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Cyclin B1/Cdk1 phosphorylation of mitochondrial p53 induces anti-apoptotic response.

Nantajit D, Fan M, Duru N, Wen Y, Reed JC, Li JJ - PLoS ONE (2010)

Bottom Line: The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis.Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL.Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53(-/-) cells resulted in an increased mitochondrial ATP production and suppression of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of California Davis, Sacramento, California, United States of America.

ABSTRACT
The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53(+/+) status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53(-/-) cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53(-/-) cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53.

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Radiation stimulated cyclin B1/Cdk1-mediated mitochondrial p53 Ser-315 phosphorylation.(A) p53 Ser-315 phosphorylation was detected by immunoblotting with anti-phospho-p53(S315) antibody with 20 µg mitochondrial fractions from irradiated HCT116 p53+/+. (B) Total protein and phosphorylated p53, cyclin B1, and Cdk1 were determined in irradiated HCT116 p53+/+ transfected with siRNAs against cyclin B1, Cdk1 or scrambled seq. (C) Structural illustration of fusion protein containing mitochondria-targeted-sequence (MTS) linked with cyclin B1-EGFP, Cdk1-RFP, as well as a mutant form delCdk1 with the deletion of catalytic domain of Cdk1 (aa 122–132). (D) Fluorescence images of HCT116 p53+/+ cells transfected with mitochondria-targeted cyclin B1-EGFP and Cdk1-RFP with Mitotracker mitochondria staining (scale bar = 5 µm). (E) Ser-315 phosphorylation of mitochondrial p53 was determined in HCT116 p53+/+ cells co-transfected with empty vectors MTS-EGFP/MTS-RFP, or with MTS-cyclin B1 fused with EGFP (MTS-cyclin B1) and MTS-Cdk1 fused with RFP (MTS-Cdk1), or with MTS-cyclin B1 fused with EGFP and MTS-delCdk1 fused with RFP (delCdk1). In addition, HCT116 p53+/+ cells without transfection was included as a control.
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pone-0012341-g003: Radiation stimulated cyclin B1/Cdk1-mediated mitochondrial p53 Ser-315 phosphorylation.(A) p53 Ser-315 phosphorylation was detected by immunoblotting with anti-phospho-p53(S315) antibody with 20 µg mitochondrial fractions from irradiated HCT116 p53+/+. (B) Total protein and phosphorylated p53, cyclin B1, and Cdk1 were determined in irradiated HCT116 p53+/+ transfected with siRNAs against cyclin B1, Cdk1 or scrambled seq. (C) Structural illustration of fusion protein containing mitochondria-targeted-sequence (MTS) linked with cyclin B1-EGFP, Cdk1-RFP, as well as a mutant form delCdk1 with the deletion of catalytic domain of Cdk1 (aa 122–132). (D) Fluorescence images of HCT116 p53+/+ cells transfected with mitochondria-targeted cyclin B1-EGFP and Cdk1-RFP with Mitotracker mitochondria staining (scale bar = 5 µm). (E) Ser-315 phosphorylation of mitochondrial p53 was determined in HCT116 p53+/+ cells co-transfected with empty vectors MTS-EGFP/MTS-RFP, or with MTS-cyclin B1 fused with EGFP (MTS-cyclin B1) and MTS-Cdk1 fused with RFP (MTS-Cdk1), or with MTS-cyclin B1 fused with EGFP and MTS-delCdk1 fused with RFP (delCdk1). In addition, HCT116 p53+/+ cells without transfection was included as a control.

Mentions: p53 Ser-315 phosphorylation has been shown to be induced by irradiation [39] and confirmed by our present study (Figure S2A). To address the question of whether the stress-induced p53 protein is, at least in part, translocated to mitochondria and its Ser-315 residue is targeted by cyclin B1/Cdk1 complex, we measured the levels of phosphorylated p53 in mitochondrial fractions isolated from HCT116 p53+/+ at different time intervals after irradiation. Although a high steady-level of radiation-induced total mitochondrial p53 was maintained throughout 24 hours after irradiation, phosphorylated p53 Ser-315 was markedly increased during the hours (Figure 3A), suggesting that the phosphorylation may happen within mitochondria after irradiation. The phosphorylation of mitochondrial p53 was further confirmed in irradiated mouse xenograft tissue as shown in Figure S2B. In agreement, inhibition of cyclin B1 or Cdk1 by short-interference siRNA knockdown drastically diminished the Ser-315 phosphorylation of mitochondrial p53 of irradiated HCT116 p53+/+ cells without altering the total mitochondrial p53 level (Figure 3B). Although the expression of cyclin B1 and Cdk1 was partially reduced in the whole cell lysate (Figure S2C), it is noteworthy that mitochondrial cyclin B1 and Cdk1 levels were markedly reduced by the specific siRNAs, suggesting that mitochondrial translocation of cyclin B1 and Cdk1 is sensitive to the overall cyclin B1 and Cdk1 cellular protein expression. In addition, siRNA knockdown of cyclin B1 did not affect mitochondrial translocation of Cdk1, and vice versa, indicating the independent mitochondrial influx of cyclin B1 and Cdk1 (Figure 3B).


Cyclin B1/Cdk1 phosphorylation of mitochondrial p53 induces anti-apoptotic response.

Nantajit D, Fan M, Duru N, Wen Y, Reed JC, Li JJ - PLoS ONE (2010)

Radiation stimulated cyclin B1/Cdk1-mediated mitochondrial p53 Ser-315 phosphorylation.(A) p53 Ser-315 phosphorylation was detected by immunoblotting with anti-phospho-p53(S315) antibody with 20 µg mitochondrial fractions from irradiated HCT116 p53+/+. (B) Total protein and phosphorylated p53, cyclin B1, and Cdk1 were determined in irradiated HCT116 p53+/+ transfected with siRNAs against cyclin B1, Cdk1 or scrambled seq. (C) Structural illustration of fusion protein containing mitochondria-targeted-sequence (MTS) linked with cyclin B1-EGFP, Cdk1-RFP, as well as a mutant form delCdk1 with the deletion of catalytic domain of Cdk1 (aa 122–132). (D) Fluorescence images of HCT116 p53+/+ cells transfected with mitochondria-targeted cyclin B1-EGFP and Cdk1-RFP with Mitotracker mitochondria staining (scale bar = 5 µm). (E) Ser-315 phosphorylation of mitochondrial p53 was determined in HCT116 p53+/+ cells co-transfected with empty vectors MTS-EGFP/MTS-RFP, or with MTS-cyclin B1 fused with EGFP (MTS-cyclin B1) and MTS-Cdk1 fused with RFP (MTS-Cdk1), or with MTS-cyclin B1 fused with EGFP and MTS-delCdk1 fused with RFP (delCdk1). In addition, HCT116 p53+/+ cells without transfection was included as a control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2925892&req=5

pone-0012341-g003: Radiation stimulated cyclin B1/Cdk1-mediated mitochondrial p53 Ser-315 phosphorylation.(A) p53 Ser-315 phosphorylation was detected by immunoblotting with anti-phospho-p53(S315) antibody with 20 µg mitochondrial fractions from irradiated HCT116 p53+/+. (B) Total protein and phosphorylated p53, cyclin B1, and Cdk1 were determined in irradiated HCT116 p53+/+ transfected with siRNAs against cyclin B1, Cdk1 or scrambled seq. (C) Structural illustration of fusion protein containing mitochondria-targeted-sequence (MTS) linked with cyclin B1-EGFP, Cdk1-RFP, as well as a mutant form delCdk1 with the deletion of catalytic domain of Cdk1 (aa 122–132). (D) Fluorescence images of HCT116 p53+/+ cells transfected with mitochondria-targeted cyclin B1-EGFP and Cdk1-RFP with Mitotracker mitochondria staining (scale bar = 5 µm). (E) Ser-315 phosphorylation of mitochondrial p53 was determined in HCT116 p53+/+ cells co-transfected with empty vectors MTS-EGFP/MTS-RFP, or with MTS-cyclin B1 fused with EGFP (MTS-cyclin B1) and MTS-Cdk1 fused with RFP (MTS-Cdk1), or with MTS-cyclin B1 fused with EGFP and MTS-delCdk1 fused with RFP (delCdk1). In addition, HCT116 p53+/+ cells without transfection was included as a control.
Mentions: p53 Ser-315 phosphorylation has been shown to be induced by irradiation [39] and confirmed by our present study (Figure S2A). To address the question of whether the stress-induced p53 protein is, at least in part, translocated to mitochondria and its Ser-315 residue is targeted by cyclin B1/Cdk1 complex, we measured the levels of phosphorylated p53 in mitochondrial fractions isolated from HCT116 p53+/+ at different time intervals after irradiation. Although a high steady-level of radiation-induced total mitochondrial p53 was maintained throughout 24 hours after irradiation, phosphorylated p53 Ser-315 was markedly increased during the hours (Figure 3A), suggesting that the phosphorylation may happen within mitochondria after irradiation. The phosphorylation of mitochondrial p53 was further confirmed in irradiated mouse xenograft tissue as shown in Figure S2B. In agreement, inhibition of cyclin B1 or Cdk1 by short-interference siRNA knockdown drastically diminished the Ser-315 phosphorylation of mitochondrial p53 of irradiated HCT116 p53+/+ cells without altering the total mitochondrial p53 level (Figure 3B). Although the expression of cyclin B1 and Cdk1 was partially reduced in the whole cell lysate (Figure S2C), it is noteworthy that mitochondrial cyclin B1 and Cdk1 levels were markedly reduced by the specific siRNAs, suggesting that mitochondrial translocation of cyclin B1 and Cdk1 is sensitive to the overall cyclin B1 and Cdk1 cellular protein expression. In addition, siRNA knockdown of cyclin B1 did not affect mitochondrial translocation of Cdk1, and vice versa, indicating the independent mitochondrial influx of cyclin B1 and Cdk1 (Figure 3B).

Bottom Line: The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis.Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL.Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53(-/-) cells resulted in an increased mitochondrial ATP production and suppression of apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University of California Davis, Sacramento, California, United States of America.

ABSTRACT
The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53(+/+) status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53(-/-) cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53(-/-) cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53.

Show MeSH
Related in: MedlinePlus