Mitochondrially localized ERK2 regulates mitophagy and autophagic cell stress: implications for Parkinson's disease.
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 (MEK2-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 1 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.
Dagda
Ruben K
RK
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Zhu
Jianhui
J
Kulich
Scott M
SM
Chu
Charleen T
CT
D000818:Animals; D001343:Autophagy / Q000502:physiology*; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line / Q000187:drug effects; D003594:Cytoplasmic Granules / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D016627:Oxidopamine / Q000494:pharmacology; D010300:Parkinson Disease / Q000378:metabolism* / Q000473:pathology; D010588:Phagosomes / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D015415:Biomarkers; D011993:Recombinant Fusion Proteins; D016627:Oxidopamine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3