Serine phosphorylation at a novel site in p62 is required for ACD in I(−) HCN cells.
A, the predicted phosphorylation motif of rat p62 Ser-293 is well conserved in mouse (Ser-296) and human (Ser-294) p62. B, expression of hp62-WT and hp62-S294E, but not hp62-S294A, restored cell death in Sh-rp62 HCN cells following insulin withdrawal. Sh-rp62 HCN cells were transfected with plasmids to express HA-tagged hp62 (WT, phosphorylation-defective mutant (S294A), or phosphomimicking mutant (S294E)). Cell death was measured 24 h after insulin withdrawal (n = 5). C, hp62-WT and hp62-S294E, but not hp62-S294A, restored autophagy flux. D, quantification of LC3-II and p62 after normalization to β-actin (n = 5). Error bars represent ±S.D. from independent assays. *, p < 0.05; **, p < 0.01.