Journal: Cell Death and Differentiation

Article Title: Hornerin mediates phosphorylation of the polo-box domain in Plk1 by Chk1 to induce death in mitosis

doi: 10.1038/s41418-023-01208-y

Figure Lengend Snippet: A – C HeLa cells treated with 0.5 µM B[a]P for 48 h were analyzed by live cell imaging for 10 h ( A , n = 1609 cells). DiM ( B , n = 356) and the fate of cells with multipolar spindles ( C , n = 257) were analyzed. D, E After treatment with the indicated genotoxic stresses, HeLa cells were stained with the indicated antibodies, and the number of prometaphase cells with multipolar spindles was determined from 300 prometaphase cells in three independent experiments. F, G Twenty-four hours after treatment with 2 µM NU6027 as an ATR inhibitor (ATRi), 20 nM KU55933 as an ATM inhibitor (ATMi), 300 nM UCN-01 as a Chk1 inhibitor (Chk1i), 1 µM BML-277 as a Chk2 inhibitor (Chk2i), or 0.5 µM KU57788 as a DNA-PK inhibitor (DNA-PKi), HeLa cells were treated with 0.5 µM B[a]P for 48 h or 0.5 µM etoposide for 24 h, and multipolar spindles were analyzed ( n = 400 centrioles from three independent experiments). Scale bars, 5 µm. Error bars, SEM. * p < 0.01 (two-tailed t test).

Article Snippet: For the Chk1 kinase assay, 200 ng of human recombinant Chk1 kinase (SignalChem) and Aurora A (purified from asynchronous sf9 cells) were incubated with 1 μg of His-Plk1 WT, T210A, S526A, S529A, or T539A mutant and 10 μM ATP in 50 μl of kinase buffer (25 mM Tris-HCl (pH 7.5), 2 mM DTT, 10 mM MgCl2, 5 mM β-glycerophosphate, and 0.1 mM Na3VO4) for 30 min at 37 °C.

Techniques: Live Cell Imaging, Staining, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Hornerin mediates phosphorylation of the polo-box domain in Plk1 by Chk1 to induce death in mitosis

doi: 10.1038/s41418-023-01208-y

Figure Lengend Snippet: A – C After transfection of plasmids and B[a]P treatment, the localization of myc-Plk1 and multipolar spindles was analyzed ( n = 300). D, E Chemically treated HeLa cells were analyzed by immunoblotting. F Cells were treated with the indicated inhibitors and B[a]P and analyzed by immunoblotting. G, H WT or mutant His-Plk1 was incubated with active Chk1 and analyzed by immunoblotting. I Recombinant Chk1 was incubated with recombinant His-Plk1 and proteins pulled down with Ni-beads were analyzed by immunoblotting. J After treatment with 0.5 µM B[a]P for 48 h or 300 ng/ml nocodazole for 16 h, cells were treated with 1 µM RO3306 as a Cdk1 inhibitor for 5 h, 1 µM BI2536 as a Plk1 inhibitor for 1 h, 5 µM VX680 as an Aurora A inhibitor for 1 h, and 2 µM Hesperadin as an Aurora B inhibitor for 5 h. Cell lysates were analyzed by immunoblotting. K His-Plk1 was incubated with Aurora A, pulled down with Ni + -beads, and analyzed by immunoblotting. L Lysates from DNA-transfected HeLa cells were incubated with recombinant His-Sgo1 and proteins pulled down with Ni-beads were analyzed by immunoblotting. Images of uncropped blots and gels are provided as a Supplementary Material file. Scale bar, 5 µm. Error bars, SEM. * p < 0.01 (two-tailed t test).

Article Snippet: For the Chk1 kinase assay, 200 ng of human recombinant Chk1 kinase (SignalChem) and Aurora A (purified from asynchronous sf9 cells) were incubated with 1 μg of His-Plk1 WT, T210A, S526A, S529A, or T539A mutant and 10 μM ATP in 50 μl of kinase buffer (25 mM Tris-HCl (pH 7.5), 2 mM DTT, 10 mM MgCl2, 5 mM β-glycerophosphate, and 0.1 mM Na3VO4) for 30 min at 37 °C.

Techniques: Transfection, Western Blot, Mutagenesis, Incubation, Recombinant, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: Hornerin mediates phosphorylation of the polo-box domain in Plk1 by Chk1 to induce death in mitosis

doi: 10.1038/s41418-023-01208-y

Figure Lengend Snippet: Upon mitotic entry with DNA damage, Aurora A phosphorylates Ser 526 for normal mitotic progression but hornerin mediates additional phosphorylations by Chk1 at Ser 529 and The 539 in Plk1 to induce multipolar spindle and concomitant mitotic catastrophe. KD kinase domain.

Article Snippet: For the Chk1 kinase assay, 200 ng of human recombinant Chk1 kinase (SignalChem) and Aurora A (purified from asynchronous sf9 cells) were incubated with 1 μg of His-Plk1 WT, T210A, S526A, S529A, or T539A mutant and 10 μM ATP in 50 μl of kinase buffer (25 mM Tris-HCl (pH 7.5), 2 mM DTT, 10 mM MgCl2, 5 mM β-glycerophosphate, and 0.1 mM Na3VO4) for 30 min at 37 °C.

Techniques:

Journal:

Article Title: ATR couples FANCD2 monoubiquitination to the DNA-damage response

doi: 10.1101/gad.1196104

Figure Lengend Snippet: ATR is required for damage-inducible monoubiquitination of FANCD2. (A) ATR siRNA A strongly inhibits MMC-induced monoubiquitination of FANCD2 in HeLa and U2OS cells. Silencing of FANCA suppresses FANCD2 monoubiquitination either with or without exposure to MMC. (B) Silencing of ATR with siRNA in U2OS cells suppresses FANCD2 monoubiquitination following exposure to HU, MMC, or IR for the indicated times. Suppression of ATR activity is demonstrated by decreased phosphorylation of Chk1 on S345 in response to silencing. (C) Silencing of ATM does not decrease FANCD2 monoubiquitination. (D) Flow cytometric analysis demonstrates that inhibition of damage-induced monoubiquitination of FANCD2 by suppression of ATR is not due to an alteration of cell-cycle progression. Histograms of DNA content are displayed, with S-phase index measured by BrdU incorporation as described (Andreassen et al. 2001), inset in each histogram.

Article Snippet: Antibodies included anti-FANCA (1:1000) and anti-FANCD2 (E35, 1: 1000; Garcia-Higuera et al. 2001 ), anti-ATR (1:1000 Santa Cruz [N19]), anti-ATM (1:500, Novus), anti-RPA1 (1:200 Oncogene Research), anti-phospho-S345-Chk1 (1:1000, Cell Signaling), and anti-Chk1 (1:500 Santa Cruz [G4]).

Techniques: Activity Assay, Inhibition, BrdU Incorporation Assay

Journal: BMC Cancer

Article Title: Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition

doi: 10.1186/s12885-015-1387-6

Figure Lengend Snippet: Enhanced TH-302 cytotoxicity by Chk1 inhibitors is only observed in p53-deficient but not p53-proficient cells

Article Snippet: Antibodies against phospho-Histone H3, phospho-Cdc2 Y15 antibody, total Chk1, phospho-Chk1 (S296) were from Cell Signaling.

Techniques:

Journal: BMC Cancer

Article Title: Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition

doi: 10.1186/s12885-015-1387-6

Figure Lengend Snippet: Chk1 inhibitors reduced TH-302-mediated HeLa cell-cycle arrest

Article Snippet: Antibodies against phospho-Histone H3, phospho-Cdc2 Y15 antibody, total Chk1, phospho-Chk1 (S296) were from Cell Signaling.

Techniques:

Journal: BMC Cancer

Article Title: Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition

doi: 10.1186/s12885-015-1387-6

Figure Lengend Snippet: Chk1 inhibitors reduced TH-302-mediated HT-29 cell-cycle arrest

Article Snippet: Antibodies against phospho-Histone H3, phospho-Cdc2 Y15 antibody, total Chk1, phospho-Chk1 (S296) were from Cell Signaling.

Techniques:

Journal: BMC Cancer

Article Title: Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition

doi: 10.1186/s12885-015-1387-6

Figure Lengend Snippet: (A) AZD7762 enhanced apoptosis in cells co-treated with TH-302. HT29 cells were exposed to TH-302 at the indicated concentrations and 0.1 μM of AZD7762 for 2 h under either normoxia (21% O 2 ) or hypoxia (N 2 ). After wash, cells were continuously cultured for additional 46 h in the presence of 0.1 μM AZD7762. Caspase 3/7 activity was detected using luminescence-based caspase activity assay kit. The specificity of caspase 3/7 activity was confirmed by including pan-caspase inhibitor ZVAD. (B) AZD7762 inhibited Chk1 autophosphorylation. HT29 cells were treated with TH-302, AZD7762, or combination of TH-302 and AZD7762 for 2 h, washed, and AZD7762 added back and incubated for an additional 46 h. Cell lysates were harvested and immunoblotted with antibodies against Chk1 autophosphorylation (S296) and total Chk1. Equal loading was confirmed by actin blot. (C) TH-302 AZD7762 co-treatment abrogated cell cycle arrest biomarkers and decreased Rad51 expression. Cells were treated as described in Figure 6B, and then immunoblotted with antibodies against cell-cycle biomarkers phospho-histone H3, phospho-Cdc2 Y15, total Cdc2 and Rad51. Equal loading was confirmed by actin blot. The blot data are the representative of two independent experiments.

Article Snippet: Antibodies against phospho-Histone H3, phospho-Cdc2 Y15 antibody, total Chk1, phospho-Chk1 (S296) were from Cell Signaling.

Techniques: Cell Culture, Activity Assay, Caspase Activity Assay, Incubation, Expressing

Journal: BMC Cancer

Article Title: Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition

doi: 10.1186/s12885-015-1387-6

Figure Lengend Snippet: Effects of AZD7762 and TH-302 on pharmacodynamic biomarkers in HT29 xenografts. (A) schematic diagram illustrating the study design. (B) representative images of γH2AX, Caspase 3 and Chk1-S345 immunostainings, respectively. Insets in γH2AX images, magnification of γH2AX positive cells showing the staining pattern was either pan-nuclear or foci-formation. (C, D and E) , morphometric analysis of γH2AX, Caspase 3 and Chk1-S345 positive cells. Scale bar, 100 μm; Each bar represents mean ± SEM of 5 animals per group. * p <0.05 vs. vehicle group; a , p <0.05 vs. AZD7762 group; b , p <0.05 vs. TH-302 group. # , p <0.05 vs. TAA group.

Article Snippet: Antibodies against phospho-Histone H3, phospho-Cdc2 Y15 antibody, total Chk1, phospho-Chk1 (S296) were from Cell Signaling.

Techniques: Staining

Journal: Cell Cycle

Article Title: Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1c β (PP1c β )

doi: 10.1080/15384101.2017.1418235

Figure Lengend Snippet: Interaction between Chk1 and MYPT1. (A) Chk1 was immunoprecipitated from HeLa cells, and samples were analyzed by SDS-PAGE with Coomassie blue staining. Chk1-interacting proteins were identified by mass spectrometry. Shown in the table are the proteins identified in the immunocomplex. The number of peptides and peptide coverage pertentage were indicated. (B) Chk1 immunoprecipitates were blotted with anti-Chk1 and anti-MYPT1 antibodies.(C) HeLa cells transfected with vector or HA-MYPT1, and Flag-Chk1 constructs were subjected to IP and IB assays using the antibodies indicated. Asterisks indicate IgG. (D) Domain structures of MYPT1. The PP1 binding motif, the ankyrin repeats, and the Leucine zipper domain, and the boundaries of MYPT1 fragments used in this study are indicated. (E) Bacterially expressed MYPT1 fragments were incubated with lysates from HeLa cells transfected with HA-Chk1. Asterisks indicate the corresponding bands.

Article Snippet: Briefly, recombinant Chk1 kinase was purchased from R & D systems (Cat. # 1630-KS), incubated with purified GST-MYPT1 with 1 M HEPES (pH 7.4), 1 M MgCl 2 , 1 M Dithiothreitol, 0.1 M Na 3 VO 4 , 0.1 mM ATP or 1 μCi of γ-[ 32 P]ATP.

Techniques: Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Transfection, Plasmid Preparation, Construct, Binding Assay, Incubation

Journal: Cell Cycle

Article Title: Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1c β (PP1c β )

doi: 10.1080/15384101.2017.1418235

Figure Lengend Snippet: Ser20 is identified as one of the Chk1-dependent phosphorylation sites. (A) Recombinant Chk1 and MYPT1 were incubated in kinase buffers containing 32P-ATP, and an IVK assay was carried out. Coomassie blue staining showed input MYPT1 proteins and autoradiography showed phosphorylated GST-MYPT1. (B) LC-MS/MS analysis identified Ser20 of MYPT1 as one of the sites phosphorylated by Chk1 in vitro. From this collision-induced dissociation spectrum, a phosphorylated peptide WIG(pS)ETDLEPPVVK of MYPT1 was identified following incubation with Chk1 in an IVK reaction. “b” and “y” ion series represent fragment ions containing the N- and C-termini of the peptide, respectively. (C-D) S20A mutant was constructed in GST-MYPT1-FL (C) and -F1 (D) fragment, and IVK assays were carried out using WT and S20A of GST-MYPT1-FL or F1 proteins. (E) A comparison between Chk1 phosphorylation consensus sites and the adjacent amino acids of MYPT1 Ser20.

Article Snippet: Briefly, recombinant Chk1 kinase was purchased from R & D systems (Cat. # 1630-KS), incubated with purified GST-MYPT1 with 1 M HEPES (pH 7.4), 1 M MgCl 2 , 1 M Dithiothreitol, 0.1 M Na 3 VO 4 , 0.1 mM ATP or 1 μCi of γ-[ 32 P]ATP.

Techniques: Phospho-proteomics, Recombinant, Incubation, Staining, Autoradiography, Liquid Chromatography with Mass Spectroscopy, In Vitro, Mutagenesis, Construct, Comparison

Journal: Cell Cycle

Article Title: Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1c β (PP1c β )

doi: 10.1080/15384101.2017.1418235

Figure Lengend Snippet: Ser20 of MYPT1 is phosphorylate by Chk1. (A) HeLa cells were transfected with Flag-Chk1 or Flag-Chk1-kinase dead (KD), and then IPed with anti-Flag antibodies. The immunoprecipitates were used in an IP-kinase assay using recombinant GST-MYPT1 as substrates. The reaction was then terminated and blotted with antibodies indicated. (B) IVK assays using recombinant His-Chk1 and GST-MYPT1 or GST-MYPT1-S20A as substrates. The products were then blotted with pS20 antibodies. (C) Ser20 of MYPT1 was phosphorylated in vivo. HeLa cells transfected with HA-MYPT1 were treated with Noc, Noc plus UV, Noc plus UV plus UCN-01 (an inhibitor of Chk1). Lysates from these cells were blotted with pS20 antibodies.

Article Snippet: Briefly, recombinant Chk1 kinase was purchased from R & D systems (Cat. # 1630-KS), incubated with purified GST-MYPT1 with 1 M HEPES (pH 7.4), 1 M MgCl 2 , 1 M Dithiothreitol, 0.1 M Na 3 VO 4 , 0.1 mM ATP or 1 μCi of γ-[ 32 P]ATP.

Techniques: Transfection, IP-Kinase Assay, Recombinant, In Vivo

Journal: Cell Cycle

Article Title: Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1c β (PP1c β )

doi: 10.1080/15384101.2017.1418235

Figure Lengend Snippet: pS20 promotes the degradation of MYPT1. (A) HeLa cells transfected with Flag-Chk1 and HA-Ub were treated with MG132 or left untreated, IPed with anti-MYPT1 antibodies and IBed with the antibodies indicated. (B) Cells were transfected with vector, WT or KD of Flag-Chk1 and HA-Ub plasmids, then analyzed as in (A). (C) Cells were treated with UCN-01 or left untreated, then analyzed as in A. (D) Cells were transfected with HA-MYPT1, treated with UCN-01 or left untreated, then incubated with CHX for the time indicated. (E) Quantitation of the results in (D). (F) Cells were transfected with WT or S20A of HA-MYPT1, then incubated with CHX. (G) Quantitation of the results in (F).

Article Snippet: Briefly, recombinant Chk1 kinase was purchased from R & D systems (Cat. # 1630-KS), incubated with purified GST-MYPT1 with 1 M HEPES (pH 7.4), 1 M MgCl 2 , 1 M Dithiothreitol, 0.1 M Na 3 VO 4 , 0.1 mM ATP or 1 μCi of γ-[ 32 P]ATP.

Techniques: Transfection, Plasmid Preparation, Incubation, Quantitation Assay

Journal: Cell Cycle

Article Title: Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1c β (PP1c β )

doi: 10.1080/15384101.2017.1418235

Figure Lengend Snippet: pS20 is essential for the interaction between MYPT1 and PP1cβ. (A) HeLa cells were transfected with HA-MYPT1-WT or S20A plasmids, then subject to IP and IB analysis as indicated. (B) Recombinant GST-MYPT1 and S20A proteins were used to pulldown transfected HA- PP1cβ. (C) HeLa cells were transfected with HA-MYPT1-WT or S20A plasmids and treated with Noc, then subject to IP with anti-HA antibodies, followed by IP-phosphatase assays using active Plk1 as substrates. The products were then blotted with anti-Plk1-pT210 antibodies. (D) A proposed model of how Chk1 inhibits Plk1 through MYPT1. Chk1 phosphorylates MYPT1 at Ser20 to promote the interaction between MYPT1 and PP1cβ. MYPT1 then targets PP1cβ to Plk1 to dephosphorylate Plk1 at pT210, and possibly other proteins.

Article Snippet: Briefly, recombinant Chk1 kinase was purchased from R & D systems (Cat. # 1630-KS), incubated with purified GST-MYPT1 with 1 M HEPES (pH 7.4), 1 M MgCl 2 , 1 M Dithiothreitol, 0.1 M Na 3 VO 4 , 0.1 mM ATP or 1 μCi of γ-[ 32 P]ATP.

Techniques: Transfection, Recombinant