Figure 5.

mTOR phosphorylates Atg13, ULK1, and ULK2. (a) Rapamycin or leucine deprivation induces dephosphorylation of ULK1, ULK2, and Atg13. HEK293T cells were treated with rapamycin or vehicle for 1 h or deprived of leucine for 2 h (−leu) or deprived of leucine for 1 h and supplemented with leucine for 1 h (−leu → +leu). The migration patterns of endogenous Atg13, ULK1, and ULK2 on SDS-PAGE were analyzed by Western blotting. (b) Rapamycin blocks phosphorylation of ULK1. Myc-tagged ULK1 was expressed in 293T cells and incubated with ³²P in phosphate-free DMEM for 4 h. Cells were treated with rapamycin (100 nM) or vehicle for 1 h. Myc-ULK1 immunoprecipitate was isolated, and the phosphorylation state of myc-ULK1 was analyzed by autoradiography. (c) Rheb induces phosphorylation of Atg13 and ULK1, whereas rapamycin suppresses the phosphorylation. Myc-tagged Atg13 was expressed with or without HA-tagged Rheb in 293T cells and incubated with ³²P in phosphate-free DMEM for 4 h. Cells were treated with rapamycin (100 nM) or vehicle for 1 h. The migration patterns and ³²P incorporation of myc-Atg13 and endogenous ULK1 isolated from myc immunoprecipitate were analyzed by Western blotting and autoradiography. (d) mTOR immunoprecipitate has the capacity to phosphorylate Atg13. Endogenous mTOR was isolated from 293T cells by mTOR immunoprecipitation and incubated with Atg13 purified from E. coli in the presence of ³²P-ATP. The reaction was analyzed by autoradiography. (e) mTOR phosphorylates Atg13 in vitro. The active form of mTOR containing residues 1362-end (Millipore) and myc-mTOR wild type (WT) and its kinase dead (KD) mutant, D2357E, were tested for their activity to phosphorylate Atg13 in vitro. The incorporation of ³²P into Atg13 and mTOR was analyzed by autoradiography. (f) mTOR phosphorylates ULK1 in vitro. Myc-tagged ULK1 M92A kinase dead (KD) mutant or ULK1 fragment containing residues 281-end was incubated with the active form of mTOR (Millipore) in the presence of ³²P-ATP. The levels of ³²P-labeled ULK1 KD, ULK1 fragment, and mTOR fragment were analyzed by autoradiography. (g) mTOR immunoprecipitate has the capacity to phosphorylate ULK1. Endogenous mTOR or myc-tagged wild type or the kinase dead mutant of mTOR was isolated from 293T cells by immunoprecipitation and incubated with E. coli—purified ULK1 fragment containing residues 651-end. The levels of ³²P-labeled mTOR and the ULK1 fragment were analyzed by autoradiography. (h) mTOR immunoprecipitate has the capacity to phosphorylate ULK2. mTOR immunoprecipitates were prepared as described in panel g and incubated with E. coli–purified ULK2 fragment containing residues 651-end. The autoradiogram was obtained to analyze the phosphorylation status of mTOR and ULK2.