Cdc7 activates replication checkpoint by phosphorylating the Chk1-binding domain of Claspin in human cells
- Tokyo Metropolitan Institute of Medical Science, Japan
Figures
Figure 1 with 2 supplements
Cdc7 is required for activation of Chk1 in response to replication stress.
(A) HCT116 and its derivative, HCT116-323, in which the promoter for the Cdc7 gene was mutated (see Figure 1—figure supplement 1 for details of the mutation), were incubated with BrdU for 20 min and …
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Figure 1—source data 1
Quantification for graph (three independent FACS experiments) in Figure 1A.
- https://cdn.elifesciences.org/articles/50796/elife-50796-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
Cdc7 is required for activation of Chk1 in HCT116 cells.
HCT116 cells were transfected with siControl or siCdc7 for 48 hr. Cells were harvested at 0, 15, 30 and 50 min after addition of 2 mM HU, and the whole cell extracts were analyzed by western …
Figure 1—figure supplement 2
Sequences of the promoter region of human Cdc7 gene and deletion introduced by CRISPR-Cas9.
The upper drawing shows the exon (white boxes) and intron (bars) organization of the Cdc7 gene. The bent arrows indicate two transcription initiation sites. The exons and introns are in red and …
Figure 2 with 1 supplement
AP (Acidic Patch) of Claspin is required for interaction with Chk1 and Chk1 activation.
(A) C-terminally Flag-tagged wild type (WT) and mutant Claspin indicated were transiently expressed in 293 T cells and were pulled down by anti-Flag beads. Immunoprecipitated proteins were analyzed …
Figure 2—figure supplement 1
Pull down assays with biotinylated CKBD phospho-oligopeptides.
Biotinylated peptides, as shown, were mixed with cell lysates (A) or purified Chk1 protein (B and C) and pulled down by streptavidin beads, and were analyzed by western blotting using anti-Chk1 …
Figure 3
Cdc7 is required for interaction of Claspin with Chk1.
(A) 293 T cells transfected with siControl or siCdc7 for 48 hr were treated with 2 mM HU for 4 hr or non-treated. Cells were lysed with CSK buffer containing 0.1% Triton-X100 and proteins were …
Figure 4 with 1 supplement
Cdc7 is required for phosphorylation of CKBD of Claspin in 293 T cells.
Phosphorylation sites of Claspin in the non-treated, HU-treated, siCdc7-treated and siCdc7+HU-treated cells were determined by mass spectrometry analyses and are shown on the drawing along the …
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Figure 4—source data 1
Primary data (1st experiment) for Figure 4.
- https://cdn.elifesciences.org/articles/50796/elife-50796-fig4-data1-v3.xlsx
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Figure 4—source data 2
Primary data (2nd experiment) for Figure 4.
- https://cdn.elifesciences.org/articles/50796/elife-50796-fig4-data2-v3.xlsx
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Figure 4—source data 3
Primary data (3rd experiment) for Figure 4.
- https://cdn.elifesciences.org/articles/50796/elife-50796-fig4-data3-v3.xlsx
Figure 4—figure supplement 1
Mass spectrometry analyses of phosphorylation sites on Clapsin in 293 T cells.
(A) 293 T cells, treated with siControl or siCdc7 for 24 hr were treated with 2 mM HU for 50 min or not treated. Cells were lysed with RIPA buffer. Claspin was pulled down by anti-Claspin antibody, …
Figure 5 with 5 supplements
Both Cdc7 and CK1γ1 are required for full activation of Chk1 in response to replication stress.
(A) U2OS cells were transfected with siControl, siCdc7, siCK1γ1 or siCdc7+siCK1γ1 for 48 hr. (B) HCT116 and HCT116-323, a Cdc7 promoter mutant cell line, were transfected with siControl or siCK1γ1 …
Figure 5—figure supplement 1
Cdc7 and CK1γ1 are required for full activation of Chk1 in response to replication stress in HeLa cells.
HeLa cells were transfected with siControl, siCdc7, siCK1γ1 or siCdc7+siCK1γ1 for 48 hr. Cells were harvested at 0, 10, 30 and 50 min after addition of 2 mM HU and whole cell extracts were analyzed …
Figure 5—figure supplement 2
Effect of CK1γ1 depletion on DNA replication in HCT116-323 cells.
(A) HCT116 and Cdc7 promotor mutant HCT116-323 were transfected with siControl or siCK1γ1for 48 hr. Cell were labeled by BrdU for 20 min and BrdU incorporation was analyzed by FACS. (B) The average …
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Figure 5—figure supplement 2—source data 1
Quantification for graph (three independent FACS experiments) in Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/50796/elife-50796-fig5-figsupp2-data1-v3.xlsx
Figure 5—figure supplement 3
Cdc7 plays a major role in prevention of replication stress-induced cell death in U2OS cells.
(A) U2OS cells were transfected with indicated siRNA for 48 hr, and then were treated with 2 mM HU for 0, 24 or 48 hr. After mixing with reagents for TUNEL assays, cells were analyzed by FACS. P1 …
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Figure 5—figure supplement 3—source data 1
Quantification for graph (three independent FACS experiments) in Figure 5—figure supplement 3.
- https://cdn.elifesciences.org/articles/50796/elife-50796-fig5-figsupp3-data1-v3.xlsx
Figure 5—figure supplement 4
CK1γ1 plays a major role for full activation of Chk1 in response to replication stress in non-cancer cells.
RPE-1 and TIG-3 cells were transfected with siControl, siCdc7, siCK1γ1 or siCdc7+siCK1γ1 for 48 hr. Cells were harvested at 1 hr after addition of 2 mM HU and whole cell extracts were analyzed by …
Figure 5—figure supplement 5
Cdc7 plays a major role for full activation of Chk1 in response to replication stress in 293 T cells 293 T cells were transfected with siControl, siCdc7, siCK1γ1 or siCdc7+siCK1γ1 for 48 hr.
Cells were harvested at 50 min after addition of 2 mM HU and whole cell extracts were analyzed by western blotting with indicated antibodies.
Figure 6 with 2 supplements
Cdc7 phosphorylates T916 and S945.
(A) Two hundred ng (34 pmole) each of wild type CKBD peptide (wild-type) or mutant peptides (CKBD-A, Others-A and Others-E) was incubated in the kinase reaction with indicated amounts of purified …
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Figure 6—source data 1
Quantification for graph (three independent FACS experiments) in Figure 6B.
- https://cdn.elifesciences.org/articles/50796/elife-50796-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
Cdc7 and CK1γ1 phosphorylate Claspin independently.
(A) Purified Claspin polypeptide #27 (aa897-1100 containing CKBD and AP) was incubated in the kinase reaction with indicated amounts of purified Cdc7-ASK and/or CK1γ1 in the presence of [γ-32P]-ATP. …
Figure 6—figure supplement 2
CK1γ1 phosphorylates Claspin in a manner independent of serine/threonine residues in the CKBD-AP segment.
(A) Purified wild type Claspin or Claspin mutant ST27A (serines and threonines in aa903-1120 substituted by alanine) was incubated in the kinase reaction with indicated amounts of purified CK1γ1 in …
Figure 7 with 3 supplements
Levels of Cdc7 protein affect the dependency on CK1γ1 for checkpoint activation.
(A) HCT116 and HCT116-323 (Cdc7 promoter mutant) cells were transfected with siControl, siCdc7, siCK1γ1 or siCdc7+ siCK1γ1 for 48 hr. Cells were then treated with 2 mM HU for 50 min. Whole cell …
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Figure 7—source data 1
Quantification for western data (three independent experiments of Figure 7A) in Figure 7B.
- https://cdn.elifesciences.org/articles/50796/elife-50796-fig7-data1-v3.xlsx
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Figure 7—source data 2
Quantification for western data (three independent experiments of Figure 7C) in Figure 7D.
- https://cdn.elifesciences.org/articles/50796/elife-50796-fig7-data2-v3.xlsx
Figure 7—figure supplement 1
Effect of Cdc7 inhibition on checkpoint activation in various cancer and normal cell lines (related to Figures 1 and 4 as well).
(A and B) Various cells indicated in the figure were treated with 10 µM XL413 (Cdc7-specific inhibitor) for 90 min and then treated with HU at the concentrations shown for the time indicated. The …
Figure 7—figure supplement 2
Estimation of the numbers of molecules of Cdc7 and CK1γ1 in various cell lines.
The whole cell extracts from 5 × 104 293T, HeLa, HCT116, U2OS and NHDF cells and purified Cdc7 and CK1γ1 proteins of the amount indicated were run on 4–20% gradient SDS-PAGE and analyzed by western …
Figure 7—figure supplement 3
Overexpression of Cdc7 in NHDF cells increases dependency on Cdc7 for checkpoint activation.
(A) NHDF cells were infected by Cdc7- or CK1γ1-expressing lentiviruses. At 2 days after infection, cells were selected by puromycin (4 µg/ml) for 2 days, followed by incubation in the absence of the …
Author response image 1
Flag-tagged wild-type (lanes 1 and 4) or APDE/A (lanes 3 and 6) or HA-tagged wild-type (lanes 2 and 5; negative control) Claspin was transiently expressed in 293T cells, and extracts were made with CSK buffer containing 0.1% Triton X-100.
Immnoprecipitates with anti-Flag antibody were analyzed for CK1γ1 (lanes 4-6). Inputs were also analyzed (lanes 1-3).
Additional files
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Supplementary file 1
Key Resources Table.
- https://cdn.elifesciences.org/articles/50796/elife-50796-supp1-v3.docx
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Supplementary file 2
List of the siRNA sequences used in this study.
- https://cdn.elifesciences.org/articles/50796/elife-50796-supp2-v3.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50796/elife-50796-transrepform-v3.docx
