Journal: Cell Death and Differentiation

Article Title: Nuclear ULK1 promotes cell death in response to oxidative stress through PARP1

doi: 10.1038/cdd.2015.88

Figure Lengend Snippet: ULK1 kinase activity promotes PARP1 activation and sensitizes cells to oxidative stress-induced death. (a) Ulk1 KO/Ulk2 shRNA MEFs27 stably expressing vector control, WT ULK1, or KI ULK1 were cotransfected with a vector containing PARP1-GFP and incubated in complete media (CM) with or without H2O2 for 20 min. PARP1 was depleted from the nucleoli of cells expressing WT ULK1 but not from those of cells expressing the KI mutant. Scale bars=10 μM. Representative immunoblots (b) and a graph (c) show the levels of PARylated proteins in the indicated MEF lines incubated in the presence or the absence of 500 μM H2O2 for 10 min. (d and e) Trypan blue-exclusion assays demonstrate that the KI-ULK1 mutant fails to promote cell death to the same extent as WT ULK1 does when stably expressed in Ulk1-deficient MEFs. (f) The 293T cells cotransfected with PARP1-GFP and either WT ULK1 or the R266A-ULK1 mutant were treated for 10 min with 100 μM H2O2. Immunoblot analyses demonstrate a decrease in PARylated proteins in cells transfected with the R266A mutant. (g) The 293T cells transfected with the indicated expression constructs were treated with 100 μM H2O2. ATP levels were analyzed in cells collected 10, 30, or 60 min after treatment with 100 μM H2O2 and normalized to levels in untreated cells. (h) Trypan blue-exclusion assays performed 6 h after treatment with the indicated dose of H2O2 demonstrate increased viability of 293T transfected with the R266A-ULK1 mutant expression construct compared with that of those transfected with the WT ULK1 construct. (i) The 293T cells were transfected with empty vector (−), WT ULK1 (WT), or the following mutant forms of ULK1: K46A (kinase-defective mutant), 4SA (AMPK-resistant, autophagy-defective mutant), or R266A (NLS mutant). Transfected cells were treated with 4.5 μM β-lapachone for 72 h and then cell viability was assessed using CellTiter-Glo assays. Signals were normalized to those in DMSO-treated controls (n=4 biologic replicates per condition), P<0.001. (j) The 293T cells transfected with the indicated expression vectors were arrayed in a 96-well format and treated with either camptothecin for 72 h (starting at a concentration equal to 7 mM followed by threefold dilutions) or DMSO (negative control). Signals (in relative light units, RLUs) from the CellTiter-Glo assays were normalized by subtracting the median RLU of the DMSO control wells and then fit to the four-parameter log-logistic function (‘Hill equation') to yield ‘activity.' EC50: log concentration at half-maximal observed response. The results demonstrate an eightfold difference between the EC50 of WT ULK1 and that of the R266A-ULK1 mutant. (k) Representative immunoblot showing the levels of ULK1 in cells transfected with the indicated constructs and used for the experiments described in i and j (n=3 biologic replicates per condition, unless otherwise indicated)

Article Snippet: Flag-tagged ULK1 (gift from Dr. Toshifumi Tomoda), pMXs-IP-EGFP-ULK1 (gift from Noboru Mizushima, Addgene plasmid 38193), 52 pMXs-IP-EGFP-ULK2 (gift from Noboru Mizushima, Addgene plasmid 38201), 52 HA-tagged ATG13 (gift from Dr. Do-Hyung Kim), 53 and PARP1-GFP (gift from Dr.

Techniques: Activity Assay, Activation Assay, shRNA, Stable Transfection, Expressing, Plasmid Preparation, Incubation, Mutagenesis, Western Blot, Transfection, Construct, Concentration Assay, Negative Control