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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 induces Che-1 degradation. (a and b) H1299 (A) or HCT116 (b) cells were transiently transfected with shControl or shHIPK2 (A), or siRNA GFP (siControl) or siRNA HIPK2 (b), and treated with 2 μM ADR. TCEs were analyzed by WB with the indicated Abs. (c) HCT116 cells were transiently transfected with GFP-HIPK2 or GFP-K221R expression vectors and treated or not treated with 10 μM MG132 for 16 h. TCEs were analyzed by WB with the indicated Abs. (d) U2OS cells were transiently transfected as in c and analyzed by immunofluorescence with anti-Che-1 antibody. (e) HCT116 cells were transiently transfected with Myc-Che-1 or Myc-Che-1T144A and increasing amounts of GFP-HIPK2. TCEs were analyzed by WB with the indicated Abs. (f and g) HCT116 cells were transiently transfected with HIPK2 (1–838) expression vector and treated or not treated with 50 μM z-VAD for 12 h (F) or 10 μM MG132 for 16 h (G). TCEs were analyzed by WB with the indicated Abs
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fig3: HIPK2 induces Che-1 degradation. (a and b) H1299 (A) or HCT116 (b) cells were transiently transfected with shControl or shHIPK2 (A), or siRNA GFP (siControl) or siRNA HIPK2 (b), and treated with 2 μM ADR. TCEs were analyzed by WB with the indicated Abs. (c) HCT116 cells were transiently transfected with GFP-HIPK2 or GFP-K221R expression vectors and treated or not treated with 10 μM MG132 for 16 h. TCEs were analyzed by WB with the indicated Abs. (d) U2OS cells were transiently transfected as in c and analyzed by immunofluorescence with anti-Che-1 antibody. (e) HCT116 cells were transiently transfected with Myc-Che-1 or Myc-Che-1T144A and increasing amounts of GFP-HIPK2. TCEs were analyzed by WB with the indicated Abs. (f and g) HCT116 cells were transiently transfected with HIPK2 (1–838) expression vector and treated or not treated with 50 μM z-VAD for 12 h (F) or 10 μM MG132 for 16 h (G). TCEs were analyzed by WB with the indicated Abs

Mentions: As described, in response to apoptotic stimuli, Che-1 binds Pin1, which, in turn, mediates conformational changes of Che-1.9 This event is required for Che-1/HDM2 interaction with consequent Che-1 degradation, and it depends on the phosphorylation at residue T144. Indeed, substitution of T144 reduces the ability of Che-1 to bind both Pin1 and HDM2, with an increase of its half-life.9 On the basis of these observations, we speculated that HIPK2 might be involved in Che-1 stabilization. To test this hypothesis, we performed a western blot analysis of the total cell extracts (TCEs) obtained from H1299 and HCT116 cells that were transiently transfected with HIPK2 shRNA or siRNA and treated with ADR. As is shown in Figures 3a and b, HIPK2 depletion strongly increased Che-1 protein levels. In agreement with these findings, HIPK2 overexpression was able to reduce Che-1 levels, whereas this effect was not observed in cells transfected with K221R mutant (Figure 3c). Notably, MG132 treatment counteracted HIPK2 activity on Che-1 (Figure 3c), thus reinforcing the notion that HIPK2 affects Che-1 stability. These results were confirmed by immunofluorescence assays that revealed the absence of Che-1 protein in U2OS cells overexpressing HIPK2 but not its K221R derivative (Figure 3d). To confirm the involvement of T144 in Che-1 degradation by HIPK2, we transiently transfected HCT116 cells with vectors carrying the Che-1 wt or Che-1T144A mutant and increasing amounts of vector carrying HIPK2. As shown in Figure 3e, HIPK2 overexpression strongly reduced Myc-Che-1 levels, whereas it produced little effect on Che-1T144A expression. Next, to evaluate whether Che-1 degradation is due to direct modification by HIPK2 and not rather the result of apoptotic induction, we treated HCT116 cells with a specific inhibitor of caspases (z-VAD) in the presence or absence of the C-terminal-deleted form of HIPK2 (1–838) that lacks the autoinhibitory domain and mimics the caspase-superactivated form of HIPK2.33 As shown in Figure 3f, a significant Che-1 degradation was observed in cells overexpressing superactivated HIPK2 (1–838) in the presence of z-VAD but not in the presence of MG132 (Figure 3g), confirming in such a way that HIPK2 is directly involved in Che-1 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 induces Che-1 degradation. (a and b) H1299 (A) or HCT116 (b) cells were transiently transfected with shControl or shHIPK2 (A), or siRNA GFP (siControl) or siRNA HIPK2 (b), and treated with 2 μM ADR. TCEs were analyzed by WB with the indicated Abs. (c) HCT116 cells were transiently transfected with GFP-HIPK2 or GFP-K221R expression vectors and treated or not treated with 10 μM MG132 for 16 h. TCEs were analyzed by WB with the indicated Abs. (d) U2OS cells were transiently transfected as in c and analyzed by immunofluorescence with anti-Che-1 antibody. (e) HCT116 cells were transiently transfected with Myc-Che-1 or Myc-Che-1T144A and increasing amounts of GFP-HIPK2. TCEs were analyzed by WB with the indicated Abs. (f and g) HCT116 cells were transiently transfected with HIPK2 (1–838) expression vector and treated or not treated with 50 μM z-VAD for 12 h (F) or 10 μM MG132 for 16 h (G). TCEs were analyzed by WB with the indicated Abs
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: HIPK2 induces Che-1 degradation. (a and b) H1299 (A) or HCT116 (b) cells were transiently transfected with shControl or shHIPK2 (A), or siRNA GFP (siControl) or siRNA HIPK2 (b), and treated with 2 μM ADR. TCEs were analyzed by WB with the indicated Abs. (c) HCT116 cells were transiently transfected with GFP-HIPK2 or GFP-K221R expression vectors and treated or not treated with 10 μM MG132 for 16 h. TCEs were analyzed by WB with the indicated Abs. (d) U2OS cells were transiently transfected as in c and analyzed by immunofluorescence with anti-Che-1 antibody. (e) HCT116 cells were transiently transfected with Myc-Che-1 or Myc-Che-1T144A and increasing amounts of GFP-HIPK2. TCEs were analyzed by WB with the indicated Abs. (f and g) HCT116 cells were transiently transfected with HIPK2 (1–838) expression vector and treated or not treated with 50 μM z-VAD for 12 h (F) or 10 μM MG132 for 16 h (G). TCEs were analyzed by WB with the indicated Abs
Mentions: As described, in response to apoptotic stimuli, Che-1 binds Pin1, which, in turn, mediates conformational changes of Che-1.9 This event is required for Che-1/HDM2 interaction with consequent Che-1 degradation, and it depends on the phosphorylation at residue T144. Indeed, substitution of T144 reduces the ability of Che-1 to bind both Pin1 and HDM2, with an increase of its half-life.9 On the basis of these observations, we speculated that HIPK2 might be involved in Che-1 stabilization. To test this hypothesis, we performed a western blot analysis of the total cell extracts (TCEs) obtained from H1299 and HCT116 cells that were transiently transfected with HIPK2 shRNA or siRNA and treated with ADR. As is shown in Figures 3a and b, HIPK2 depletion strongly increased Che-1 protein levels. In agreement with these findings, HIPK2 overexpression was able to reduce Che-1 levels, whereas this effect was not observed in cells transfected with K221R mutant (Figure 3c). Notably, MG132 treatment counteracted HIPK2 activity on Che-1 (Figure 3c), thus reinforcing the notion that HIPK2 affects Che-1 stability. These results were confirmed by immunofluorescence assays that revealed the absence of Che-1 protein in U2OS cells overexpressing HIPK2 but not its K221R derivative (Figure 3d). To confirm the involvement of T144 in Che-1 degradation by HIPK2, we transiently transfected HCT116 cells with vectors carrying the Che-1 wt or Che-1T144A mutant and increasing amounts of vector carrying HIPK2. As shown in Figure 3e, HIPK2 overexpression strongly reduced Myc-Che-1 levels, whereas it produced little effect on Che-1T144A expression. Next, to evaluate whether Che-1 degradation is due to direct modification by HIPK2 and not rather the result of apoptotic induction, we treated HCT116 cells with a specific inhibitor of caspases (z-VAD) in the presence or absence of the C-terminal-deleted form of HIPK2 (1–838) that lacks the autoinhibitory domain and mimics the caspase-superactivated form of HIPK2.33 As shown in Figure 3f, a significant Che-1 degradation was observed in cells overexpressing superactivated HIPK2 (1–838) in the presence of z-VAD but not in the presence of MG132 (Figure 3g), confirming in such a way that HIPK2 is directly involved in Che-1 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