recombinant chk1 kinase  (R&D Systems)

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: Fig. 1 DNA damage causes multipolar spindle formation via the DDR. 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, 10mM MgCl2, 5 mM β-glycerophosphate, and 0.1 mM Na3VO4) for 30min 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: Fig. 6 Hornerin mediates Plk1 phosphorylation by Chk1. A–D siRNA-transfected HeLa cells were treated with B[a]P and analyzed by IP- immunoblotting (A and E), immunostaining (B and C; n = 300), and immunoblotting (D). E siRNA-transfected HeLa cells were treated with B[a] P and analyzed by IP-immunoblotting. F Twenty-four hours after transfection of WT or mutant Plk1, the cell lysates were incubated with recombinant His-Plk1 and proteins pulled down with Ni-beads were analyzed by immunoblotting. G HeLa cells transfected with the indicated plasmid were subjected to IP-immunoblotting. H Hornerin-Plk1 3D proximity by in situ PLA in HeLa cells transfected with myc-Plk1 WT or mutants (n = 30). I HeLa cells were stained with hornerin (green), γ-tubulin (red), and DAPI (blue). Images of uncropped blots and gels are provided as a Supplementary Material file. AU, arbitrary units, 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, 10mM MgCl2, 5 mM β-glycerophosphate, and 0.1 mM Na3VO4) for 30min at 37 °C.

Techniques: Phospho-proteomics, Transfection, Western Blot, Immunostaining, Mutagenesis, Incubation, Recombinant, Plasmid Preparation, In Situ, Staining, Two Tailed Test

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1cβ (PP1cβ).

doi: 10.1080/15384101.2017.1418235

Figure Lengend Snippet: Figure 1. Interaction between Chk1 and MYPT1. (A) Chk1 was immunoprecipitated from HeLa cells, and samples were analyzed by SDS-PAGE with Coomassie blue stain- ing. 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 MgCl2, 1 M Dithiothreitol, 0.1 M Na3VO4, 0.1 mM ATP or 1 mCi of g-[ 32P]ATP.

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

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1cβ (PP1cβ).

doi: 10.1080/15384101.2017.1418235

Figure Lengend Snippet: Figure 3. 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 antibod- ies. 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 MgCl2, 1 M Dithiothreitol, 0.1 M Na3VO4, 0.1 mM ATP or 1 mCi of g-[ 32P]ATP.

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

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1cβ (PP1cβ).

doi: 10.1080/15384101.2017.1418235

Figure Lengend Snippet: Figure 2. 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 MgCl2, 1 M Dithiothreitol, 0.1 M Na3VO4, 0.1 mM ATP or 1 mCi of g-[ 32P]ATP.

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

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1cβ (PP1cβ).

doi: 10.1080/15384101.2017.1418235

Figure Lengend Snippet: Figure 4. 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 MgCl2, 1 M Dithiothreitol, 0.1 M Na3VO4, 0.1 mM ATP or 1 mCi of g-[ 32P]ATP.

Techniques: Transfection, Plasmid Preparation, Incubation, Quantitation Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1cβ (PP1cβ).

doi: 10.1080/15384101.2017.1418235

Figure Lengend Snippet: Figure 5. pS20 is essential for the interaction between MYPT1 and PP1cb. (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- PP1cb. (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 PP1cb. MYPT1 then targets PP1cb 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 MgCl2, 1 M Dithiothreitol, 0.1 M Na3VO4, 0.1 mM ATP or 1 mCi of g-[ 32P]ATP.

Techniques: Transfection, Recombinant