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NORE1A is a Ras senescence effector that controls the apoptotic/senescent balance of p53 via HIPK2.

Donninger H, Calvisi DF, Barnoud T, Clark J, Schmidt ML, Vos MD, Clark GJ - J. Cell Biol. (2015)

Bottom Line: We show that NORE1A is a powerful Ras senescence effector and that down-regulation of NORE1A suppresses senescence induction by Ras and enhances Ras transformation.NORE1A acts to suppress its proapoptotic phosphorylation of p53 but enhance its prosenescent acetylation of p53.Thus, we identify a major new Ras signaling pathway that links Ras to the control of specific protein acetylation and show how NORE1A allows Ras to qualitatively modify p53 function to promote senescence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Department of Biochemistry and Molecular Biology, Department of Pharmacology and Toxicology, J.G. Brown Cancer Center, Molecular Targets Group, University of Louisville, Louisville, KY 40202.

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The interaction with HIPK2 is essential for NORE1A-mediated senescence. (A) HIPK2 knockdown blocks NORE1A induced senescence. A549 cells were stably transfected with shRNA constructs against HIPK2 or a scrambled control. The cells were then transfected with NORE1A and the induction of senescence measured by β-galactosidase assay. HIPK2 and NORE1A expression was assessed by Western blot (inset). (B) Loss of HIPK2 reduces baseline senescence in HBEC-3KT cells. HBEC-3KT cells were transfected with the same HIPK2 shRNA constructs and 72 h later senescence measured by β-galactosidase assay. (C) A HIPK2 binding mutant of NORE1A is defective for senescence induction. A mutant form of NORE1A designated NORE1AR3A was generated by mutating the Arginines at residues 92–94 to Alanines. The mutant was co-transfected with HIPK2 in HEK-293T cells and its ability to co-immunoprecipitate with HIPK2 compared with the WT protein (left). The NORE1AR3A mutant was then assayed for the relative ability to induce senescence in A549 cells (right). (D) NORE1AR3A retains the ability to bind MST1 and induce apoptosis. The NORE1AR3A mutant was then examined to determine if it retained WT MST1 binding activity (left) and apoptosis inducing capacity, using a pCaspase-3-sensor fluorescent protein indicator assay (right). Indicator cells were COS-7 and the assay was scored 48 h after transfection. Data represent the mean ± SD of triplicate experiments. P < 0.05 compared with vector transfected cells.
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fig6: The interaction with HIPK2 is essential for NORE1A-mediated senescence. (A) HIPK2 knockdown blocks NORE1A induced senescence. A549 cells were stably transfected with shRNA constructs against HIPK2 or a scrambled control. The cells were then transfected with NORE1A and the induction of senescence measured by β-galactosidase assay. HIPK2 and NORE1A expression was assessed by Western blot (inset). (B) Loss of HIPK2 reduces baseline senescence in HBEC-3KT cells. HBEC-3KT cells were transfected with the same HIPK2 shRNA constructs and 72 h later senescence measured by β-galactosidase assay. (C) A HIPK2 binding mutant of NORE1A is defective for senescence induction. A mutant form of NORE1A designated NORE1AR3A was generated by mutating the Arginines at residues 92–94 to Alanines. The mutant was co-transfected with HIPK2 in HEK-293T cells and its ability to co-immunoprecipitate with HIPK2 compared with the WT protein (left). The NORE1AR3A mutant was then assayed for the relative ability to induce senescence in A549 cells (right). (D) NORE1AR3A retains the ability to bind MST1 and induce apoptosis. The NORE1AR3A mutant was then examined to determine if it retained WT MST1 binding activity (left) and apoptosis inducing capacity, using a pCaspase-3-sensor fluorescent protein indicator assay (right). Indicator cells were COS-7 and the assay was scored 48 h after transfection. Data represent the mean ± SD of triplicate experiments. P < 0.05 compared with vector transfected cells.

Mentions: HIPK2 is a tumor suppressor that binds p53 and modulates its activity both directly and indirectly (Puca et al., 2010). Thus, HIPK2 is a good candidate for a downstream component of NORE1A senescence signaling. To determine the importance of HIPK2 for NORE1A-mediated senescence, we generated stable clones of A549 cells transfected with shRNAs against HIPK2. Compared with the scrambled shRNA control, the HIPK2 knockdown cells were resistant to NORE1A induced senescence (Fig. 6 a). Suppression of HIPK2 similarly reduced baseline senescence in HBEC-3KT cells (Fig. 6 b). To further confirm the biological importance of the interaction between NORE1A and HIPK2, we used deletion mutagenesis followed by point mutagenesis to identify the minimal domain required for the interaction of NORE1A with HIPK2. Ultimately, Arginines 92–94 of NORE1A were mutated to Alanines. The resultant NORE1AR92-94A mutant (NORE1AR3A) was defective for the interaction with HIPK2 and defective for the induction of senescence in A549 cells (Fig. 6 c) and showed a reduced ability to stabilize HIPK2 (Fig. S1 a). However, it retained WT binding to MST1 and retained WT apoptosis capacity (Fig. 6 d).


NORE1A is a Ras senescence effector that controls the apoptotic/senescent balance of p53 via HIPK2.

Donninger H, Calvisi DF, Barnoud T, Clark J, Schmidt ML, Vos MD, Clark GJ - J. Cell Biol. (2015)

The interaction with HIPK2 is essential for NORE1A-mediated senescence. (A) HIPK2 knockdown blocks NORE1A induced senescence. A549 cells were stably transfected with shRNA constructs against HIPK2 or a scrambled control. The cells were then transfected with NORE1A and the induction of senescence measured by β-galactosidase assay. HIPK2 and NORE1A expression was assessed by Western blot (inset). (B) Loss of HIPK2 reduces baseline senescence in HBEC-3KT cells. HBEC-3KT cells were transfected with the same HIPK2 shRNA constructs and 72 h later senescence measured by β-galactosidase assay. (C) A HIPK2 binding mutant of NORE1A is defective for senescence induction. A mutant form of NORE1A designated NORE1AR3A was generated by mutating the Arginines at residues 92–94 to Alanines. The mutant was co-transfected with HIPK2 in HEK-293T cells and its ability to co-immunoprecipitate with HIPK2 compared with the WT protein (left). The NORE1AR3A mutant was then assayed for the relative ability to induce senescence in A549 cells (right). (D) NORE1AR3A retains the ability to bind MST1 and induce apoptosis. The NORE1AR3A mutant was then examined to determine if it retained WT MST1 binding activity (left) and apoptosis inducing capacity, using a pCaspase-3-sensor fluorescent protein indicator assay (right). Indicator cells were COS-7 and the assay was scored 48 h after transfection. Data represent the mean ± SD of triplicate experiments. P < 0.05 compared with vector transfected cells.
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fig6: The interaction with HIPK2 is essential for NORE1A-mediated senescence. (A) HIPK2 knockdown blocks NORE1A induced senescence. A549 cells were stably transfected with shRNA constructs against HIPK2 or a scrambled control. The cells were then transfected with NORE1A and the induction of senescence measured by β-galactosidase assay. HIPK2 and NORE1A expression was assessed by Western blot (inset). (B) Loss of HIPK2 reduces baseline senescence in HBEC-3KT cells. HBEC-3KT cells were transfected with the same HIPK2 shRNA constructs and 72 h later senescence measured by β-galactosidase assay. (C) A HIPK2 binding mutant of NORE1A is defective for senescence induction. A mutant form of NORE1A designated NORE1AR3A was generated by mutating the Arginines at residues 92–94 to Alanines. The mutant was co-transfected with HIPK2 in HEK-293T cells and its ability to co-immunoprecipitate with HIPK2 compared with the WT protein (left). The NORE1AR3A mutant was then assayed for the relative ability to induce senescence in A549 cells (right). (D) NORE1AR3A retains the ability to bind MST1 and induce apoptosis. The NORE1AR3A mutant was then examined to determine if it retained WT MST1 binding activity (left) and apoptosis inducing capacity, using a pCaspase-3-sensor fluorescent protein indicator assay (right). Indicator cells were COS-7 and the assay was scored 48 h after transfection. Data represent the mean ± SD of triplicate experiments. P < 0.05 compared with vector transfected cells.
Mentions: HIPK2 is a tumor suppressor that binds p53 and modulates its activity both directly and indirectly (Puca et al., 2010). Thus, HIPK2 is a good candidate for a downstream component of NORE1A senescence signaling. To determine the importance of HIPK2 for NORE1A-mediated senescence, we generated stable clones of A549 cells transfected with shRNAs against HIPK2. Compared with the scrambled shRNA control, the HIPK2 knockdown cells were resistant to NORE1A induced senescence (Fig. 6 a). Suppression of HIPK2 similarly reduced baseline senescence in HBEC-3KT cells (Fig. 6 b). To further confirm the biological importance of the interaction between NORE1A and HIPK2, we used deletion mutagenesis followed by point mutagenesis to identify the minimal domain required for the interaction of NORE1A with HIPK2. Ultimately, Arginines 92–94 of NORE1A were mutated to Alanines. The resultant NORE1AR92-94A mutant (NORE1AR3A) was defective for the interaction with HIPK2 and defective for the induction of senescence in A549 cells (Fig. 6 c) and showed a reduced ability to stabilize HIPK2 (Fig. S1 a). However, it retained WT binding to MST1 and retained WT apoptosis capacity (Fig. 6 d).

Bottom Line: We show that NORE1A is a powerful Ras senescence effector and that down-regulation of NORE1A suppresses senescence induction by Ras and enhances Ras transformation.NORE1A acts to suppress its proapoptotic phosphorylation of p53 but enhance its prosenescent acetylation of p53.Thus, we identify a major new Ras signaling pathway that links Ras to the control of specific protein acetylation and show how NORE1A allows Ras to qualitatively modify p53 function to promote senescence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Department of Biochemistry and Molecular Biology, Department of Pharmacology and Toxicology, J.G. Brown Cancer Center, Molecular Targets Group, University of Louisville, Louisville, KY 40202.

Show MeSH
Related in: MedlinePlus