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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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Cyclin B1/Cdk1-mediated p53 phosphorylation inhibited mitochondria-mediated apoptosis.(A) HCT116 p53+/+, HCT116 p53−/− and HCT116 p53−/− transfectants of wild-type, or mutant S315A, S315D p53 were co-transfected with MTS-EGFP and MTS-RFP, MTS-cyclin B1 and MTS-Cdk1, or MTS-cyclin B1 and MTS-delCdk1 for 24 h and irradiated with 5 Gy. Apoptotic cells with sub G0/G1 DNA content were measured 24 h post-irradiation by flow cytometry (n = 3; mean ± SEM; **P<0.01). (B) Cytosolic and mitochondrial cytochrome c in the same set of cells was measured 24 h after 5 Gy irradiation (α-tubulin and Cox4 included as loading controls). (C) Cyclin B1/Cdk1-mediated p53 phosphorylation inhibited the interaction of p53 to Bcl2 or Bcl-xL p53. HCT116 p53+/+ cells transfected with MTS-cyclin B1/MTS-Cdk1 or siRNAs against cyclin B1/Cdk1 for 24 h followed by 5 Gy irradiation. Cell lysates were immunoprecipitated with p53 antibody (IP) followed by immunoblotted (IB) using Bcl-2, Bcl-xL or p53 antibodies. HCT116 p53−/− cell lysates were included as negative control.
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pone-0012341-g006: Cyclin B1/Cdk1-mediated p53 phosphorylation inhibited mitochondria-mediated apoptosis.(A) HCT116 p53+/+, HCT116 p53−/− and HCT116 p53−/− transfectants of wild-type, or mutant S315A, S315D p53 were co-transfected with MTS-EGFP and MTS-RFP, MTS-cyclin B1 and MTS-Cdk1, or MTS-cyclin B1 and MTS-delCdk1 for 24 h and irradiated with 5 Gy. Apoptotic cells with sub G0/G1 DNA content were measured 24 h post-irradiation by flow cytometry (n = 3; mean ± SEM; **P<0.01). (B) Cytosolic and mitochondrial cytochrome c in the same set of cells was measured 24 h after 5 Gy irradiation (α-tubulin and Cox4 included as loading controls). (C) Cyclin B1/Cdk1-mediated p53 phosphorylation inhibited the interaction of p53 to Bcl2 or Bcl-xL p53. HCT116 p53+/+ cells transfected with MTS-cyclin B1/MTS-Cdk1 or siRNAs against cyclin B1/Cdk1 for 24 h followed by 5 Gy irradiation. Cell lysates were immunoprecipitated with p53 antibody (IP) followed by immunoblotted (IB) using Bcl-2, Bcl-xL or p53 antibodies. HCT116 p53−/− cell lysates were included as negative control.

Mentions: To determine the potential effect of cyclin B1/Cdk1-phosphorylated p53 in mitochondria-mediated apoptosis, we measured the apoptosis in irradiated HCT116 p53+/+ and HCT116 p53−/− cells with different p53 status. Little differences of the radiation-induced apoptosis were detected among HCT116 cells regardless of their p53 status (Figure S5B), which agreed with the previous report [47]. However, inhibition of cyclin B1 by siRNA knockdown along with irradiation dramatically increased the ratio of apoptotic population in cells with wild-type p53, but not in the cells with mutant p53 (Figure S5C). These results suggest that mitochondrial cyclin B1/Cdk1 is required for anti-apoptotic response mediated by p53. Co-overexpression of mitochondrial cyclin B1/Cdk1 also significantly decreased the apoptotic ratio by approximately 20% in cells expressing wild-type p53 after exposure to radiation (Figure 6A). Since initiation of mitochondria-mediated apoptosis starts with permeabilization of the mitochondrial outer membrane and the release of mitochondrial cytochrome c that triggers subsequent activation of caspase cascades [48], we measured levels of cytosolic and mitochondrial cytochrome c and observed a strikingly less cytosolic cytochrome c released from mitochondria after irradiation in cells expressing wild-type p53 transfected with MTS-cyclin B1/MTS-Cdk1, but not in cells with deficient or mutant p53 (Figure 6B).


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)

Cyclin B1/Cdk1-mediated p53 phosphorylation inhibited mitochondria-mediated apoptosis.(A) HCT116 p53+/+, HCT116 p53−/− and HCT116 p53−/− transfectants of wild-type, or mutant S315A, S315D p53 were co-transfected with MTS-EGFP and MTS-RFP, MTS-cyclin B1 and MTS-Cdk1, or MTS-cyclin B1 and MTS-delCdk1 for 24 h and irradiated with 5 Gy. Apoptotic cells with sub G0/G1 DNA content were measured 24 h post-irradiation by flow cytometry (n = 3; mean ± SEM; **P<0.01). (B) Cytosolic and mitochondrial cytochrome c in the same set of cells was measured 24 h after 5 Gy irradiation (α-tubulin and Cox4 included as loading controls). (C) Cyclin B1/Cdk1-mediated p53 phosphorylation inhibited the interaction of p53 to Bcl2 or Bcl-xL p53. HCT116 p53+/+ cells transfected with MTS-cyclin B1/MTS-Cdk1 or siRNAs against cyclin B1/Cdk1 for 24 h followed by 5 Gy irradiation. Cell lysates were immunoprecipitated with p53 antibody (IP) followed by immunoblotted (IB) using Bcl-2, Bcl-xL or p53 antibodies. HCT116 p53−/− cell lysates were included as negative control.
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pone-0012341-g006: Cyclin B1/Cdk1-mediated p53 phosphorylation inhibited mitochondria-mediated apoptosis.(A) HCT116 p53+/+, HCT116 p53−/− and HCT116 p53−/− transfectants of wild-type, or mutant S315A, S315D p53 were co-transfected with MTS-EGFP and MTS-RFP, MTS-cyclin B1 and MTS-Cdk1, or MTS-cyclin B1 and MTS-delCdk1 for 24 h and irradiated with 5 Gy. Apoptotic cells with sub G0/G1 DNA content were measured 24 h post-irradiation by flow cytometry (n = 3; mean ± SEM; **P<0.01). (B) Cytosolic and mitochondrial cytochrome c in the same set of cells was measured 24 h after 5 Gy irradiation (α-tubulin and Cox4 included as loading controls). (C) Cyclin B1/Cdk1-mediated p53 phosphorylation inhibited the interaction of p53 to Bcl2 or Bcl-xL p53. HCT116 p53+/+ cells transfected with MTS-cyclin B1/MTS-Cdk1 or siRNAs against cyclin B1/Cdk1 for 24 h followed by 5 Gy irradiation. Cell lysates were immunoprecipitated with p53 antibody (IP) followed by immunoblotted (IB) using Bcl-2, Bcl-xL or p53 antibodies. HCT116 p53−/− cell lysates were included as negative control.
Mentions: To determine the potential effect of cyclin B1/Cdk1-phosphorylated p53 in mitochondria-mediated apoptosis, we measured the apoptosis in irradiated HCT116 p53+/+ and HCT116 p53−/− cells with different p53 status. Little differences of the radiation-induced apoptosis were detected among HCT116 cells regardless of their p53 status (Figure S5B), which agreed with the previous report [47]. However, inhibition of cyclin B1 by siRNA knockdown along with irradiation dramatically increased the ratio of apoptotic population in cells with wild-type p53, but not in the cells with mutant p53 (Figure S5C). These results suggest that mitochondrial cyclin B1/Cdk1 is required for anti-apoptotic response mediated by p53. Co-overexpression of mitochondrial cyclin B1/Cdk1 also significantly decreased the apoptotic ratio by approximately 20% in cells expressing wild-type p53 after exposure to radiation (Figure 6A). Since initiation of mitochondria-mediated apoptosis starts with permeabilization of the mitochondrial outer membrane and the release of mitochondrial cytochrome c that triggers subsequent activation of caspase cascades [48], we measured levels of cytosolic and mitochondrial cytochrome c and observed a strikingly less cytosolic cytochrome c released from mitochondria after irradiation in cells expressing wild-type p53 transfected with MTS-cyclin B1/MTS-Cdk1, but not in cells with deficient or mutant p53 (Figure 6B).

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