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Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway.

Antunes AT, Goos YJ, Pereboom TC, Hermkens D, Wlodarski MW, Da Costa L, MacInnes AW - PLoS Genet. (2015)

Bottom Line: In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells.Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53.By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53.

View Article: PubMed Central - PubMed

Affiliation: Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.

ABSTRACT
Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes.

No MeSH data available.


Related in: MedlinePlus

p53 protein stabilization is impaired independently of p53 mRNA levels.A) qPCR analysis measuring levels of p53 mRNA in wild type or rpS7 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. B) qPCR analysis of p53 mRNA levels in wild type or rpL11 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. *p<0.05. C) Western blot analysis of p53 protein levels and the quantification of the p53:actin ratio of in rpS7 or rpL11 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. * indicates either a p53-specific isoform or a degradation product.
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pgen.1005326.g003: p53 protein stabilization is impaired independently of p53 mRNA levels.A) qPCR analysis measuring levels of p53 mRNA in wild type or rpS7 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. B) qPCR analysis of p53 mRNA levels in wild type or rpL11 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. *p<0.05. C) Western blot analysis of p53 protein levels and the quantification of the p53:actin ratio of in rpS7 or rpL11 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. * indicates either a p53-specific isoform or a degradation product.

Mentions: The increased transcription of the zebrafish p53 gene and its p53Δ113 isoform (a target gene of stabilized p53) has been described in several models of RP loss and likely reflects the early response of p53 that triggers an up regulation of its own transcription and the transcription of p53Δ113 [15,35,38–41]. In line with these results, we found using real-time quantitative PCR analysis with primers that amplify both full-length p53 and p53Δ113 that p53 mRNA levels were significantly higher in rpS7 and rpL11 mutants at 1 and 2 dpf both in the presence and absence of ionizing radiation compared to untreated wild type embryos (Fig 3A and 3B). Semi-quantitative PCR analysis of p53 mRNA levels in several other RP-mutant embryos (rpS3a, rpL23a, and rpL36) at 2 dpf similarly revealed equivalent levels of p53 transcription in the mutants compared to wild types (S3 Fig). However, when we performed western blotting analysis using a zebrafish p53-specific antibody, we were unable to detect any appreciable amount of p53 protein in the rpS7 or rpL11 mutants in either the presence or absence of ionizing radiation at either 1 or 2 dpf (Fig 3C). This is the case in all the mutant RP lines we tested including in rpS3a, rpL23a, and rpL36 (S3 Fig). We often observe what may be a p53-specific isoform such as p53Δ113 on the western blots, but this may also be a p53 degradation product and in this work we cannot be certain of its exact identity. Taken together, the results suggest that although the p53 response to the RP mutation on a transcriptional level may function normally, an additional level of p53 post-translational regulation exists in the presence of RP mutations that serves to reduce p53 protein.


Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway.

Antunes AT, Goos YJ, Pereboom TC, Hermkens D, Wlodarski MW, Da Costa L, MacInnes AW - PLoS Genet. (2015)

p53 protein stabilization is impaired independently of p53 mRNA levels.A) qPCR analysis measuring levels of p53 mRNA in wild type or rpS7 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. B) qPCR analysis of p53 mRNA levels in wild type or rpL11 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. *p<0.05. C) Western blot analysis of p53 protein levels and the quantification of the p53:actin ratio of in rpS7 or rpL11 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. * indicates either a p53-specific isoform or a degradation product.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488577&req=5

pgen.1005326.g003: p53 protein stabilization is impaired independently of p53 mRNA levels.A) qPCR analysis measuring levels of p53 mRNA in wild type or rpS7 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. B) qPCR analysis of p53 mRNA levels in wild type or rpL11 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. *p<0.05. C) Western blot analysis of p53 protein levels and the quantification of the p53:actin ratio of in rpS7 or rpL11 mutants at 1 or 2 dpf either untreated or exposed to 25 Gy ionizing radiation. * indicates either a p53-specific isoform or a degradation product.
Mentions: The increased transcription of the zebrafish p53 gene and its p53Δ113 isoform (a target gene of stabilized p53) has been described in several models of RP loss and likely reflects the early response of p53 that triggers an up regulation of its own transcription and the transcription of p53Δ113 [15,35,38–41]. In line with these results, we found using real-time quantitative PCR analysis with primers that amplify both full-length p53 and p53Δ113 that p53 mRNA levels were significantly higher in rpS7 and rpL11 mutants at 1 and 2 dpf both in the presence and absence of ionizing radiation compared to untreated wild type embryos (Fig 3A and 3B). Semi-quantitative PCR analysis of p53 mRNA levels in several other RP-mutant embryos (rpS3a, rpL23a, and rpL36) at 2 dpf similarly revealed equivalent levels of p53 transcription in the mutants compared to wild types (S3 Fig). However, when we performed western blotting analysis using a zebrafish p53-specific antibody, we were unable to detect any appreciable amount of p53 protein in the rpS7 or rpL11 mutants in either the presence or absence of ionizing radiation at either 1 or 2 dpf (Fig 3C). This is the case in all the mutant RP lines we tested including in rpS3a, rpL23a, and rpL36 (S3 Fig). We often observe what may be a p53-specific isoform such as p53Δ113 on the western blots, but this may also be a p53 degradation product and in this work we cannot be certain of its exact identity. Taken together, the results suggest that although the p53 response to the RP mutation on a transcriptional level may function normally, an additional level of p53 post-translational regulation exists in the presence of RP mutations that serves to reduce p53 protein.

Bottom Line: In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells.Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53.By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53.

View Article: PubMed Central - PubMed

Affiliation: Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.

ABSTRACT
Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes.

No MeSH data available.


Related in: MedlinePlus