Fig. 3: R170me2s activates ULK1 by facilitating ULK1 T180 autophosphorylation.

b–e, h–k Immunoblot were performed with the indicated antibodies. a Indicated cells were incubated under 1% oxygen for 12 h. Flag-ULK1 proteins were precipitated, and ULK1 kinase activity was analyzed. Data represent the mean ± SD from three independent experiments. P-values are from the two-sided t-tests. Bonferroni correction was used for multiple hypothesis correction. b Indicated LN229 cells incubated under 1% oxygen for 12 h. Exo, exogenous; Endo, endogenous. c Purified ULK1 proteins was immobilized on beads, and mixed with purified His-PRMT5 protein for an in vitro methylation assay. Immobilized ULK1 proteins were washed, and mixed with purified GST-Atg13 or His-Beclin 1 proteins for an in vitro kinase assay. d LN229 cells with expression of Flag-ULK1 were incubated under 1% oxygen for 12 h. Flag-ULK1 proteins were pulldown, washed and mixed with purified WT His-KDM5C or His-KDM5C H514A protein for an in vitro demethylation assay under normoxia condition. The ULK1 precipitates were washed, and mixed with purified GST-Atg13 or His-Beclin 1 proteins for an in vitro kinase assay. e Indicated cells were incubated under 1% oxygen for 12 h. VPS34 activity in the precipitates were measured. Data represent the mean ± SD from three independent experiments. P-value is from the two-sided t-test. f Indicated cells were incubated under 1% oxygen for 12 h. EGFP-FYVE puncta formation was analyzed. Scale bar, 6 µm. Data represent the mean ± SD from three independent experiments. P-value is from the two-sided t-test. g Human ULK1 structure (PDB code: 4WNP) shows an activation loop domain (yellow). The surrounding areas of R170 and T180 are boxed and enlarged. For the side chain of R170 and T180, nitrogen, oxygen and carbon atoms were shown in purple, red and green, respectively. h LN229 cells were incubated under 1% oxygen. i Indicated cells were incubated under 1% oxygen for 12 h. j, k Purified WT Flag-ULK1 (j) or/and Flag-ULK1 R170K (k) proteins were subjected to a His-PRMT5 protein-mediated in vitro methylation assay and an in vitro autophosphorylation assay. Source data are provided as a Source data file.