Atg13 interacts with ULK1, ULK2, and FIP200. (a) ULK1 and ULK2 are coimmunoprecipitated with Atg13. HA-tagged ULK1 was expressed with myc-tagged Atg13 or control proteins (S6K1 and tubulin) in 293T cells. Anti-myc immune complexes were assessed for the presence of HA-ULK1 and HA-ULK2 by Western blotting. (b) Atg13 binds ULK1 and ULK2. Myc-tagged Atg13 was expressed with HA-tagged ULK1, ULK2, or control proteins (S6K1, tubulin) in 293T cells. Anti-HA immune complexes were isolated and the amount of Atg13 was assessed by Western blotting. (c) Recombinant Atg13 is coimmunoprecipitated with ULK1. Myc-tagged Atg13 or control proteins was expressed in 293T cells. Anti-myc immune complexes were isolated, and the presence of endogenous ULK1 was assessed by Western blotting. (d) Endogenous Atg13 is coimmunoprecipitated with ULK1. ULK1 immunoprecipitate was obtained from 293T cells in the presence (+pep) or absence (−pep) of ULK1 antibody epitope peptides and the amounts of Atg13 in the immune complexes were assayed by Western blotting. (e) Atg13 interacts with FIP200. Myc-tagged Atg13 or tubulin (control) was coexpressed with HA-FIP200 in 293T cells. Anti-myc immune complexes were analyzed for the presence of FIP200 on Western blots. (f) FIP200 interacts with Atg13, ULK1, and ULK2. HA-tagged Atg13, ULK1, and ULK2 were coexpressed with myc-tagged FIP200 in 293T cells. HA immunoprecipitates were analyzed for the amount of myc-FIP200 on Western blots.

Atg13, ULK1, and ULK2 are important for autophagy. (a) Knockdown of Atg13 or ULK2 or knockout of ULK1 inhibits rapamycin-induced autophagic flux of LC3-II. HEK293T cells stably transduced by lentiviral shRNAs or MEF cells were treated with rapamycin (100 nM) or vehicle for 4 h in the presence or absence of lysosomal inhibitors, pepstatin A (10 μg/ml) and E-64 (10 μg/ml), and the indicated proteins in cell lysates were analyzed by Western blotting. (b) Quantitative analysis of relative amounts of LC3-II from two independent measurements (mean ± SD). (c) Knockdown of Atg13, ULK1, or ULK2 or knockout of ULK1 inhibits rapamycin-induced degradation of p62 and enhances S6K1 phosphorylation. The shRNA-transduced HEK293T cells or MEF cells were treated with rapamycin or vehicle for 4 h, and the indicated proteins were analyzed by Western blotting. (d) Knockdown of Atg13, ULK1, or ULK2 inhibits the formation of LC3-positive autophagosomes. HeLa cells stably transduced by lentiviral shRNAs were treated with rapamycin (100 nM) or vehicle for 24 h and stained with anti-LC3 antibody (green) and DAPI (violet). (e) Quantitative analysis of the number of cells with LC3-positive autophagosome structures relative to the total number of cells (mean ± SD). Three or four areas of microscopic images were captured and analyzed by counting the stained cells (Fig S2). (f) Knockdown of ULK2 inhibits the formation of LC3-positive autophagosomes. HeLa cells stably transduced by lentiviral shRNA were treated with rapamycin (100 nM) or vehicle for 2 h or 5 h in the presence of pepstatin (10 μg/ml) and stained with anti-LC3 antibody (green) and DAPI (violet). (g) Quantitative analysis of the number of cells with LC3-positive autophagosome structures relative to the total number of cells (mean ± SD).

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.

Atg13 positively regulates ULK activity and is required for starvation-induced phosphorylation of FIP200. (a) Atg13 increases the kinase activity of ULK1 in vitro. Myc-tagged ULK1 was isolated from 293T cells and incubated with purified Atg13 at various amounts (0.5, 1, 2, 5 μg) and 1 μg of MBP in the presence of ³²P-ATP. (b) Quantitative analysis of ULK1 activity from panel a. (c) Coexpression of Atg13 with ULK1 increases the kinase activity of ULK1. Myc-tagged ULK1 was coexpressed with HA-Atg13 at different amounts (0, 0.05, 0.5, and 1.5 μg of DNA) in 293T cells. Myc-ULK1 immunoprecipitate was isolated and incubated with 1 μg of MBP in the presence of ³²P-ATP and the levels of ³²P-labeled MBP were analyzed by autoradiography. (d) Quantitative analysis of ULK1 activity from panel c. (e) Atg13 is important for the stability of ULK1 and the rapamycin- and starvation-induced phosphorylation of FIP200. HeLa or HEK293T cells stably transduced by lentiviral shRNAs were treated with rapamycin (100 nM) or vehicle for 1 h or incubated in the presence or absence of leucine (52 μg/ml) for 1 h. The expression levels and the mobility shifts of endogenous FIP200, ULK1, ULK2, and Atg13 were analyzed by Western blotting. (f) Model: The ULK-Atg13-FIP200 complexes mediate mTOR signaling downstream to the autophagy machinery. Starvation suppresses mTOR-mediated phosphorylation of ULK and Atg13 that have inhibitory effects on the kinase activity of ULK, resulting in ULK-mediated phosphorylations of Atg13, FIP200, and ULK itself.

Atg13 interacts with ULK C-terminus and mediates the interaction between ULKs and FIP200. (a and b) Atg13 isoforms 1 and 2, but not isoform 3, bind ULK1 and ULK2. Myc-tagged Atg13 isoforms were expressed alone (a) or with HA-ULK2 (b) in 293T cells, and the amounts of endogenous ULK1 or HA-ULK2 in myc immunoprecipitaes was analyzed by Western blotting. (c) ULK1, ULK2, and FIP200 interact with Atg13 C-terminus. Myc-tagged fragments of Atg13 were coexpressed with HA-tagged ULK1, ULK2, or FIP200 in 293T cells. The levels of HA-constructs coimmunoprecipitated with myc-tagged Atg13 fragments were analyzed on Western blots. (d) Atg13 interacts with ULK C-terminus. Myc-tagged fragments of ULK1 or ULK2 were coexpressed with HA-Atg13 in 293T cells. The amount of HA-Atg13 recovered with myc-tagged constructs was analyzed on Western blots. (e) Atg13 directly interacts with ULK1. GST alone or GST-tagged ULK1 or ULK2 fragments were incubated with either E. coli–purified Atg13 or myc-Atg13 expressed in 293T cells, and the amounts of Atg13 recovered with GST-tagged proteins were analyzed by Western blotting. (f) Atg13 interacts with FIP200 directly and mediates the interaction between FIP200 and ULKs. GST-tagged ULK1 and ULK2 C-terminal fragments purified from E. coli were incubated with myc-tagged FIP200 expressed in 293T cells in the presence or absence of Atg13 purified from E. coli. The amounts of myc-FIP200 and Atg13 recovered with GST-tagged proteins were analyzed by Western blotting. (g) Atg13 is necessary for the interaction between ULK and FIP200. Myc-tagged FIP200 was coexpressed with HA-tagged ULK1 in 293T cells transduced with either scrambled shRNA or Atg13 shRNA. The amount of myc-tagged FIP200 coimmunoprecipitated with HA-tagged ULK1 in HA immunoprecipitate was analyzed by Western blotting.

ULK1 and ULK2 phosphorylate Atg13 and FIP200. (a) ULK1 induces phosphorylation of Atg13. Myc-tagged Atg13 was coexpressed with HA-tagged ULK1 in 293T cells. Myc immunoprecipitate was obtained and treated with lambda phosphatase for 30 min. The migration patterns of Atg13 and ULK1 isolated from myc immunoprecipitation were analyzed on SDS-PAGE. (b) ULK1 induces ³²P labeling of Atg13. Myc-tagged Atg13 was coexpressed with HA-tagged ULK1 or control proteins in 293T cells. Transduced cells were incubated with ³²P in phosphate-free DMEM for 4 h. Myc-Atg13 immunoprecipitate was isolated and analyzed for its phosphorylation state by autoradiography. (c) The kinase activity of ULK1 and ULK2 is required for phosphorylation of Atg13. HA-tagged Atg13 was expressed together with ULK1 wild type, its kinase-dead mutant M92A, or ULK2 wild type in 293T cells. The migration patterns of HA-Atg13 and myc-ULK on SDS-PAGE were analyzed by Western blotting. (d) ULK1 and ULK2 directly phosphorylate Atg13. Myc-tagged ULK1 or ULK2 was isolated by myc immunoprecipitation from 293T cells and incubated with Atg13 purified from E. coli in the presence of ³²P-ATP in vitro. The incorporation of ³²P into ULK1, ULK2, and Atg13 was analyzed by autoradiography. (e) The kinase activity of ULK1 and ULK2 is required for phosphorylation of FIP200. Myc-tagged FIP200 was coexpressed with ULK1 wild type or M92A mutant (kinase dead) or ULK2 wild type in 293T cells, and the migration patterns of myc-FIP200 in cell lysate was analyzed on SDS-PAGE. (f) ULK1 and ULK2 directly phosphorylate FIP200. Myc-tagged ULK1 wild type or M92A mutant or ULK2 was isolated from 293T cells by immunoprecipitation using anti-myc antibody and incubated in the presence of ³²P-ATP with the FIP200 fragment containing residues 860-end that was purified from E. coli. The incorporation of ³²P into ULK and FIP200 was analyzed by autoradiography. (g) ULK1 and ULK2 are important for Atg13 and FIP200 phosphorylation in living cells. ULK1 MEF cells or shRNA-transduced 293T cells were harvested and the phosphorylation states of FIP200, ULK1, ULK2, and Atg13 were analyzed on SDS-PAGE by Western blotting.

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.