Figure 6.

mTOR negatively regulates the kinase activity of ULK. (a) Rapamycin increases the kinase activity of ULK1. 293T cells were treated with rapamycin or vehicle for 1 h. ULK1 immunoprecipitate was isolated using anti-ULK1 antibody and incubated with MBP (1 μg) in the presence of ³²P-ATP. The levels of ³²P-labeled MBP were analyzed by autoradiography. (b) Quantitative analysis of ULK1 kinase activity from two independent experiments (mean ± SD). (c) Leucine starvation enhances the kinase activity of ULK1. 293T cells were incubated in the presence or absence of leucine (52 μg/ml) for 1 h. ULK1 immunoprecipitate was isolated and analyzed for its kinase activity toward phosphorylation of MBP in the presence of ³²P-ATP. (d) Quantitative analysis of leucine-dependent ULK1 kinase activity. (e) Rheb overexpression inhibits ULK1 kinase activity. Myc-tagged ULK1 was expressed alone or with HA-tagged Rheb in 293T cells. Myc-ULK1 was isolated by immunoprecipitation using anti-myc antibody and analyzed for the kinase activity as described in panel a. (f) Quantitative analysis of the ULK1 kinase activity from two independent measurements (mean ± SD). (g) Rapamycin enhances the kinase activity of ULK2. Myc-tagged ULK2 was expressed in 293T cells and treated with rapamycin or vehicle for 1 h. Myc-ULK2 was isolated by immunoprecipitation using anti-myc antibody and incubated with either MBP or the FIP200 fragment (aa 860-end) purified from E. coli in the presence of ³²P-ATP. The levels of ³²P-labeled MBP and FIP200 were analyzed by autoradiography. (h) Quantitative analysis of ULK2 kinase activity toward MBP and FIP200. (i) Rapamycin or starvation does not have any significant effect on the interaction between ULK1, Atg13, and FIP200 but induces phosphorylation of ULK1 during a prolonged rapamycin treatment. HEK293T cells were treated with rapamycin or vehicle for 1 or 24 h or incubated in the presence or absence of leucine for 1, 2, or 24 h, and ULK1 immunoprecipitate was isolated using anti-ULK1 antibody. The amounts of Atg13 and FIP200 coimmunoprecipitated with ULK1 and in cell lysate were analyzed by Western blotting. (j) ULK1 and ULK2 are important for rapamycin- and starvation-induced phosphorylation of FIP200. ULK1 MEF cells or shRNA-transduced 293T cells were treated with rapamycin or vehicle or incubated in the presence or absence of leucine for 1 h, and the phosphorylation states of FIP200, ULK, and Atg13 were analyzed on SDS-PAGE by Western blotting.