[논문]Dephosphorylation of p53 Ser 392 Enhances Trimethylation of Histone H3 Lys 9 via SUV39h1 Stabilization in CK2 Downregulation-Mediated Senescence

Park, Jeong-Woo; Bae, Young-Seuk

doi:10.14348/molcells.2019.0018

Dephosphorylation of p53 Ser 392 Enhances Trimethylation of Histone H3 Lys 9 via SUV39h1 Stabilization in CK2 Downregulation-Mediated Senescence 원문보기

Molecules and cells, v.42 no.11, 2019년, pp.773 - 782

Park, Jeong-Woo (School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University) , Bae, Young-Seuk (School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University)

Abstract ▼ AI-Helper

Cellular senescence is an irreversible form of cell cycle arrest. Senescent cells have a unique gene expression profile that is frequently accompanied by senescence-associated heterochromatic foci (SAHFs). Protein kinase CK2 (CK2) downregulation can induce trimethylation of histone H3 Lys 9 (H3K9me3) and SAHFs formation by activating SUV39h1. Here, we present evidence that the PI3K-AKT-mTOR-reactive oxygen species-p53 pathway is necessary for CK2 downregulation-mediated H3K9me3 and SAHFs formation. CK2 downregulation promotes SUV39h1 stability by inhibiting its proteasomal degradation in a p53-dependent manner. Moreover, the dephosphorylation status of Ser 392 on p53, a possible CK2 target site, enhances the nuclear import and subsequent stabilization of SUV39h1 by inhibiting the interactions between p53, MDM2, and SUV39h1. Furthermore, $p21^{Cip1/WAF1}$ is required for CK2 downregulation-mediated H3K9me3, and dephosphorylation of Ser 392 on p53 is important for efficient transcription of $p21^{Cip1/WAF}$. Taken together, these results suggest that CK2 downregulation induces dephosphorylation of Ser 392 on p53, which subsequently increases the stability of SUV39h1 and the expression of $p21^{Cip1/WAF1}$, leading to H3K9me3 and SAHFs formation.

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그림 Fig. 1. Induction of H3K9me3 and SUV39h1 after CK2 downregulation requires PI3K-AKT-mTOR-ROS-p53 pathway. (A and B) Cells were treated with CK2α siRNA for 48 h in the presence of 100 nM wortmannin or 10 μM triciribine (A) and 5 mM NAC or 100 nM rapamycin (B). Cell extracts were then visualized by immunoblotting (upper panels). Representative data from three independent experiments are shown. The graph shows the quantification of each protein relative to a-tubulin (A) or b-actin (B) (bottom panels). Values indicate mean ± SEM. Bars that do not share a common letter (a, b, or c) are significantly different between groups at P < 0.05. (C and D) Essential role of p53 in inducing H3K9me3 and SUV39h1 upon CK2 downregulation. Wild-type p53 cells (HCT116 and MCF-7) and mutant p53 cells (HCT116 p53^-/- and MDA-MB-435) were treated with CK2α siRNA, and cell extracts were visualized by immunoblotting. The values below each band represent the mean fold differences (n = 3) in expression levels compared to the control, which was assigned a value of 100.
그림 Fig. 2. p53-dependent induction of SAHFs formation after CK2 downregulation. (A) Confocal immunofluorescent images (×800 magnification) showing the co-localization of chromatin foci with H3K9me3 (red) and HP1γ (green) in cells treated with CK2α siRNA in the presence of the p53 inhibitor pifithrin-α (30 μM). DAPI staining (blue) was used to visualize DNA foci. Fluorescence intensity was quantified using ImageJ software (right panels). Arbitrary intensity values for H3K9me3 (red), HP1γ (green), or DAPI (blue) are shown relative to the reference line (white) used for the analysis. (B and C) The effect of nutlin-3 on the expression of p53, SUV39h1, and H3K9me3. (B) Cells were treated with the MDM2 inhibitor nutlin-3 (0.5 to 4 μM) for 48 h. (C) Cells were treated with pcDNA3.1-HACK2α in the absence or presence of nutlin-3 (0.5 μM) for 48 h. Cell lysates were visualized by immunoblotting (upper panels). The graph shows the quantification of each protein relative to β-actin (bottom panels). The values indicate mean ± SEM. Bars that do not share a common letter (a, b, or c) are significantly different between groups at P < 0.05.
그림 Fig. 3. The role of CK2 in regulating SUV39h1 expression. (A and B) p53-dependent destabilization of SUV39h1 protein after CK2 upregulation. Wild-type p53 cells (HCT116 and MCF-7) (A) and p53-null cells (HCT116 p53^-/-) (B) were treated with the protein synthesis inhibitor cycloheximide (CHX, 50 μg/ml) for the indicated times in the absence or presence of pcDNA3.1-HA-CK2α. The cell lysates were visualized by immunoblotting. The values below each band represent the mean fold differences (n = 3) in expression levels compared to the control, which was assigned a value of 100. (C) Cells were treated with CK2α siRNA or pcDNA3.1-HA-CK2α in the absence or presence of the proteasome inhibitor MG132 (10 μM) for 6 h. Cell lysates were visualized by immunoblotting (upper panel). The graph shows the quantification of each protein relative to β-actin (bottom panel). (D) CK2 overexpression compromises the p53-mediated induction of SUV39h1. Immunoblot analysis of cells treated with CK2a siRNA or pcDNA3.1-p53 (upper panel). The graph shows the quantification of SUV39h1 and H3K9me3 relative to β-actin (bottom panel). The values indicate mean ± SEM. Bars that do not share a common letter (a, b, or c) are significantly different between groups at P < 0.05.
그림 Fig. 4. The effect of p53 S392 phosphorylation on the stability of SUV39h1. (A) CK2 phosphorylates S392 on p53. Immunoblot analysis of cells treated with CK2a siRNA or pcDNA3.1-p53 (upper panel). The graph shows the quantification of phospho-p53 S392 relative to p53 (bottom panel). The values indicate means ± SEM. Bars that do not share a common letter (a, b, or c) are significantly different between groups at P < 0.05. (B-D) Cells were treated with pcDNA3.1-p53 wild-type (WT) or -p53 mutants (S392A and S392E). (B) Induction of SUV39h1 and H3K9me3 by p53^S392A relative to WT p53 or p53^S392E. Protein extracts were visualized by immunoblotting (upper panels). The graph shows the quantification of SUV39h1 and H3K9me3 relative to b-actin (bottom panels). (C) Confocal immunofluorescent images (×800 magnification) of co-localization of chromatin foci with H3K9me3 (red) and HP1γ (green). DAPI staining (blue) was used to visualize DNA foci. The fluorescence intensity was quantified using ImageJ software (right panels). Arbitrary intensity values for H3K9me3 (red), HP1γ (green), and DAPI (blue) are shown relative to the reference line (white) used for the analysis. (D) Cells were treated with or without MG132 (10 μM) for 6 h. Cell lysates were visualized by immunoblotting (upper panel). The graph shows the quantification of each protein relative to b-actin (bottom panel). The values indicate means ± SEM. Bars that do not share a common letter (a, b, c, or d) are significantly different between groups at P < 0.05.
그림 Fig. 5. The effect of p53 S392 phosphorylation on the interactions between p53, MDM2, and SUV39h1, and the nuclear localization of SUV39h1. Cells were treated with CK2α siRNA or pcDNA3.1-CK2 (A and C) or with pcDNA3.1-p53 variants (B and D). (A and B) The inhibitory effect of CK2 downregulation on the interactions between p53, MDM2, and SUV39h1 (A) and the induction of p53, MDM2, and SUV39h1 interactions by p53^S392E relative to wild-type (WT) p53 and p53^S392A (B). Cell lysates were immunoprecipitated (IP) with anti-p53 antibodies followed by immunoblotting with anti-MDM2 and SUV39h1 antibodies. Cell lysate IP with IgG heavy chain served as a loading control. The same membranes were reprobed with anti-p53 antibodies and TrueBlot reagent, which eliminates the detection of the immunoprecipitated antibodies. Representative data from three independent experiments are shown. (C and D) The inhibitory effect of CK2 upregulation on the nuclear localization of p53 and SUV39h1 (C) and the induction of SUV39h1 nuclear localization by p53^S392A relative to WT p53 and p53^S392E (D). Cytoplasm and nuclei were isolated from p53-null cells overexpressing variants of p53 and were visualized by immunoblotting. β-actin (cytoplasmic marker) and histone H3 (nuclear marker) were quantified as loading controls (upper panels). The levels of CK2a protein in whole cells extracts are shown (left on the C panel). The graph shows the quantification of p53 and SUV39h1 relative to the subcellular markers (bottom panels). The values indicate mean ± SE. Bars that do not share a common letter (a, b, or c) are significantly different between groups at P < 0.05.
그림 Fig. 6. p21^Cip1/WAF1 is required for CK2 downregulation-mediated H3K9me3, and dephosphorylation of S392 on p53 is important for efficient transcription of p21^Cip1/WAF. (A) p21^Cip1/WAF1 is essential in CK2 downregulation-mediated induction of H3K9me3, but not for the induction of SUV39h1. Immunoblotting analysis was conducted on HCT116 p21^-/- cells treated with CK2α siRNA or pcDNA3.1-HA-CK2α (upper panel). The graph shows the quantification of each protein relative to the levels of b-actin (bottom panel). The values indicate mean ± SEM. **P < 0.01, ***P < 0.001. (B) Induction of p21^Cip1/WAF1 mRNA levels by p53^S392A relative to wild-type (WT) p53 or p53^S392E. RT-PCR analyses were performed using specific primers for p53 and p21^Cip1/WAF1 (upper panel). The graph shows the quantification of p21^Cip1/WAF1 mRNA levels relative to those of b-actin (bottom panel). The values indicate mean ± SE. Bars that do not share a common letter (a, b, or c) were significantly different between groups at P < 0.05. (C) A model possibly illustrating CK2 downregulation-induced SAHFs formation. CK2 downregulation induces not only p53 accumulation through increased ROS, but also dephosphorylation of S392 on p53. This process elevates H3K9me3 by impeding the interactions between p53, MDM2, and SUV39h1, which causes the nuclear import and subsequent stabilization of SUV39h1, and by enhancing p21^Cip1/WAF1 expression, which results in Rb activation and subsequent recruitment of SUV39h1 and HP1γ.

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