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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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NORE1A forms a Ras regulated complex with HIPK2. (A) NORE1A is primarily nuclear. NCI-H1792 cells (that retain NORE1A expression) were fractionated into nuclear and cytoplasmic fractions to determine the sub-cellular localization of endogenous NORE1A. The distribution of TFIIH and Actin served as controls for effective fractionation. (B) Exogenously expressed NORE1A forms nuclear speckles with HIPK2. (top) COS-7 cells were transfected with GFP-NORE1A which localized to the nucleus and in some cells to nuclear speckles. (bottom) RFP-NORE1A co-localized with GFP-HIPK2 speckles in the nuclei of COS-7 cells. A representative nucleus is shown. Bar, 40 µM. (C) NORE1A and HIPK2 can be detected in endogenous complex. HepG2 cells and HuH6 cells were immunoprecipitated (IP) for NORE1A and immunoblotted (IB) for HIPK2. (D) Activated Ras enhances the association of NORE1A and HIPK2. HEK-293T cells were cotransfected with expression constructs for HIPK2, NORE1A and activated H-Ras or activated K-Ras for 24 h, lysed and equal amounts of protein immunoprecipitated with anti-GFP. The immunoprecipitates were analyzed by Western blotting with anti-HA and anti-GFP antibodies. (E) Activated Ras enhances the interaction between endogenous NORE1A and HIPK2. HBEC-3KT cells expressing inducible activated H-Ras (Fig. 2) were left untreated (−) or treated (+) with 2 µg/ml doxycycline for 72 h to induce Ras expression and lysed, and equal amounts of protein were immunoprecipitated with an anti-NORE1A antibody. The immunoprecipitates were analyzed by Western blotting with anti-NORE1A, anti-HIPK2, and anti–H-Ras antibodies. (left) Immunoprecipitation; (right) inputs.
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fig4: NORE1A forms a Ras regulated complex with HIPK2. (A) NORE1A is primarily nuclear. NCI-H1792 cells (that retain NORE1A expression) were fractionated into nuclear and cytoplasmic fractions to determine the sub-cellular localization of endogenous NORE1A. The distribution of TFIIH and Actin served as controls for effective fractionation. (B) Exogenously expressed NORE1A forms nuclear speckles with HIPK2. (top) COS-7 cells were transfected with GFP-NORE1A which localized to the nucleus and in some cells to nuclear speckles. (bottom) RFP-NORE1A co-localized with GFP-HIPK2 speckles in the nuclei of COS-7 cells. A representative nucleus is shown. Bar, 40 µM. (C) NORE1A and HIPK2 can be detected in endogenous complex. HepG2 cells and HuH6 cells were immunoprecipitated (IP) for NORE1A and immunoblotted (IB) for HIPK2. (D) Activated Ras enhances the association of NORE1A and HIPK2. HEK-293T cells were cotransfected with expression constructs for HIPK2, NORE1A and activated H-Ras or activated K-Ras for 24 h, lysed and equal amounts of protein immunoprecipitated with anti-GFP. The immunoprecipitates were analyzed by Western blotting with anti-HA and anti-GFP antibodies. (E) Activated Ras enhances the interaction between endogenous NORE1A and HIPK2. HBEC-3KT cells expressing inducible activated H-Ras (Fig. 2) were left untreated (−) or treated (+) with 2 µg/ml doxycycline for 72 h to induce Ras expression and lysed, and equal amounts of protein were immunoprecipitated with an anti-NORE1A antibody. The immunoprecipitates were analyzed by Western blotting with anti-NORE1A, anti-HIPK2, and anti–H-Ras antibodies. (left) Immunoprecipitation; (right) inputs.

Mentions: The aforementioned experiments implicate NORE1A as a key Ras senescence effector that acts via p53 but does not use the canonical Hippo pathway. While attempting to determine the mechanism behind the effect, we examined the subcellular localization of endogenous NORE1A and found that it is primarily localized to the nucleus (Fig. 4 a). Transient transfections of NORE1A tagged with GFP confirmed a mainly nuclear localization but also revealed that some cells demonstrated NORE1A in nuclear speckles (Fig. 4 b, top). Systematic analysis of a series of fluorescently tagged known nuclear speckle proteins showed that NORE1A specifically co-localized with the kinase HIPK2, a known regulator of p53 (Puca et al., 2010; Fig. 4 b, bottom). Further analysis confirmed that endogenous NORE1A could be co-immunoprecipitated with endogenous HIPK2 from HuH6 and HepG2 liver cancer cell lines (Fig. 4 c).


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)

NORE1A forms a Ras regulated complex with HIPK2. (A) NORE1A is primarily nuclear. NCI-H1792 cells (that retain NORE1A expression) were fractionated into nuclear and cytoplasmic fractions to determine the sub-cellular localization of endogenous NORE1A. The distribution of TFIIH and Actin served as controls for effective fractionation. (B) Exogenously expressed NORE1A forms nuclear speckles with HIPK2. (top) COS-7 cells were transfected with GFP-NORE1A which localized to the nucleus and in some cells to nuclear speckles. (bottom) RFP-NORE1A co-localized with GFP-HIPK2 speckles in the nuclei of COS-7 cells. A representative nucleus is shown. Bar, 40 µM. (C) NORE1A and HIPK2 can be detected in endogenous complex. HepG2 cells and HuH6 cells were immunoprecipitated (IP) for NORE1A and immunoblotted (IB) for HIPK2. (D) Activated Ras enhances the association of NORE1A and HIPK2. HEK-293T cells were cotransfected with expression constructs for HIPK2, NORE1A and activated H-Ras or activated K-Ras for 24 h, lysed and equal amounts of protein immunoprecipitated with anti-GFP. The immunoprecipitates were analyzed by Western blotting with anti-HA and anti-GFP antibodies. (E) Activated Ras enhances the interaction between endogenous NORE1A and HIPK2. HBEC-3KT cells expressing inducible activated H-Ras (Fig. 2) were left untreated (−) or treated (+) with 2 µg/ml doxycycline for 72 h to induce Ras expression and lysed, and equal amounts of protein were immunoprecipitated with an anti-NORE1A antibody. The immunoprecipitates were analyzed by Western blotting with anti-NORE1A, anti-HIPK2, and anti–H-Ras antibodies. (left) Immunoprecipitation; (right) inputs.
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fig4: NORE1A forms a Ras regulated complex with HIPK2. (A) NORE1A is primarily nuclear. NCI-H1792 cells (that retain NORE1A expression) were fractionated into nuclear and cytoplasmic fractions to determine the sub-cellular localization of endogenous NORE1A. The distribution of TFIIH and Actin served as controls for effective fractionation. (B) Exogenously expressed NORE1A forms nuclear speckles with HIPK2. (top) COS-7 cells were transfected with GFP-NORE1A which localized to the nucleus and in some cells to nuclear speckles. (bottom) RFP-NORE1A co-localized with GFP-HIPK2 speckles in the nuclei of COS-7 cells. A representative nucleus is shown. Bar, 40 µM. (C) NORE1A and HIPK2 can be detected in endogenous complex. HepG2 cells and HuH6 cells were immunoprecipitated (IP) for NORE1A and immunoblotted (IB) for HIPK2. (D) Activated Ras enhances the association of NORE1A and HIPK2. HEK-293T cells were cotransfected with expression constructs for HIPK2, NORE1A and activated H-Ras or activated K-Ras for 24 h, lysed and equal amounts of protein immunoprecipitated with anti-GFP. The immunoprecipitates were analyzed by Western blotting with anti-HA and anti-GFP antibodies. (E) Activated Ras enhances the interaction between endogenous NORE1A and HIPK2. HBEC-3KT cells expressing inducible activated H-Ras (Fig. 2) were left untreated (−) or treated (+) with 2 µg/ml doxycycline for 72 h to induce Ras expression and lysed, and equal amounts of protein were immunoprecipitated with an anti-NORE1A antibody. The immunoprecipitates were analyzed by Western blotting with anti-NORE1A, anti-HIPK2, and anti–H-Ras antibodies. (left) Immunoprecipitation; (right) inputs.
Mentions: The aforementioned experiments implicate NORE1A as a key Ras senescence effector that acts via p53 but does not use the canonical Hippo pathway. While attempting to determine the mechanism behind the effect, we examined the subcellular localization of endogenous NORE1A and found that it is primarily localized to the nucleus (Fig. 4 a). Transient transfections of NORE1A tagged with GFP confirmed a mainly nuclear localization but also revealed that some cells demonstrated NORE1A in nuclear speckles (Fig. 4 b, top). Systematic analysis of a series of fluorescently tagged known nuclear speckle proteins showed that NORE1A specifically co-localized with the kinase HIPK2, a known regulator of p53 (Puca et al., 2010; Fig. 4 b, bottom). Further analysis confirmed that endogenous NORE1A could be co-immunoprecipitated with endogenous HIPK2 from HuH6 and HepG2 liver cancer cell lines (Fig. 4 c).

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