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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: 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.

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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HIPK2 promotes Che-1 ubiquitylation. (a) HCT116 cells were transiently transfected with GFP-HIPK2 or GFP-K221R expression vectors. TCEs were immunoprecipitated with anti-MDM2 antibody and analyzed by WB with the indicated Abs. (b) HEK293 cells were co-transfected with Myc-Che-1, Ha-Ubiquitin and GFP-HIPK2 or GFP-K221R and treated with 10 μM MG132 for 16 h. TCEs were immunoprecipitated with anti-Myc monoclonal antibody and analyzed by WB with the indicated Abs. (c) HEK293 cells transfected with Myc-Che-1, Ha-Ubiquitin and GFP-HIPK2 or GFP-HIPK2 (1–838) expression vectors and treated with MG132 as in B. TCEs were immunoprecipitated as in B and analyzed by WB with the indicated Abs. (d) HEK293 cells were transiently transfected with indicated Che-1 deletion mutants, Ha-Ubiquitin and GFP-HIPK2, and treated with MG132 as in B. TCEs were immunoprecipitated as in b and analyzed by WB with the indicated Abs. In the right panel, a schematic representation of full-length Che-1 protein and two of its deletion mutants is provided
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fig4: HIPK2 promotes Che-1 ubiquitylation. (a) HCT116 cells were transiently transfected with GFP-HIPK2 or GFP-K221R expression vectors. TCEs were immunoprecipitated with anti-MDM2 antibody and analyzed by WB with the indicated Abs. (b) HEK293 cells were co-transfected with Myc-Che-1, Ha-Ubiquitin and GFP-HIPK2 or GFP-K221R and treated with 10 μM MG132 for 16 h. TCEs were immunoprecipitated with anti-Myc monoclonal antibody and analyzed by WB with the indicated Abs. (c) HEK293 cells transfected with Myc-Che-1, Ha-Ubiquitin and GFP-HIPK2 or GFP-HIPK2 (1–838) expression vectors and treated with MG132 as in B. TCEs were immunoprecipitated as in B and analyzed by WB with the indicated Abs. (d) HEK293 cells were transiently transfected with indicated Che-1 deletion mutants, Ha-Ubiquitin and GFP-HIPK2, and treated with MG132 as in B. TCEs were immunoprecipitated as in b and analyzed by WB with the indicated Abs. In the right panel, a schematic representation of full-length Che-1 protein and two of its deletion mutants is provided

Mentions: As previously described, Che-1 half-life is tightly regulated by the proteasome pathway, and in response to apoptotic stimuli HDM2 protein negatively regulates Che-1 by promoting its ubiquitin-mediated degradation.9 On the basis of the above results, we evaluated whether HIPK2 could affect Che-1/HDM2 interaction by coimmunoprecipitation with anti-HDM2 antibody in HCT116 cells overexpressing HIPK2 or its K221R mutant and treated with ADR. As shown in Figure 4a, HCT116 cells overexpressing HIPK2 wt showed an increase of Che-1 bound to HDM2, whereas this effect was not observed in cells transfected with the K221R mutant. Consistent with these findings, HIPK2 overexpression produced a strong increase in the ubiquitylation of Che-1, which is not found in cells overexpressing the K221R mutant (Figure 4b), indicating that the kinase activity of HIPK2 is required for the interaction between Che-1 and HDM2 and Che-1 ubiquitylation. Notably, the superactive form of HIPK2 (1–838) was found to be more active in Che-1 ubiquitylation (Figure 4c), reinforcing the involvement of HIPK2 in Che-1 degradation in response to apoptotic induction. To further characterize the role of HIPK2 in Che-1 degradation, we transiently transfected HCT116 cells with HIPK2 and Che-1 in the wt form or two different deletion mutants (1–163 and 164–558). In agreement with previous results, we found that exogenous HIPK2 expression produces a strong ubiquitylation of Che-1 wt and 1–163 mutant, whereas the polypeptide 164–558 lacking T144 did not exhibit any ubiquitylation (Figure 4d). Taken together, these results support the hypothesis that upon induction of apoptosis HIPK2 phosphorylates Che-1, thereby increasing its affinity for HDM2, its ubiquitylation and its degradation.


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)

HIPK2 promotes Che-1 ubiquitylation. (a) HCT116 cells were transiently transfected with GFP-HIPK2 or GFP-K221R expression vectors. TCEs were immunoprecipitated with anti-MDM2 antibody and analyzed by WB with the indicated Abs. (b) HEK293 cells were co-transfected with Myc-Che-1, Ha-Ubiquitin and GFP-HIPK2 or GFP-K221R and treated with 10 μM MG132 for 16 h. TCEs were immunoprecipitated with anti-Myc monoclonal antibody and analyzed by WB with the indicated Abs. (c) HEK293 cells transfected with Myc-Che-1, Ha-Ubiquitin and GFP-HIPK2 or GFP-HIPK2 (1–838) expression vectors and treated with MG132 as in B. TCEs were immunoprecipitated as in B and analyzed by WB with the indicated Abs. (d) HEK293 cells were transiently transfected with indicated Che-1 deletion mutants, Ha-Ubiquitin and GFP-HIPK2, and treated with MG132 as in B. TCEs were immunoprecipitated as in b and analyzed by WB with the indicated Abs. In the right panel, a schematic representation of full-length Che-1 protein and two of its deletion mutants is provided
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: HIPK2 promotes Che-1 ubiquitylation. (a) HCT116 cells were transiently transfected with GFP-HIPK2 or GFP-K221R expression vectors. TCEs were immunoprecipitated with anti-MDM2 antibody and analyzed by WB with the indicated Abs. (b) HEK293 cells were co-transfected with Myc-Che-1, Ha-Ubiquitin and GFP-HIPK2 or GFP-K221R and treated with 10 μM MG132 for 16 h. TCEs were immunoprecipitated with anti-Myc monoclonal antibody and analyzed by WB with the indicated Abs. (c) HEK293 cells transfected with Myc-Che-1, Ha-Ubiquitin and GFP-HIPK2 or GFP-HIPK2 (1–838) expression vectors and treated with MG132 as in B. TCEs were immunoprecipitated as in B and analyzed by WB with the indicated Abs. (d) HEK293 cells were transiently transfected with indicated Che-1 deletion mutants, Ha-Ubiquitin and GFP-HIPK2, and treated with MG132 as in B. TCEs were immunoprecipitated as in b and analyzed by WB with the indicated Abs. In the right panel, a schematic representation of full-length Che-1 protein and two of its deletion mutants is provided
Mentions: As previously described, Che-1 half-life is tightly regulated by the proteasome pathway, and in response to apoptotic stimuli HDM2 protein negatively regulates Che-1 by promoting its ubiquitin-mediated degradation.9 On the basis of the above results, we evaluated whether HIPK2 could affect Che-1/HDM2 interaction by coimmunoprecipitation with anti-HDM2 antibody in HCT116 cells overexpressing HIPK2 or its K221R mutant and treated with ADR. As shown in Figure 4a, HCT116 cells overexpressing HIPK2 wt showed an increase of Che-1 bound to HDM2, whereas this effect was not observed in cells transfected with the K221R mutant. Consistent with these findings, HIPK2 overexpression produced a strong increase in the ubiquitylation of Che-1, which is not found in cells overexpressing the K221R mutant (Figure 4b), indicating that the kinase activity of HIPK2 is required for the interaction between Che-1 and HDM2 and Che-1 ubiquitylation. Notably, the superactive form of HIPK2 (1–838) was found to be more active in Che-1 ubiquitylation (Figure 4c), reinforcing the involvement of HIPK2 in Che-1 degradation in response to apoptotic induction. To further characterize the role of HIPK2 in Che-1 degradation, we transiently transfected HCT116 cells with HIPK2 and Che-1 in the wt form or two different deletion mutants (1–163 and 164–558). In agreement with previous results, we found that exogenous HIPK2 expression produces a strong ubiquitylation of Che-1 wt and 1–163 mutant, whereas the polypeptide 164–558 lacking T144 did not exhibit any ubiquitylation (Figure 4d). Taken together, these results support the hypothesis that upon induction of apoptosis HIPK2 phosphorylates Che-1, thereby increasing its affinity for HDM2, its ubiquitylation and its degradation.

Bottom Line: 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.

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