by protein-specific antibodies on the western blots. (B), Sf9 purified BUB1 WT (1 mg in lanes 2 &3, 2 mg in lane 4, 500 ng lane 5), BUB1-KD (1 mg), or BUB1-E (1 mg) were used as substrates while baculovirally purified GST-TGFBR2 (200 ng/lane in all lanes except lane 5 where 500 ng) was used as a kinase in in vitro kinase reactions run at 37 C for 3 h with 5–10 mCi hot-ATP. A representative radio-phosphoimage is shown. Numbers below the lanes show average fold enrichment (of BUB1-KD phosphorylation) from three different replicates. Arrows point to the proteins detected with specific antibodies in western- blots. Bub3 and BUB1-E are undetectable in radio-phosphoimages. (C) In vitro kinase assay where BUB1-WT (200 ng), GST-TGFBR2 (1 mg) along with BUB1 inhibitor 2OH-BNPP1 (10 mM) was used. The in vitro kinase reaction was performed in same conditions as in Fig. 1A and B. (D) In vitro kinase assay where purified BUB1-WT (100 ng) was used as a kinase and histone-H2A (500 ng) was used as a substrate in the presence of 10 mM 2OH- BNPP1. Radio-phosphoimage shows 2OH-BNPP1 inhibits BUB1 kinase activity in this assay.

Article Snippet: BUB1 (#TA306432) was from Origene.

Techniques: In Vitro, Kinase Assay, Purification, SDS Page, Western Blot, Phospho-proteomics, Activity Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling.

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: Fig. 3. Phosphorylation of BUB1 at Ser318 causes reduction in interaction with TGFBR1 and SMAD2. (A) HEK293T cells were transfected with Myc- BUB1-WT, S318A, S318D mutants and HA-tagged TGFBR2, serum starved and treated for an hour with TGF-b (5 ng/mL). Lysates were made 40– 48 h post-transfections. Immunoprecipitation was performed using Myc-tag antibodies and blots were probed with TGFBR2 and Myc-tag antibodies. (B) IP for TGFBRI and then blotting for Myc in lysates from HEK293T cells transfected with Myc-BUB1-WT, S318A, S318D mutants and His-tagged TGFBR1, serum-starved, and treated with TGF-b (5 ng/mL) for 1 h. (C) IP for FLAG and then blotting for Myc in lysates from HEK293T cells transfected with BUB1-WT, S318A and S318D mutants and FL-SMAD2, serum starved and treated with TGF-b (5 ng/mL) for 1 h.

Article Snippet: BUB1 (#TA306432) was from Origene.

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation

Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling.

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: Fig. 4. BUB1 may have multiple contact points for TGF-b signaling components. (A) IP for Myc and then blotting four TGFBR2 in lysates from HEK293T cells transfected with Myc-BUB1 truncation mutants (1–241, 242–481 and 482–723) and HA-TGFBR2, serum starved and treated with TGF-b (5 ng/mL) for 1 h. (B) IP for Myc and then blotting four His in lysates from HEK293T cells transfected with Myc-BUB1 truncation mutants and His-TGFBR1 cytoplasmic tail, synchronized in G2/M by nocodazole. (C) IP for FLAG and then blotting for Myc in lysates from HEK293T cells transfected with BUB1 truncation mutants and FL-SMAD2, serum starved and left untreated or treated with TGF-b (5 ng/mL) for 1 h.

Article Snippet: BUB1 (#TA306432) was from Origene.

Techniques: Transfection

Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling.

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: Fig. 5. TGFBR2 mediated phosphorylation of BUB1 reduces its interaction with TGFBR2 and SMAD2. (A) IP for Myc and then blotting for TGFBR2 in lysates from A549 cells transfected with Myc-BUB1 truncation mutant 241–282 and phospho-deficient (S318A) or phospho-mimicking (S318D) mutants along with HA-TGFBR2. Cells were serum starved and treated with TGF-b (5 ng/mL for 1 h) before harvesting. (B) IP for Myc and then blotting for SMAD2 in lysates from HEK293T cells transfected with Myc-BUB1 truncation mutant 241–282 and phospho-deficient (S318A) or phospho-mimicking (S318D) mutants along with FL-SMAD2. Cells were serum starved and treated with TGF-b (5 ng/mL for 1 hour) before harvesting.

Article Snippet: BUB1 (#TA306432) was from Origene.

Techniques: Phospho-proteomics, Transfection, Mutagenesis

Journal: Neoplasia (New York, N.Y.)

Article Title: TGFBR2 mediated phosphorylation of BUB1 at Ser-318 is required for transforming growth factor-β signaling.

doi: 10.1016/j.neo.2020.02.001

Figure Lengend Snippet: Fig. 7. A schematic representing the proposed model with steps involved in BUB1 mediated regulation of activated TGF-b signaling complex. (i) BUB1 is recruited to TGFBR1-TGFBR2 complex in response to ligand, (ii) BUB1 participates in the recruitment of SMAD2/3 to the receptor, and (iii) TGFBR2 phosphorylates BUB1 at S318, which triggers the disassembly of the activated complex.

Article Snippet: BUB1 (#TA306432) was from Origene.

Techniques:

Journal: Cancer research

Article Title: Pharmacologic abrogation of the mitotic spindle checkpoint by an indolocarbazole discovered by cellular screening efficiently kills cancer cells.

doi: 10.1158/0008-5472.CAN-08-3597

Figure Lengend Snippet: Figure 2. Go¨6976 is an inhibitor of the mitotic spindle checkpoint. A, Go¨6976 induces mitotic exit and proteasome-dependent proteolysis of cyclin B and securin. HCT116 cells were treated with nocodazole (N) or Taxol (T) and incubated with DMSO (D) or 2 Amol/L Go¨6976 (G) in the presence or absence of 200 Amol/L ALLN (A). The mitotic index was determined (left) and the protein levels of cyclin B and securin were determined on Western blot (right). B, Go¨6976 induces premature sister chromatid separation. Nocodazole-arrested HCT116 cells were treated with DMSO or Go¨6976 for 10 and 15 min and metaphase spreads were prepared. Examples of typical metaphase spreads and the quantitation of cells showing premature sister chromatid separations (n = 400) are shown. C, Go¨6976 reduces kinetochore localization of Bub1 and BubR1. HCT116 cells were treated with nocodazole to arrest cells in mitosis followed by incubation with 20 Amol/L MG132 and 2 Amol/L Go¨6976 for 2 h. Cells were fixed and immunofluorescence staining was performed for Bub1, BubR1, and CREST and chromosomes were stained with Hoechst dye. Pixel intensities for Bub1 and BubR1 at kinetochores were quantified relative to the CREST signals. The graphs represent quantitations from 80 kinetochores from six cells. D, Go¨6976 inhibits phosphorylation of Bub1, Mps1, Aurora-A, and Aurora-B. Cells were treated as in C in the presence or absence of ALLN and the indicated mitotic proteins were detected on Western blots. For each cell population, the mitotic index was determined.

Article Snippet: Cell lysates were prepared, and SDS-PAGE, semidry Western blotting, and detection were performed as described (11) using the following antibodies: Bub1 (Bethyl; Stephen Taylor, University of Manchester, Manchester, United Kingdom), BubR1 (Chemicon; Stephen Taylor), Bub3 (BD Biosciences), Mps1 (Santa Cruz Biotechnology), Plk1 (Santa Cruz Biotechnology), Aurora-A (Santa Cruz Biotechnology; pT288: Cell Signaling), Aurora-B (BD Biosciences; pT232: Cell Signaling), cyclin B (Santa Cruz Biotechnology), tubulin (Sigma), actin (Sigma), securin (NeoMarkers), histone H3 (pS-10; Cell Signaling), and poly(ADP-ribose) polymerase (PARP; Pharmingen).

Techniques: Incubation, Western Blot, Quantitation Assay, Immunofluorescence, Staining, Phospho-proteomics