The CDK-PLK1 axis targets the DNA damage checkpoint sensor protein RAD9 to promote cell proliferation and tolerance to genotoxic stress
- Radiation Biology Center, Kyoto University, Japan
- Graduate School of Bioresources, Mie University, Japan
- Graduate School of Medicine, Kyoto University, Japan
- Mukogawa Women’s University, Japan
- Kyoto University, Japan
- Mie University, Japan
- Graduate School of Biostudies, Kyoto University, Japan
Figures
Figure 1 with 1 supplement
CDK phosphorylates threonine 292 of RAD9.
(A) Schematic of the aim of this manuscript. (B) The recombinant GST-tagged C-terminal (a.a. 266–391) portion of RAD9 was mixed with the purified active CDK2-CyclinA2 complex. Western blotting was …
Figure 1—figure supplement 1
CDK phosphorylates threonine 292, and construction of the RAD9-WT and -S291A/T292A(T292A) expressing stable cell lines.
(A) HEK293A cells were transfected with a plasmid that expresses RAD9-WT-mH (WT) or RAD9-T292A-mH (T292A) under the CMV promoter, cultured in the presence or absence of RO3306 (5 µM, indicated as 330…
Figure 2 with 1 supplement
CDK-dependent phosphorylation of threonine 292 of RAD9 accommodates and activates PLK1.
(A) The RAD9 C-terminus co-precipitates with PLK1. GST-RAD9 (3A: S277A, S328A, S336G) (30 pmol) was pre-incubated with CDK2-Cyclin A2 (0.5 pmol) in 15 µl of kinase buffer at 30 ˚C for 30 min, …
Figure 2—figure supplement 1
PLK1 interacts with and phosphorylates RAD9.
(A) Yeast two hybrid assays were performed using the wild type (WT) or T292A-mutated (T292A) C-terminus of pLEX-RAD9 (pLEX-RAD9C) as the bait, and the PBD of PLK1-5, which was cloned into pGADT7, as …
Figure 3 with 3 supplements
PLK1 phosphorylates RAD9 on Thr 313 and Ser 326 in vivo and in vitro.
(A) PLK1 phosphorylates CDK-phosphorylated RAD9. PLK1 or PLK1-KR (K82R) (0.3 pmol) was incubated with GST-RAD9 (3A) (10 pmol; phosphorylated with the CDK complex (0.5 pmol) for 30 min at 30 ˚C), at …
Figure 3—figure supplement 1
PLK1 phosphorylates RAD9 on Thr 313 and Ser 326.
(A) An alignment of the RAD9 C-terminal region is shown. RAD9 (RAD9A) proteins from Human (H. sapiens), Mouse, Opossum, Chicken, Frog (Xenopus) and Zebrafish were aligned. Identical amino acids for …
Figure 3—figure supplement 2
PLK1 phosphorylates RAD9 on Thr 313 and Ser 326 in vivo.
(A) 293A T-REx cells, stably expressing the wild type or S291A/T292A-, T313A- or S326A- mutated RAD9-mH from the FRT locus, were transfected with CMV-PLK1 or CMV-PLK1-KR. Calyculin A (0.5 µM, Santa …
Figure 3—figure supplement 3
The phosphorylation status of RAD9 under various genotoxic stresses.
(A), (B) Cell lysates were subjected to immunoprecipitation, using α-myc antibody-coated agarose beads. Western blotting analyses of the immunoprecipitates (myc-IP) were performed using α-myc …
Figure 4 with 6 supplements
Enhanced checkpoint signaling when PLK1 fails to phosphorylate RAD9.
(A) A chromatin fractionation assay (see Materials and methods) was performed in HEK293A-T-REx cell lines stably expressing RAD9-WT-mH (RAD9-WT), RAD9- T313A-mH (RAD9-T313A) or RAD9- S326A-mH …
Figure 4—figure supplement 1
U2OS stable cell line that expresses RAD9-mycHis.
Western blotting analyses were performed to monitor the expression levels of myc-tagged RAD9 (RAD9-WT-mH (WT), RAD9-S291A/T292A (T292A), and RAD9-T313A-mH (T313A). RAD9-S326A (S326A)) was expressed …
Figure 4—figure supplement 2
DNA damage signaling was increased when PLK1 failed to phosphorylate RAD9.
U2OS cells were treated with hydroxyurea (0.2 mM, 24 hr) and/or BI2536 (1 µM, 15 min) before the harvest. A chromatin fractionation assay was performed. Western blotting analyses were performed …
Figure 4—figure supplement 3
dNTP incorporation was decreased in HU treated cells in which PLK1-dependent phosphorylation of RAD9 was defective.
(A) An EdU incorporation assay was performed. An example of the raw data of the dNTP incorporation presented in Figure 4B is shown. The fluorescent value (Y-axis) of the enclosed area in the scatter …
Figure 4—figure supplement 4
The analysis using CMV-RAD9-expressing U2OS cell line under endogenous RAD9 knock down.
(A) U2OS T-REx cells were subjected to siRNA treatment against RAD9 (siRAD9: s11719 (Ambion)). Cells were harvested 48 hr after the siRNA treatments. The antibodies used were α-RAD9 (RAD9), …
Figure 4—figure supplement 5
Decreased dNTP incorporation was observed in telomerase-positive HEK293A cells that expressed CDK-PLK1-dependent phosphorylation defective RAD9.
(A) An EdU incorporation assay was performed. Scatter plots showing EdU incorporation and histograms showing DNA content (propidium iodide staining) are presented. Cells (RAD9-WT, RAD9-T292A, …
Figure 4—figure supplement 6
EdU incorporation assay under Aphidicolin- and MMC-induced stress.
(A), (B) An EdU incorporation assay was performed. Scatter plots showing EdU incorporation and histograms showing DNA content (propidium iodide staining) are presented. Cells were incubated without …
Figure 5 with 2 supplements
The effect of origin firing and DNA replication fork integrity of PLK1-dependent phosphorylation on RAD9.
(A) A schematic of the time course applied for the combing assay analysis. U2OS cells expressing WT- or T313A-RAD9 from genomic FRT sites were used for the analysis. (B) Examples of DNA fibers seen …
Figure 5—figure supplement 1
The DNA combing assay in aphidicolin-treated cells.
The combing assay was performed. In this assay, the cells (U2OS T-REx cells expressing RAD9-WT or RAD9-T313A) were first labeled with IdU for 30 min, followed by CldU labeling for 30 min at the end …
Figure 5—figure supplement 2
Enhanced RAD9-CLASPIN complex formation when CDK or PLK1 failed to phosphorylate RAD9.
293A T-REx cells were cultured in HU (0.2 mM) containing media for 24 hr, and the cell lysates were subjected to immunoprecipitation using α-myc antibody-coated agarose beads. HEK293A cells stably …
Figure 6 with 1 supplement
PLK1-dependent phosphorylation of RAD9 is required for proliferation under replicative stress.
(A) Cell growth was monitored using the Presto-Blue Cell viability reagent (see Materials and methods). U2OS cells stably expressing RAD9-WT-mH (WT), RAD9-S291A/T292A-mH (T292A), and RAD9-T313A-mH …
Figure 6—figure supplement 1
Reduced cell proliferation by expression of non-phosphorylatable RAD9 in telomerase-positive HEK293A cells.
HEK293A T-REx cells expressing RAD9-WT, RAD9-T292A or RAD9-T313A were grown in 96 well plates in the presence of 0.2 mM or 0.4 mM hydroxyurea for 4 days. siRNA (s11719: Ambion) was transfected 48 hr …
Figure 7
A schematic of PLK1-dependent damage tolerance.
(Left) The collaboration of CDK and PLK1 targets and suppresses the DNA damage detection machinery in the DNA damage checkpoint process, and promotes the S-phase progression to support cell …
Additional files
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Supplementary file 1
Primers used in this study
Primers used in this study was listed.
- https://doi.org/10.7554/eLife.29953.023
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Supplementary file 2
Statistic data to construct Figures 4B and 6A,B
An excel formatted file was presented.
- https://doi.org/10.7554/eLife.29953.024
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Supplementary file 3
The expanded photos of DNA fibers used in Figure 5B
The field in white squares are region presented in Figure 5B.
- https://doi.org/10.7554/eLife.29953.025
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Transparent reporting form
- https://doi.org/10.7554/eLife.29953.026
