PARP inhibition induces Akt-mediated cytoprotective effects through the formation of a mitochondria-targeted phospho-ATM-NEMO-Akt-mTOR signalosome

Antal Tapodi, Zita Bognar, Csaba Szabo, Ferenc Gallyas, Balázs Sumegi, Enikő Hocsak

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Purpose: The cytoprotective effect of poly(ADP-ribose) polymerase 1 (PARP1) inhibition is well documented in various cell types subjected to oxidative stress. Previously, we have demonstrated that PARP1 inhibition activates Akt, and showed that this response plays a critical role in the maintenance of mitochondrial integrity and in cell survival. However, it has not yet been defined how nuclear PARP1 signals to cytoplasmic Akt. Methods: WRL 68, HeLa and MCF7 cells were grown in culture. Oxidative stress was induced with hydrogen peroxide. PARP was inhibited with the PARP inhibitor PJ34. ATM, mTOR- and NEMO were silenced using specific siRNAs. Cell viability assays were based on the MTT assay. PARP-ATM pulldown experiments were conducted; each protein was visualized by Western blotting. Immunoprecipitation of ATM, phospho-ATM and NEMO was performed from cytoplasmic and mitochondrial cell fractions and proteins were detected by Western blotting. In some experiments, a continually active Akt construct was introduced. Nuclear to cytoplasmic and mitochondrial translocation of phospho-Akt was visualized by confocal microscopy. Results: Here we present evidence for a PARP1 mediated, PARylation-dependent interaction between ATM and NEMO, which is responsible for the cytoplasmic transport of phosphorylated (thus, activated) ATM kinase. In turn, the cytoplasmic p-ATM and NEMO forms complex with mTOR and Akt, yielding the phospho-ATM-NEMO-Akt-mTOR signalosome, which is responsible for the PARP-inhibition induced Akt activation. The phospho-ATM-NEMO-Akt-mTOR signalosome localizes to the mitochondria and is essential for the PARP-inhibition-mediated cytoprotective effects in oxidatively stressed cells. When the formation of the signalosome is prevented, the cytoprotective effects diminish, but cells can be rescued by constantly active Akt1, further confirming the critical role of Akt activation in cytoprotection. Conclusions: Taken together, the data presented in the current paper are consistent with the hypothesis that PARP inhibition suppresses the PARylation of ATM, which, in turn, forms an ATM-NEMO complex, which exits the nucleus, and combines in the cytosol with mTOR and Act, resulting in Act phosphorylation (i.e. activation), which, in turn, produces the cytoprotective action via the induction of Akt-mediated survival pathways. This mechanism can be important in the protective effect of PARP inhibitor in various diseases associated with oxidative stress. Moreover, disruption of the formation or action of the phospho-ATM-NEMO-Akt-mTOR signalosome may offer potential future experimental therapeutic checkpoints.

Original languageEnglish (US)
JournalBiochemical Pharmacology
DOIs
StateAccepted/In press - Jan 1 2018
Externally publishedYes

Fingerprint

Mitochondria
Automatic teller machines
Oxidative Stress
Cell Survival
Poly(ADP-ribose) Polymerases
Western Blotting
Oxidative stress
Cytoprotection
MCF-7 Cells
HeLa Cells
Immunoprecipitation
Confocal Microscopy
Cytosol
Hydrogen Peroxide
Proteins
Phosphotransferases
Chemical activation
Maintenance
Phosphorylation
Poly (ADP-Ribose) Polymerase-1

Keywords

  • Akt
  • Cytotoxicity
  • Mitochondrion
  • Oxidative stress
  • Poly(ADP)ribose polymerase
  • Signaling

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

Cite this

PARP inhibition induces Akt-mediated cytoprotective effects through the formation of a mitochondria-targeted phospho-ATM-NEMO-Akt-mTOR signalosome. / Tapodi, Antal; Bognar, Zita; Szabo, Csaba; Gallyas, Ferenc; Sumegi, Balázs; Hocsak, Enikő.

In: Biochemical Pharmacology, 01.01.2018.

Research output: Contribution to journalArticle

@article{15426d48a9da426b82bb6c693845616f,
title = "PARP inhibition induces Akt-mediated cytoprotective effects through the formation of a mitochondria-targeted phospho-ATM-NEMO-Akt-mTOR signalosome",
abstract = "Purpose: The cytoprotective effect of poly(ADP-ribose) polymerase 1 (PARP1) inhibition is well documented in various cell types subjected to oxidative stress. Previously, we have demonstrated that PARP1 inhibition activates Akt, and showed that this response plays a critical role in the maintenance of mitochondrial integrity and in cell survival. However, it has not yet been defined how nuclear PARP1 signals to cytoplasmic Akt. Methods: WRL 68, HeLa and MCF7 cells were grown in culture. Oxidative stress was induced with hydrogen peroxide. PARP was inhibited with the PARP inhibitor PJ34. ATM, mTOR- and NEMO were silenced using specific siRNAs. Cell viability assays were based on the MTT assay. PARP-ATM pulldown experiments were conducted; each protein was visualized by Western blotting. Immunoprecipitation of ATM, phospho-ATM and NEMO was performed from cytoplasmic and mitochondrial cell fractions and proteins were detected by Western blotting. In some experiments, a continually active Akt construct was introduced. Nuclear to cytoplasmic and mitochondrial translocation of phospho-Akt was visualized by confocal microscopy. Results: Here we present evidence for a PARP1 mediated, PARylation-dependent interaction between ATM and NEMO, which is responsible for the cytoplasmic transport of phosphorylated (thus, activated) ATM kinase. In turn, the cytoplasmic p-ATM and NEMO forms complex with mTOR and Akt, yielding the phospho-ATM-NEMO-Akt-mTOR signalosome, which is responsible for the PARP-inhibition induced Akt activation. The phospho-ATM-NEMO-Akt-mTOR signalosome localizes to the mitochondria and is essential for the PARP-inhibition-mediated cytoprotective effects in oxidatively stressed cells. When the formation of the signalosome is prevented, the cytoprotective effects diminish, but cells can be rescued by constantly active Akt1, further confirming the critical role of Akt activation in cytoprotection. Conclusions: Taken together, the data presented in the current paper are consistent with the hypothesis that PARP inhibition suppresses the PARylation of ATM, which, in turn, forms an ATM-NEMO complex, which exits the nucleus, and combines in the cytosol with mTOR and Act, resulting in Act phosphorylation (i.e. activation), which, in turn, produces the cytoprotective action via the induction of Akt-mediated survival pathways. This mechanism can be important in the protective effect of PARP inhibitor in various diseases associated with oxidative stress. Moreover, disruption of the formation or action of the phospho-ATM-NEMO-Akt-mTOR signalosome may offer potential future experimental therapeutic checkpoints.",
keywords = "Akt, Cytotoxicity, Mitochondrion, Oxidative stress, Poly(ADP)ribose polymerase, Signaling",
author = "Antal Tapodi and Zita Bognar and Csaba Szabo and Ferenc Gallyas and Bal{\'a}zs Sumegi and Enikő Hocsak",
year = "2018",
month = "1",
day = "1",
doi = "10.1016/j.bcp.2018.10.005",
language = "English (US)",
journal = "Biochemical Pharmacology",
issn = "0006-2952",
publisher = "Elsevier Inc.",

}

TY - JOUR

T1 - PARP inhibition induces Akt-mediated cytoprotective effects through the formation of a mitochondria-targeted phospho-ATM-NEMO-Akt-mTOR signalosome

AU - Tapodi, Antal

AU - Bognar, Zita

AU - Szabo, Csaba

AU - Gallyas, Ferenc

AU - Sumegi, Balázs

AU - Hocsak, Enikő

PY - 2018/1/1

Y1 - 2018/1/1

N2 - Purpose: The cytoprotective effect of poly(ADP-ribose) polymerase 1 (PARP1) inhibition is well documented in various cell types subjected to oxidative stress. Previously, we have demonstrated that PARP1 inhibition activates Akt, and showed that this response plays a critical role in the maintenance of mitochondrial integrity and in cell survival. However, it has not yet been defined how nuclear PARP1 signals to cytoplasmic Akt. Methods: WRL 68, HeLa and MCF7 cells were grown in culture. Oxidative stress was induced with hydrogen peroxide. PARP was inhibited with the PARP inhibitor PJ34. ATM, mTOR- and NEMO were silenced using specific siRNAs. Cell viability assays were based on the MTT assay. PARP-ATM pulldown experiments were conducted; each protein was visualized by Western blotting. Immunoprecipitation of ATM, phospho-ATM and NEMO was performed from cytoplasmic and mitochondrial cell fractions and proteins were detected by Western blotting. In some experiments, a continually active Akt construct was introduced. Nuclear to cytoplasmic and mitochondrial translocation of phospho-Akt was visualized by confocal microscopy. Results: Here we present evidence for a PARP1 mediated, PARylation-dependent interaction between ATM and NEMO, which is responsible for the cytoplasmic transport of phosphorylated (thus, activated) ATM kinase. In turn, the cytoplasmic p-ATM and NEMO forms complex with mTOR and Akt, yielding the phospho-ATM-NEMO-Akt-mTOR signalosome, which is responsible for the PARP-inhibition induced Akt activation. The phospho-ATM-NEMO-Akt-mTOR signalosome localizes to the mitochondria and is essential for the PARP-inhibition-mediated cytoprotective effects in oxidatively stressed cells. When the formation of the signalosome is prevented, the cytoprotective effects diminish, but cells can be rescued by constantly active Akt1, further confirming the critical role of Akt activation in cytoprotection. Conclusions: Taken together, the data presented in the current paper are consistent with the hypothesis that PARP inhibition suppresses the PARylation of ATM, which, in turn, forms an ATM-NEMO complex, which exits the nucleus, and combines in the cytosol with mTOR and Act, resulting in Act phosphorylation (i.e. activation), which, in turn, produces the cytoprotective action via the induction of Akt-mediated survival pathways. This mechanism can be important in the protective effect of PARP inhibitor in various diseases associated with oxidative stress. Moreover, disruption of the formation or action of the phospho-ATM-NEMO-Akt-mTOR signalosome may offer potential future experimental therapeutic checkpoints.

AB - Purpose: The cytoprotective effect of poly(ADP-ribose) polymerase 1 (PARP1) inhibition is well documented in various cell types subjected to oxidative stress. Previously, we have demonstrated that PARP1 inhibition activates Akt, and showed that this response plays a critical role in the maintenance of mitochondrial integrity and in cell survival. However, it has not yet been defined how nuclear PARP1 signals to cytoplasmic Akt. Methods: WRL 68, HeLa and MCF7 cells were grown in culture. Oxidative stress was induced with hydrogen peroxide. PARP was inhibited with the PARP inhibitor PJ34. ATM, mTOR- and NEMO were silenced using specific siRNAs. Cell viability assays were based on the MTT assay. PARP-ATM pulldown experiments were conducted; each protein was visualized by Western blotting. Immunoprecipitation of ATM, phospho-ATM and NEMO was performed from cytoplasmic and mitochondrial cell fractions and proteins were detected by Western blotting. In some experiments, a continually active Akt construct was introduced. Nuclear to cytoplasmic and mitochondrial translocation of phospho-Akt was visualized by confocal microscopy. Results: Here we present evidence for a PARP1 mediated, PARylation-dependent interaction between ATM and NEMO, which is responsible for the cytoplasmic transport of phosphorylated (thus, activated) ATM kinase. In turn, the cytoplasmic p-ATM and NEMO forms complex with mTOR and Akt, yielding the phospho-ATM-NEMO-Akt-mTOR signalosome, which is responsible for the PARP-inhibition induced Akt activation. The phospho-ATM-NEMO-Akt-mTOR signalosome localizes to the mitochondria and is essential for the PARP-inhibition-mediated cytoprotective effects in oxidatively stressed cells. When the formation of the signalosome is prevented, the cytoprotective effects diminish, but cells can be rescued by constantly active Akt1, further confirming the critical role of Akt activation in cytoprotection. Conclusions: Taken together, the data presented in the current paper are consistent with the hypothesis that PARP inhibition suppresses the PARylation of ATM, which, in turn, forms an ATM-NEMO complex, which exits the nucleus, and combines in the cytosol with mTOR and Act, resulting in Act phosphorylation (i.e. activation), which, in turn, produces the cytoprotective action via the induction of Akt-mediated survival pathways. This mechanism can be important in the protective effect of PARP inhibitor in various diseases associated with oxidative stress. Moreover, disruption of the formation or action of the phospho-ATM-NEMO-Akt-mTOR signalosome may offer potential future experimental therapeutic checkpoints.

KW - Akt

KW - Cytotoxicity

KW - Mitochondrion

KW - Oxidative stress

KW - Poly(ADP)ribose polymerase

KW - Signaling

UR - http://www.scopus.com/inward/record.url?scp=85054653927&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85054653927&partnerID=8YFLogxK

U2 - 10.1016/j.bcp.2018.10.005

DO - 10.1016/j.bcp.2018.10.005

M3 - Article

JO - Biochemical Pharmacology

JF - Biochemical Pharmacology

SN - 0006-2952

ER -