Figure 6.

ULK1 kinase activity is required for nuclear localization of ULK1 and its interaction with PARP1. (a and b) The 293T cells cotransfected with PARP1-GFP and untagged WT ULK1 were incubated in complete media (CM) in the absence (a) or the presence (b) of H₂O₂ for 30 min. The percentage of cells showing clear nuclear localization of ULK1 increased from 1% to nearly 30% after H₂O₂ treatment. Representative images and line scans indicate the degree of colocalization between PARP1-GFP and ULK1 in cells treated with or without H₂O₂. (c) The 293T cells cotransfected with PARP1-GFP and the nuclear localization-defective R266A-ULK1 mutant were treated for 30 min with 500 μM H₂O₂ before imaging. Representative images and a line scan show the degree of colocalization of R266A-ULK1 and PARP1-GFP, highlighting the nuclear localization defect of the R266A mutant. Scale bars=10 μM. (d) Representative immunoblots of nuclear and cytoplasmic fractions prepared from 293T cells transfected with WT or mutant ULK1 (R266A, KI, or 4SA) and incubated in CM with or without 500 μM H₂O₂ for 30 min. The levels of WT ULK1 and the 4SA mutant in nuclear fractions increase after H₂O₂ treatment, but those of the KI and R66A mutants do not. (e) Whole-cell extracts prepared from 293 T cells cotransfected with PARP1-GFP and untagged WT or mutant (KI, R266A, or 4SA) ULK1 were subjected to IP using anti-GFP-conjugated beads before SDS-PAGE and immunoblot analyses. Immunoblot analyses demonstrate a decrease in PARylated proteins in cells transfected with the R266A mutant. (f) The 293T cells were transfected with HA-tagged ATG13 and the indicated ULK1 expression constructs. Whole-cell extracts were analyzed by SDS-PAGE followed by immunoblot analyses. ATG13 phosphorylated at S318 was detected only in lanes containing WT ULK1 or the AMPK-resistant mutant (4SA)