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Autoregulatory control of the p53 response by caspase-mediated processing of HIPK2.

ABSTRACT: The serine/threonine kinase HIPK2 phosphorylates the p53 protein at Ser 46, thus promoting p53-dependent gene expression and subsequent apoptosis. Here, we show that DNA damaging chemotherapeutic drugs cause degradation of endogenous HIPK2 dependent on the presence of a functional p53 protein. Early induced p53 allows caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977. The resulting C-terminally truncated HIPK2 forms show an enhanced induction of the p53 response and cell death, thus allowing the rapid amplification of the p53-dependent apoptotic program during the initiation phase of apoptosis by a regulatory feed-forward loop. The active HIPK2 fragments are further degraded during the execution and termination phase of apoptosis, thus ensuring the occurrence of HIPK2 signaling only during the early phases of apoptosis induction.

SUBMITTER: Gresko E

PROVIDER: S-EPMC1456935 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Autoregulatory control of the p53 response by caspase-mediated processing of HIPK2.

Gresko Ekaterina E Roscic Ana A Ritterhoff Stefanie S Vichalkovski Anton A del Sal Giannino G Schmitz M Lienhard ML

The EMBO journal 20060406 9

The serine/threonine kinase HIPK2 phosphorylates the p53 protein at Ser 46, thus promoting p53-dependent gene expression and subsequent apoptosis. Here, we show that DNA damaging chemotherapeutic drugs cause degradation of endogenous HIPK2 dependent on the presence of a functional p53 protein. Early induced p53 allows caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977. The resulting C-terminally truncated HIPK2 forms show an enhanced induction of the p53 response and cell de ...[more]

PMID: 16601678

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Project description:BackgroundActivation of p53-mediated gene transcription is a critical cellular response to DNA damage and involves a phosphorylation-acetylation cascade of p53. The discovery of differences in the response to different agents raises the question whether some of the p53 oncosuppressor functions might be exerted by different posttranslational modifications. Stress-induced homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates p53 at serine-46 (Ser46) for p53 apoptotic activity; p53 acetylation at different C-terminus lysines including p300-mediated lysine-382 (Lys382) is also required for full activation of p53 transcriptional activity. The purpose of the current study was to evaluate the interplay among HIPK2, p300, and p53 in p53 acetylation and apoptotic transcriptional activity in response to drug by using siRNA interference, p300 overexpression or deacetylase inhibitors, in cancer cells.ResultsKnockdown of HIPK2 inhibited both adriamycin-induced Ser46 phosphorylation and Lys382 acetylation in p53 protein; however, while combination of ADR and zinc restored Ser46 phosphorylation it did not recover Lys382 acetylation. Chromatin immunoprecipitation studies showed that HIPK2 was required in vivo for efficient p300/p53 co-recruitment onto apoptotic promoters and that both p53 modifications at Ser46 and Lys382 were necessary for p53 apoptotic transcription. Thus, p53Lys382 acetylation in HIPK2 knockdown as well as p53 apoptotic activity in response to drug could be rescued by p300 overexpression. Similar effect was obtained with the Sirt1-inhibitor nicotinamide. Interestingly trichostatin A (TSA), the inhibitor of histone deacetylase complexes (HDAC) did not have effect, suggesting that Sirt1 was the deacetylase involved in p53 deacetylation in HIPK2 knockdown.ConclusionThese results reveal a novel role for HIPK2 in activating p53 apoptotic transcription. Our results indicate that HIPK2 may regulate the balance between p53 acetylation and deacetylation, by stimulating on one hand co-recruitment of p300 and p53Lys382 on apoptotic promoters and on the other hand by inhibiting Sirt1 deacetylase activity. We attempted to reactivate p53 apoptotic transcriptional activity by rescuing both Ser46 and Lys382 modification in response to drug. Our data propose combination strategies for the treatment of tumors with dysfunctional p53 and/or HIPK2 that include classical chemotherapy with pharmacological or natural agents such as Sirt1-deacetylase inhibitors or zinc, respectively.

Project description:Dysregulation of the mitochondrial signaling pathway of apoptosis induction represents a major hurdle in tumor therapy. The objective of the presented work was to investigate the role of the intrinsic (mitochondrial) apoptotic pathway in the non-small lung cancer cell line NCI-H460 upon induction of apoptosis using the highly bioactive TRAIL derivative Db-scTRAIL. NCI-H460 cells were TRAIL sensitive but an only about 3 fold overexpression of Bcl-2 was sufficient to induce a highly TRAIL resistant phenotype, confirming that the mitochondrial pathway is crucial for TRAIL-induced apoptosis induction. TRAIL resistance was paralleled by a strong inhibition of caspase-8, -9 and -3 activities and blocked their full processing. Notably, especially the final cleavage steps of the initiator caspase-8 and the executioner caspase-3 were effectively blocked by Bcl-2 overexpression. Caspase-9 knockdown failed to protect NCI-H460 cells from TRAIL-induced cell death, suggesting a minor role of this initiator caspase in this apoptotic pathway. Rather, knockdown of the XIAP antagonist Smac resulted in enhanced caspase-3 degradation after stimulation of cells with TRAIL. Of note, downregulation of XIAP had only limited effects on TRAIL sensitivity of wild-type NCI-H460 cells, but resensitized Bcl-2 overexpressing cells for TRAIL-induced apoptosis. In particular, XIAP knockdown in combination with TRAIL allowed the final cleavage step of caspase-3 to generate the catalytically active p17 fragment, whose production was otherwise blocked in Bcl-2 overexpressing cells. Together, our data strongly suggest that XIAP-mediated inhibition of final caspase-3 processing is the last and major hurdle in TRAIL-induced apoptosis in NCI-H460 cells, which can be overcome by Smac in a Bcl-2 level dependent manner. Quantitative investigation of the XIAP/Smac interplay using a mathematical model approach corroborates our experimental data strengthening the suggested roles of XIAP and Smac as critical determinants for TRAIL sensitivity.

Dataset Information

Autoregulatory control of the p53 response by caspase-mediated processing of HIPK2.

Publications

Autoregulatory control of the p53 response by caspase-mediated processing of HIPK2.

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