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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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Mitochondrial Cdk1 interacted and phosphorylated p53.(A) Mitochondria fractions (100 µg) from control (sham-irradiated) and irradiated HCT116 p53+/+ cells (HCT116 p53−/− cells included as a negative control) were immunoprecipitated (IP) with p53 antibody followed by immunoblotting (IB) with Cdk1 antibody (top panel); IP with Cdk1 antibody followed by IB with p53 antibody (bottom panel). (B) Kinase assay was performed with Cdk1 purified with IP from total 2.4 mg of mitochondrial fraction isolated from irradiated HCT116 p53+/+ and incubated with 8 µg of GST-p53 (wild-type or mutants at S315A or S315D). The same amount of histone H1 was included as a positive phosphorylation substrate for Cdk1, and GST-protein only served as a negative control (mitochondrial Cdk1 mediated p53 phosphorylation was estimated by densitometry in comparison to the phosphorylation level of histone H1).
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pone-0012341-g002: Mitochondrial Cdk1 interacted and phosphorylated p53.(A) Mitochondria fractions (100 µg) from control (sham-irradiated) and irradiated HCT116 p53+/+ cells (HCT116 p53−/− cells included as a negative control) were immunoprecipitated (IP) with p53 antibody followed by immunoblotting (IB) with Cdk1 antibody (top panel); IP with Cdk1 antibody followed by IB with p53 antibody (bottom panel). (B) Kinase assay was performed with Cdk1 purified with IP from total 2.4 mg of mitochondrial fraction isolated from irradiated HCT116 p53+/+ and incubated with 8 µg of GST-p53 (wild-type or mutants at S315A or S315D). The same amount of histone H1 was included as a positive phosphorylation substrate for Cdk1, and GST-protein only served as a negative control (mitochondrial Cdk1 mediated p53 phosphorylation was estimated by densitometry in comparison to the phosphorylation level of histone H1).

Mentions: To determine whether mitochondrial cyclin B1/Cdk1 complex is capable to phosphorylate p53 in mitochondria, mitochondrial fractionation were performed from irradiated HCT116 p53+/+ cells and interaction between mitochondrial Cdk1 and p53 was detected by co-immunoprecipitation. Robust Cdk1/p53 interaction was detected in the mitochondrial fraction of irradiated HCT 116 p53+/+ cells (Figure 2A); this increased Cdk1/p53 interaction started as early as 2 to 4 hours after irradiation (Figure S1A). To verify whether mitochondria-translocated Cdk1 is active, in vivo kinase assay was performed using immune-isolated Cdk1 from mitochondrial fraction of irradiated HCT 116 p53+/+ cells with GST-tagged synthesized p53 (wild-type, Ser-315 to Ala or Ser-315 to Asp mutants) and Histone protein (H1) as the target substrates. Results in Figure 2B demonstrated that mitochondrial Cdk1 indeed demonstrated its kinase activity and phosphorylated wild-type p53 and the positive control Histone H1; in contrast, Cdk1 mediated phosphorylation was markedly reduced in the mutant p53 (S315A, phosphorylation inhibit) but not the phosphorylation counterfeit mutant p53 (S315D) [38]. The substrate specificity of the kinase assay was confirmed in Figure S1B. Together, these results demonstrate that mitochondrial translocated Cdk1 retains its kinase activity and is able to phosphorylate p53 at Ser-315.


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)

Mitochondrial Cdk1 interacted and phosphorylated p53.(A) Mitochondria fractions (100 µg) from control (sham-irradiated) and irradiated HCT116 p53+/+ cells (HCT116 p53−/− cells included as a negative control) were immunoprecipitated (IP) with p53 antibody followed by immunoblotting (IB) with Cdk1 antibody (top panel); IP with Cdk1 antibody followed by IB with p53 antibody (bottom panel). (B) Kinase assay was performed with Cdk1 purified with IP from total 2.4 mg of mitochondrial fraction isolated from irradiated HCT116 p53+/+ and incubated with 8 µg of GST-p53 (wild-type or mutants at S315A or S315D). The same amount of histone H1 was included as a positive phosphorylation substrate for Cdk1, and GST-protein only served as a negative control (mitochondrial Cdk1 mediated p53 phosphorylation was estimated by densitometry in comparison to the phosphorylation level of histone H1).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0012341-g002: Mitochondrial Cdk1 interacted and phosphorylated p53.(A) Mitochondria fractions (100 µg) from control (sham-irradiated) and irradiated HCT116 p53+/+ cells (HCT116 p53−/− cells included as a negative control) were immunoprecipitated (IP) with p53 antibody followed by immunoblotting (IB) with Cdk1 antibody (top panel); IP with Cdk1 antibody followed by IB with p53 antibody (bottom panel). (B) Kinase assay was performed with Cdk1 purified with IP from total 2.4 mg of mitochondrial fraction isolated from irradiated HCT116 p53+/+ and incubated with 8 µg of GST-p53 (wild-type or mutants at S315A or S315D). The same amount of histone H1 was included as a positive phosphorylation substrate for Cdk1, and GST-protein only served as a negative control (mitochondrial Cdk1 mediated p53 phosphorylation was estimated by densitometry in comparison to the phosphorylation level of histone H1).
Mentions: To determine whether mitochondrial cyclin B1/Cdk1 complex is capable to phosphorylate p53 in mitochondria, mitochondrial fractionation were performed from irradiated HCT116 p53+/+ cells and interaction between mitochondrial Cdk1 and p53 was detected by co-immunoprecipitation. Robust Cdk1/p53 interaction was detected in the mitochondrial fraction of irradiated HCT 116 p53+/+ cells (Figure 2A); this increased Cdk1/p53 interaction started as early as 2 to 4 hours after irradiation (Figure S1A). To verify whether mitochondria-translocated Cdk1 is active, in vivo kinase assay was performed using immune-isolated Cdk1 from mitochondrial fraction of irradiated HCT 116 p53+/+ cells with GST-tagged synthesized p53 (wild-type, Ser-315 to Ala or Ser-315 to Asp mutants) and Histone protein (H1) as the target substrates. Results in Figure 2B demonstrated that mitochondrial Cdk1 indeed demonstrated its kinase activity and phosphorylated wild-type p53 and the positive control Histone H1; in contrast, Cdk1 mediated phosphorylation was markedly reduced in the mutant p53 (S315A, phosphorylation inhibit) but not the phosphorylation counterfeit mutant p53 (S315D) [38]. The substrate specificity of the kinase assay was confirmed in Figure S1B. Together, these results demonstrate that mitochondrial translocated Cdk1 retains its kinase activity and is able to phosphorylate p53 at Ser-315.

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