NEDD4L ubiquitylates ULK1 at lysine 925 and lysine 933. (A) HeLa cells were transfected with ULK1^WT, ULK1^K925R, and ULK1^K933R HA-tagged proteins and treated with CHX (50 µM) for different time periods. Levels of ULK1 and TUBULIN were detected by WB. Densitometric analysis of ULK1 over TUBULIN is also shown (right graph). Data are expressed as the mean ± SEM (n = 3). (B and C) HeLa cells were transfected ULK1^WT, ULK1^K925R, ULK1^K933R, and ULK1^K925R+K9333R HA-tagged proteins together with NEDD4L-HA in the presence or not of MG132 (4 h) and all were treated with CHX for 3 h. Levels of ULK1, NEDD4L, and TUBULIN were detected by WB. (B) Densitometric analysis of ULK1+MG132/ULK1 control over TUBULIN bands is also shown. Data are expressed as the mean ± SEM (n = 3). (D and E) HeLa cells were transfected with ULK1^WT, ULK1^K925R, ULK1^K933R, and ULK1^K925R+K933R HA-tagged proteins in the presence or not of MG132 and autophagy was induced with EBSS for 4 h. The levels of ULK1 and TUBULIN were detected by WB. (D) Densitometric analysis of ULK1+MG132/ULK1 control over TUBULIN bands is also shown. Data are expressed as the mean ± SEM (n = 3). (F) HeLa cells were transfected with a vector encoding a 6xHIS-tag ubiquitin together with ULK1^WT, ULK1^K925R and ULK1^K933R HA-tagged proteins in the presence of NEDD4L. Protein extracts were prepared in a denaturing urea buffer and subjected to Ni-NTA purification. The amount of ubiquitylated ULK1 copurified with 6xHIS-ubiquitin was valuated by WB. The intensity of ULK1 bands is shown. Data are the mean ± SEM (n = 3). (G) HeLa cells were transfected with a vector encoding a 6xHIS-tag ubiquitin together with ULK1^WT and ULK1^K925R+K933R mCherry-tagged proteins in the presence of NEDD4L. Protein extracts were prepared in a denaturing urea buffer and subjected to Ni-NTA purification. The amount of ubiquitylated ULK1 copurified with 6xHIS-ubiquitin was evaluated by WB. (H) Endogenous ULK1 was silenced by RNAi; cells were then transfected with ULK1^WT or ULK1^K925R+K933R mCherry-tagged plasmids. Cells were nutrient-starved for the indicated time periods, fixed and labeled with anti-ATG16L antibody (green puncta), and visualized by confocal microscopy. Analysis of the number of ATG16L occurrence per cell is shown in the graph. Data are expressed as the mean value± SEM (n = 3). Representative images are shown. Bars, 20 µm (>50 cells analyzed per sample). In A, B, D, and F, data were analyzed by one-way ANOVA followed by Tukey post hoc test. *, P < 0.05; ****, P < 0.0001. In H, data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparison post test. **, P < 0.01; ****, P < 0.0001. For all ULK1 mutant constructs the same amount of DNA is used for the transfection; the differences are caused by the increased stability of these mutants.