Figure 7.

Phosphorylated p62 translocates to mitochondria in an AMPK-dependent manner in I(−) HCN cells. A, co-transfection of Sh-rp62 HCN cells with plasmids encoding DsRed2-Mito and HA-hp62-WT, -S294A mutant, or -S294E mutant. Translocation of p62 to mitochondria was examined by immunocytochemistry 5 h after insulin withdrawal. CC (0.5 μm) was added as indicated. B, analysis of A. Quantification of mitochondrial localization of p62 (n = 9) is shown. C, live confocal imaging for monitoring mitochondrial translocation of GFP-p62 upon insulin withdrawal. Arrowheads indicate the immobile p62 puncta in I(+) HCN cells; arrows indicate the dynamic p62 puncta that overlap with the mitochondria and change their size over time in I(−) HCN cells. D, AMPK is required for p62 translocation to mitochondria. Insulin withdrawal-induced translocation of hp62-WT, but not its phosphomimicking S294E form, was prevented in AMPK α2 KO HCN cells. E, analysis of D. Quantification of mitochondrial localization of p62 (n = 10) is shown. F, detection of phosphorylated p62 in the mitochondrial fraction following insulin withdrawal for 5 h. Subcellular fractions were analyzed by Western blotting with p62 Ser-293 phosphospecific antibody. Blots shown are representative of three independent experiments, which yielded similar results. Error bars represent ±S.D. from independent assays. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bar, 10 μm. Cyto, cytosolic fraction; Mito, mitochondrial fraction; COX, cytochrome c oxidase; p-p62, phosphorylated p62; t-p62, total p62.