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: Molecular cancer research : MCR

Article Title: AXL inhibition induces DNA damage and replication stress in non-small cell lung cancer cells and promotes sensitivity to ATR inhibitors

doi: 10.1158/1541-7786.MCR-20-0414

Figure Lengend Snippet: A. Sensitivity of a panel of lung cancer cell lines to BGB324 (ranked by IC50). Dashed line indicates median IC50 (2μM). AXL mRNA levels, AXL protein expression determined by RPPA, and TP53 mutation status are indicated below. B. Expression of markers of DNA damage (γH2Ax) and RS (pCHK1, pRA32) by western blotting, following treatment with indicated concentrations of BGB324 for 24 h, in TP53-deficient lung cancer cell lines. β-Actin was used as loading control. Representative immunoblots of at least 2 independent experiments are shown. C. AXL was silenced in Calu-1, H2250 and H1299 cells using 3 different siRNA sequences. Increase in CHK1 phosphorylation and γH2Ax accumulation 48 h post-AXL depletion was observed by western blotting. D. Calu-1 and H1299 cells were treated with 1μM BGB324 for indicated durations. Expression of the different DDR pathway mediators and Vinculin (loading control) was analyzed by western blotting. Relative band intensities, quantified by Image J and normalized to loading control, are indicated below the blot. E. Inhibition of AXL phosphorylation in Calu-1 and H1299 cells by BGB324. Phosphorylated proteins were immunoprecipitated using Phospho tyrosine antibody and immunoblotted for AXL. F. Immunoblots show limited γH2Ax induction and CHK1 phosphorylation in TP53-wildtype lung cancer cell lines.

Article Snippet: Antibodies for western blotting purchased from Cell signaling (Danvers, MA): phospho-CHK1 (S345) (2348, 1:1000), CHK1 (2360, 1:1000), γH 2 A x (9718, 1:1000), AXL (8661, 1:1000), Cyclin B1 (4138), phospho-cdc2 (Y15) (4539), phospho-Histone H3 (S10) (53348), phospho-AKT (S473) (4060), AKT (9272), phospho-S6 (S240/244) (2215), S6 (2217), p21 (2946); Bethyl laboratories : RPA32 (A300–244, 1:1000), phospho-RPA32 (S4/S8) (A300–245, 1:1000), phospho-RPA32 (S33) (A300–246, 1:1000) and phospho-KAP1 (S824) (A300–246, 1:1000); Santa Cruz Biotechnology (Dallas, TX) : β-actin (sc-47778, 1:2000), GAPDH (sc-20357, 1:2000).

Techniques: Expressing, Mutagenesis, Western Blot, Inhibition, Immunoprecipitation

Journal: Molecular cancer research : MCR

Article Title: AXL inhibition induces DNA damage and replication stress in non-small cell lung cancer cells and promotes sensitivity to ATR inhibitors

doi: 10.1158/1541-7786.MCR-20-0414

Figure Lengend Snippet: A. Comparison of AXL protein expression between lung cancer cell lines sensitive (IC50<3μM) and resistant (IC50>3μM) to the ATR inhibitors, VX-970 and AZD6738. Fold change, F.C. and p value by Welch’s t-test are indicated. B. Relative proliferation of Calu-1, H2250 and H1299 cell lines following 4–5 day treatment with BGB324, VX-970 or their combination at indicated concentrations, as measured by CellTiter-Glo viability assay. Data are mean±s.e.m. ΔAUC value denoting shift in dose response curve of the drug combination beyond the predicted additive effect of the single agents was calculated using the BLISS independence model. ΔAUC<−0.1 indicate a greater than additive effect of the combination. C. Clonogenic survival of Calu-1 and H1299 cells following treatment with DMSO, BGB324, VX-970 or their combination. Colonies stained with 025% crystal violet after 14 days. Representative image from two independent experiments shown. D. Heatmap depicts the effect of combination (calculated as ΔAUC by BLISS model) of BGB324 with various DDR inhibitors – ATM inhibitor (AZD0156), CHK1 inhibitor (LY2606368), WEE1 inhibitor (AZD1775) and DNAPKC inhibitor (NU7441) in Calu-1 and H1299 cells. E. Heatmap shows proteins in the AXL/PI3K/AKT/mTOR signaling pathway, determined by RPPA, following 24 h treatment of Calu-1 cells with DMSO, BGB324, VX-970 or their combination (1μM), significantly altered by ANOVA comparison at FDR=0.01 cutoff.

Article Snippet: Antibodies for western blotting purchased from Cell signaling (Danvers, MA): phospho-CHK1 (S345) (2348, 1:1000), CHK1 (2360, 1:1000), γH 2 A x (9718, 1:1000), AXL (8661, 1:1000), Cyclin B1 (4138), phospho-cdc2 (Y15) (4539), phospho-Histone H3 (S10) (53348), phospho-AKT (S473) (4060), AKT (9272), phospho-S6 (S240/244) (2215), S6 (2217), p21 (2946); Bethyl laboratories : RPA32 (A300–244, 1:1000), phospho-RPA32 (S4/S8) (A300–245, 1:1000), phospho-RPA32 (S33) (A300–246, 1:1000) and phospho-KAP1 (S824) (A300–246, 1:1000); Santa Cruz Biotechnology (Dallas, TX) : β-actin (sc-47778, 1:2000), GAPDH (sc-20357, 1:2000).

Techniques: Expressing, Viability Assay, Staining

Journal: PLoS ONE

Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

doi: 10.1371/journal.pone.0011994

Figure Lengend Snippet: ( A ) H1229 cells treated with empty vector and IC 20 of cisplatin (3.0 µM), NPRL2, or NPRL2+ with IC 20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment and analyzed for expression of Chk1, P-Chk1 (Ser345), Chk2, and P-Chk2 (Thr68). In cells treated with empty vector and IC 20 dose of cisplatin or NPRL2, P-Chk1 increased in a time-dependent manner. In contrast, in those treated with NPRL2 and IC 20 dose of cisplatin, P-Chk1 was dramatically enhanced. Although P-Chk2 was not detected in H1299 cells treated with empty vector and IC 20 dose of cisplatin, it was clearly increased in a time-dependent manner in those treated with NPRL2 or NPRL2+ IC 20 dose of cisplatin. ( B ) P-Chk2 expression was immunohistochemically analyzed in an orthotopic model of H322 pleural dissemination, as described in the section. P-Chk2 expression was slightly detected in pleural tumor cells from mice treated with NPRL2 nanoparticles, but not in those treated with LacZ or LacZ + cisplatin. In contrast, treatment by NPRL2 nanoparticles and cisplatin induced high phospho-Chk2 expression in tumor cells. Magnification: ×400. ( C ) The kinase activity of Chk1 and Chk2 in H1299 cells treated with empty vector + IC 20 dose of cisplatin, NPRL2 or NPRL2+ IC 20 dose of cisplatin was analyzed with use of a K-LISA Checkpoint Activity Kit, an enzyme-linked immunosorbent assay (ELISA)-based activity assay. Chk1 kinase activity was greater in cells treated with NPRL2+ cisplatin than in cells treated with empty vector or empty vector + cisplatin ( P <0.003). Chk2 kinase activity was more strongly enhanced in cells treated with NPRL2 or NPRL2+ cisplatin than in cells treated with empty vector or empty vector + cisplatin ( P <0.0001). Bars, SDs of the mean in four individual experiments.

Article Snippet: Anti-phospho-Chk1 (Ser345), anti-phospho-Chk2 (Thr68), anti-phospho-Chk1 (Ser345), anti-NBS1, anti-phospho-NBS1 (Ser343), anti-SMC1, anti-phospho-Cdc25C (Ser216), anti-phospho-Cdc2 (Tyr15), anti-caspase-3, and anti-caspase-9 were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Plasmid Preparation, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

doi: 10.1371/journal.pone.0011994

Figure Lengend Snippet: NPRL2+ cisplatin ( cis -diamminedichloroplatinum (II) [CDDP]) treatment effector caspase, caspase-3, is activated and PARP is cleaved. Therefore, our result that the combination treatment of NPRL2 and CDDP activates a caspase cascade and hyperphosphorylates H2AX suggests that this combination treatment can strongly enhance the apoptotic pathway. Adding NPRL2 treatment to CDDP significantly enhanced Chk2 and Chk1 kinase activity, and NPRL2+ CDDP treatment remarkably degraded the interaction of Cdc2 and cyclin B1, leading to the inactivation of the Cdc2/cyclin B1 complex and arrest in G2/M. The cells arrested at the G2/M phase ultimately promote apoptosis with characteristic nuclear fragmentation. This combination treatment strongly inactivates Cdc25A and Cdc25C, and phospho-Cdc2 (Tyr-15), an inactivated type of Cdc2, is clearly increased in NPRL2+ CDDP treatment.

Article Snippet: Anti-phospho-Chk1 (Ser345), anti-phospho-Chk2 (Thr68), anti-phospho-Chk1 (Ser345), anti-NBS1, anti-phospho-NBS1 (Ser343), anti-SMC1, anti-phospho-Cdc25C (Ser216), anti-phospho-Cdc2 (Tyr15), anti-caspase-3, and anti-caspase-9 were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activity 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: 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