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HIPK2 sustains apoptotic response by phosphorylating Che-1/AATF and promoting its degradation.

De Nicola F, Catena V, Rinaldo C, Bruno T, Iezzi S, Sorino C, Desantis A, Camerini S, Crescenzi M, Floridi A, Passananti C, Soddu S, Fanciulli M - Cell Death Dis (2014)

Bottom Line: This event strongly increases HDM2/Che-1 interaction and degradation of Che-1 protein via ubiquitin-dependent proteasomal system.In agreement with these findings, we found that HIPK2 depletion strongly decreases Che-1 ubiquitylation and degradation.Notably, Che-1 overexpression strongly counteracts HIPK2-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Epigenetics Laboratory, Regina Elena National Cancer Institute, Rome, Italy.

ABSTRACT
Che-1/AATF is an RNA polymerase II-binding protein that is involved in the regulation of gene transcription, which undergoes stabilization and accumulation in response to DNA damage. We have previously demonstrated that following apoptotic induction, Che-1 protein levels are downregulated through its interaction with the E3 ligase HDM2, which leads to Che-1 degradation by ubiquitylation. This interaction is mediated by Pin1, which determines a phosphorylation-dependent conformational change. Here we demonstrate that HIPK2, a proapoptotic kinase, is involved in Che-1 degradation. HIPK2 interacts with Che-1 and, upon genotoxic stress, phosphorylates it at specific residues. This event strongly increases HDM2/Che-1 interaction and degradation of Che-1 protein via ubiquitin-dependent proteasomal system. In agreement with these findings, we found that HIPK2 depletion strongly decreases Che-1 ubiquitylation and degradation. Notably, Che-1 overexpression strongly counteracts HIPK2-induced apoptosis. Our results establish Che-1 as a new HIPK2 target and confirm its important role in the cellular response to DNA damage.

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Che-1 interacts with HIPK2 in response to apoptosis. (a) HCT116 cells were transiently transfected with siRNA (siControl) or siRNA HIPK2 (siHIPK2), and 24 h later the total cell extracts (TCEs) were analyzed by western blotting (WB) with the indicated antibodies (Abs). (b and c) HCT116 cells were treated for 24 h with 2 μM ADR. TCEs were immunoprecipitated with anti-HIPK2 (b) or anti-Che-1 (c) antibodies and analyzed by WB using the indicated antibodies. (d) HCT116 cells were transiently transfected with expression vectors Myc-Che-1, GFP-HIPK2 and its deletion mutant. TCEs were immunoprecipitated with anti-Myc antibody and analyzed by WB using the indicated Abs. (e) HCT116 cells were transfected with expression vectors GFP-HIPK2, Myc-Che-1 and its deletion mutants. TCEs were immunoprecipitated and analyzed as in d. The bottom panel shows a schematic representation of full-length Che-1 protein and its deletion mutants
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fig1: Che-1 interacts with HIPK2 in response to apoptosis. (a) HCT116 cells were transiently transfected with siRNA (siControl) or siRNA HIPK2 (siHIPK2), and 24 h later the total cell extracts (TCEs) were analyzed by western blotting (WB) with the indicated antibodies (Abs). (b and c) HCT116 cells were treated for 24 h with 2 μM ADR. TCEs were immunoprecipitated with anti-HIPK2 (b) or anti-Che-1 (c) antibodies and analyzed by WB using the indicated antibodies. (d) HCT116 cells were transiently transfected with expression vectors Myc-Che-1, GFP-HIPK2 and its deletion mutant. TCEs were immunoprecipitated with anti-Myc antibody and analyzed by WB using the indicated Abs. (e) HCT116 cells were transfected with expression vectors GFP-HIPK2, Myc-Che-1 and its deletion mutants. TCEs were immunoprecipitated and analyzed as in d. The bottom panel shows a schematic representation of full-length Che-1 protein and its deletion mutants

Mentions: As described above, Che-1 phosphorylation at T144 seems to have an important role in the apoptosis process, and as this residue is a canonical motif for HIPK2 phosphorylation we wondered whether Che-1 could be a new HIPK2 target during apoptosis activation. For this reason, we started to analyze the role of HIPK2 in this modification by testing whether the endogenous HIPK2 and Che-1 proteins physically interact. As the anti-HIPK2 antibody shows several unspecific background bands,32 we performed HIPK2 depletion in HCT116 cells by siRNA to identify the specific signal (Figure 1a). As shown in Figure 1b, Che-1 co-precipitates with HIPK2 in control cells and, notably, the amount of Che-1 co-precipitating with HIPK2 strongly increases in response to an apoptotic dose of ADR. As expected, an increase of HIPK2 expression and a concomitant decrease of Che-1 protein levels occur in HCT116 cells after ADR treatment9, 23 (Figure 1b). In agreement, a coimmunoprecipitation performed with anti-Che-1 antibody confirmed Che-1/HIPK2 interaction (Figure 1c). Furthermore, we observed that exogenous Myc-tagged Che-1 co-precipitates with exogenous GFP-tagged HIPK2 and, consistent with the results obtained with endogenous protein, exogenous Che-1 showed a greater affinity for the caspases-cleaved, active form of HIPK2 (1–838)33 compared with wt (Figure 1d) Finally, an analysis performed with several Che-1 mutants revealed that the region 1–163 of this protein is required for its interaction with HIPK2 (Figure 1e). Together, these findings indicate that the two proteins belong to the same complex and that apoptotic induction increases the presence of Che-1 in this complex.


HIPK2 sustains apoptotic response by phosphorylating Che-1/AATF and promoting its degradation.

De Nicola F, Catena V, Rinaldo C, Bruno T, Iezzi S, Sorino C, Desantis A, Camerini S, Crescenzi M, Floridi A, Passananti C, Soddu S, Fanciulli M - Cell Death Dis (2014)

Che-1 interacts with HIPK2 in response to apoptosis. (a) HCT116 cells were transiently transfected with siRNA (siControl) or siRNA HIPK2 (siHIPK2), and 24 h later the total cell extracts (TCEs) were analyzed by western blotting (WB) with the indicated antibodies (Abs). (b and c) HCT116 cells were treated for 24 h with 2 μM ADR. TCEs were immunoprecipitated with anti-HIPK2 (b) or anti-Che-1 (c) antibodies and analyzed by WB using the indicated antibodies. (d) HCT116 cells were transiently transfected with expression vectors Myc-Che-1, GFP-HIPK2 and its deletion mutant. TCEs were immunoprecipitated with anti-Myc antibody and analyzed by WB using the indicated Abs. (e) HCT116 cells were transfected with expression vectors GFP-HIPK2, Myc-Che-1 and its deletion mutants. TCEs were immunoprecipitated and analyzed as in d. The bottom panel shows a schematic representation of full-length Che-1 protein and its deletion mutants
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4225224&req=5

fig1: Che-1 interacts with HIPK2 in response to apoptosis. (a) HCT116 cells were transiently transfected with siRNA (siControl) or siRNA HIPK2 (siHIPK2), and 24 h later the total cell extracts (TCEs) were analyzed by western blotting (WB) with the indicated antibodies (Abs). (b and c) HCT116 cells were treated for 24 h with 2 μM ADR. TCEs were immunoprecipitated with anti-HIPK2 (b) or anti-Che-1 (c) antibodies and analyzed by WB using the indicated antibodies. (d) HCT116 cells were transiently transfected with expression vectors Myc-Che-1, GFP-HIPK2 and its deletion mutant. TCEs were immunoprecipitated with anti-Myc antibody and analyzed by WB using the indicated Abs. (e) HCT116 cells were transfected with expression vectors GFP-HIPK2, Myc-Che-1 and its deletion mutants. TCEs were immunoprecipitated and analyzed as in d. The bottom panel shows a schematic representation of full-length Che-1 protein and its deletion mutants
Mentions: As described above, Che-1 phosphorylation at T144 seems to have an important role in the apoptosis process, and as this residue is a canonical motif for HIPK2 phosphorylation we wondered whether Che-1 could be a new HIPK2 target during apoptosis activation. For this reason, we started to analyze the role of HIPK2 in this modification by testing whether the endogenous HIPK2 and Che-1 proteins physically interact. As the anti-HIPK2 antibody shows several unspecific background bands,32 we performed HIPK2 depletion in HCT116 cells by siRNA to identify the specific signal (Figure 1a). As shown in Figure 1b, Che-1 co-precipitates with HIPK2 in control cells and, notably, the amount of Che-1 co-precipitating with HIPK2 strongly increases in response to an apoptotic dose of ADR. As expected, an increase of HIPK2 expression and a concomitant decrease of Che-1 protein levels occur in HCT116 cells after ADR treatment9, 23 (Figure 1b). In agreement, a coimmunoprecipitation performed with anti-Che-1 antibody confirmed Che-1/HIPK2 interaction (Figure 1c). Furthermore, we observed that exogenous Myc-tagged Che-1 co-precipitates with exogenous GFP-tagged HIPK2 and, consistent with the results obtained with endogenous protein, exogenous Che-1 showed a greater affinity for the caspases-cleaved, active form of HIPK2 (1–838)33 compared with wt (Figure 1d) Finally, an analysis performed with several Che-1 mutants revealed that the region 1–163 of this protein is required for its interaction with HIPK2 (Figure 1e). Together, these findings indicate that the two proteins belong to the same complex and that apoptotic induction increases the presence of Che-1 in this complex.

Bottom Line: This event strongly increases HDM2/Che-1 interaction and degradation of Che-1 protein via ubiquitin-dependent proteasomal system.In agreement with these findings, we found that HIPK2 depletion strongly decreases Che-1 ubiquitylation and degradation.Notably, Che-1 overexpression strongly counteracts HIPK2-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Epigenetics Laboratory, Regina Elena National Cancer Institute, Rome, Italy.

ABSTRACT
Che-1/AATF is an RNA polymerase II-binding protein that is involved in the regulation of gene transcription, which undergoes stabilization and accumulation in response to DNA damage. We have previously demonstrated that following apoptotic induction, Che-1 protein levels are downregulated through its interaction with the E3 ligase HDM2, which leads to Che-1 degradation by ubiquitylation. This interaction is mediated by Pin1, which determines a phosphorylation-dependent conformational change. Here we demonstrate that HIPK2, a proapoptotic kinase, is involved in Che-1 degradation. HIPK2 interacts with Che-1 and, upon genotoxic stress, phosphorylates it at specific residues. This event strongly increases HDM2/Che-1 interaction and degradation of Che-1 protein via ubiquitin-dependent proteasomal system. In agreement with these findings, we found that HIPK2 depletion strongly decreases Che-1 ubiquitylation and degradation. Notably, Che-1 overexpression strongly counteracts HIPK2-induced apoptosis. Our results establish Che-1 as a new HIPK2 target and confirm its important role in the cellular response to DNA damage.

Show MeSH
Related in: MedlinePlus