Mitochondrially localized ERK2 regulates mitophagy and autophagic cell stress
Mitochondrially localized ERK2 regulates mitophagy and autophagic cell stress
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DOI：
10.4161/auto.6458
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发表时间：
2008-08-16
期刊：
影响因子：
13.3
通讯作者：
Chu, Charleen T.
中科院分区：
文献类型：
--
作者：
Dagda, Ruben K.;Zhu, Jianhui;Chu, Charleen T.
帕金森病（PD）患者脑的退化神经元表现出磷酸化细胞外信号调节蛋白激酶1/2（ERK 1/2）的颗粒，其定位于自噬线粒体。在这里，我们发现，6-羟基多巴胺（6-OHDA）eliminating活性相关的ERK 1/2在SH-SY 5 Y细胞的线粒体定位，这些事件与诱导自噬和线粒体降解之前。瞬时转染野生型（WT）ERK 2或组成型活性MAPK/ERK激酶2（NIEK 2-CA）足以诱导线粒体自噬的程度与6-OHDA引起的相当，而组成型活性ERK 2（ERK 2-CA）有更大的影响。我们开发了WT、CA和激酶缺陷（KD）ERK 2的绿色荧光蛋白（GFP）融合构建体，以研究ERK 2定位在调节线粒体自噬和细胞死亡中的作用。在基础条件下，用GFP-ERK 2-WT或GFP-ERK 2-CA转染而非GFP-ERK 2-KD转染的细胞显示离散的细胞质ERK 2颗粒，其中显著部分与线粒体和自噬溶酶体成熟的标记物共定位。共定位的GFP-ERK 2/线粒体颗粒被6-OHDA进一步增加，并经历自噬降解，因为自溶酶体降解的抑制剂巴非霉素-A强烈地增加了它们的检测。有趣的是，增加ERK 2-WT或ERK 2-CA表达足以促进可比较的自噬水平，如通过分析自噬标志物微管相关蛋白I轻链3（LC 3）所评估的。相比之下，线粒体自噬水平与ERK活性水平更紧密相关，这可能是由于与ERK 2-WT相比，ERK 2-CA更大程度地定位于线粒体。这些数据表明，ERK 2活性的线粒体定位足以概括6-OHDA对线粒体自噬和自噬细胞死亡的影响。
Degenerating neurons of Parkinson's disease (PD) patient brains exhibit granules of phosphorylated extracellular signal-regulated protein kinase 1/2 (ERK1/2) that localize to autophagocytosed mitochondria. Here we show that 6-hydroxydopamine (6-OHDA) elicits activity-related localization of ERK1/2 in mitochondria of SH-SY5Y cells, and these events coincide with induction of autophagy and precede mitochondrial degradation. Transient transfection of wildtype (WT) ERK2 or constitutively active MAPK/ERK Kinase 2 (NIEK2-CA) was sufficient to induce mitophagy to a degree comparable with that elicited by 6-OHDA, while constitutively active ERK2 (ERK2-CA) had a greater effect. We developed green fluorescent protein (GFP) fusion constructs of WT, CA, and kinase-deficient (KD) ERK2 to study the role of ERK2 localization in regulating mitophagy and cell death. Under basal conditions, cells transfected with GFP-ERK2-WT or GFP-ERK2-CA, but not GFP-ERK2-KD, displayed discrete cytoplasmic ERK2 granules of which a significant fraction colocalized with mitochondria and markers of autophagolysosomal maturation. The colocalizing GFP-ERK2/mitochondria granules are further increased by 6-OHDA and undergo autophagic degradation, as bafilomycin-A, an inhibitor of autolysosomal degradation, robustly increased their detection. Interestingly, increasing ERK2-WT or ERK2-CA expression was sufficient to promote comparable levels of macroautophagy as assessed by analysis of the autophagy marker microtubule-associated protein I light chain 3 (LC3). In contrast, the level of mitophagy was more tightly correlated with ERK activity levels, potentially explained by the greater localization of ERK2-CA to mitochondria compared to ERK2-WT. These data indicate that mitochondrial localization of ERK2 activity is sufficient to recapitulate the effects of 6-OHDA on mitophagy and autophagic cell death.