Department of Biochemistry and Medical Chemistry, University of Pécs, Medical School, Szigeti Street 12, 7624 Pécs, Hungary.
Department of Biochemistry and Medical Chemistry, University of Pécs, Medical School, Szigeti Street 12, 7624 Pécs, Hungary; Department of Medicine, University of Fribourg, Switzerland.
Biochem Pharmacol. 2019 Apr;162:98-108. doi: 10.1016/j.bcp.2018.10.005. Epub 2018 Oct 6.
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.
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.
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.
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.
聚（ADP-核糖）聚合酶 1（PARP1）抑制在各种受到氧化应激的细胞类型中具有很好的细胞保护作用，这一点已得到充分证明。先前，我们已经证明 PARP1 抑制可激活 Akt，并表明该反应在维持线粒体完整性和细胞存活方面起着关键作用。然而，核 PARP1 如何向细胞质 Akt 发出信号仍未确定。
在培养物中培养 WRL 68、HeLa 和 MCF7 细胞。用过氧化氢诱导氧化应激。用 PARP 抑制剂 PJ34 抑制 PARP。使用特异性 siRNA 沉默 ATM、mTOR 和 NEMO。细胞活力测定基于 MTT 测定。进行 PARP-ATM 下拉实验；通过 Western blot 检测每种蛋白质。从细胞质和线粒体细胞部分进行 ATM、磷酸化 ATM 和 NEMO 的免疫沉淀，并通过 Western blot 检测蛋白质。在一些实验中，引入了持续活跃的 Akt 构建体。通过共聚焦显微镜可视化磷酸化 Akt 的核质和线粒体易位。
在这里，我们提供了 PARP1 介导的、依赖 PAR 化的 ATM 和 NEMO 之间相互作用的证据，该相互作用负责磷酸化（即激活）ATM 激酶的细胞质转运。反过来，细胞质 p-ATM 和 NEMO 与 mTOR 和 Akt 形成复合物，产生磷酸化 ATM-NEMO-Akt-mTOR 信号体，负责 PARP 抑制诱导的 Akt 激活。磷酸化 ATM-NEMO-Akt-mTOR 信号体定位于线粒体，对于氧化应激细胞中 PARP 抑制介导的细胞保护作用至关重要。当信号体的形成被阻止时，细胞保护作用减弱，但通过持续活跃的 Akt1 可以挽救细胞，进一步证实了 Akt 激活在细胞保护中的关键作用。
综上所述，本文提出的研究结果与 PARP 抑制抑制 ATM 的 PAR 化，进而形成 ATM-NEMO 复合物，该复合物离开细胞核，在细胞质中与 mTOR 和 Akt 结合，导致 Akt 磷酸化（即激活）的假说一致，这反过来又通过诱导 Akt 介导的存活途径产生细胞保护作用。该机制在各种与氧化应激相关的疾病中 PARP 抑制剂的保护作用中可能很重要。此外，破坏磷酸化 ATM-NEMO-Akt-mTOR 信号体的形成或作用可能为未来的实验治疗检查点提供潜在的靶点。
