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. 2009 May 1;284(18):12297-305.
doi: 10.1074/jbc.M900573200. Epub 2009 Mar 3.

ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy

Ian G Ganley  1 , Du H Lam, Junru Wang, Xiaojun Ding, She Chen, Xuejun Jiang
Affiliations

ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy

Ian G Ganley et al. J Biol Chem. .

Abstract

Autophagy is a degradative process that recycles long-lived and faulty cellular components. It is linked to many diseases and is required for normal development. ULK1, a mammalian serine/threonine protein kinase, plays a key role in the initial stages of autophagy, though the exact molecular mechanism is unknown. Here we report identification of a novel protein complex containing ULK1 and two additional protein factors, FIP200 and ATG13, all of which are essential for starvation-induced autophagy. Both FIP200 and ATG13 are critical for correct localization of ULK1 to the pre-autophagosome and stability of ULK1 protein. Additionally, we demonstrate by using both cellular experiments and a de novo in vitro reconstituted reaction that FIP200 and ATG13 can enhance ULK1 kinase activity individually but both are required for maximal stimulation. Further, we show that ATG13 and ULK1 are phosphorylated by the mTOR pathway in a nutrient starvation-regulated manner, indicating that the ULK1.ATG13.FIP200 complex acts as a node for integrating incoming autophagy signals into autophagosome biogenesis.

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Figures

FIGURE 1.
FIGURE 1.
ATG13 interacts with both ULK1 and FIP200 in vivo. A, GFP-ATG13 immunoprecipitations. MEF cells, depleted of the indicated proteins by RNAi, were transduced with retrovirus expressing GFP-ATG13. Following a 1-h incubation in complete growth medium or amino acid-free medium (starve), cells were lysed, and GFP-ATG13 immunoprecipitated using mouse anti-GFP. Co-immunoprecipitated proteins were analyzed by immunoblotting as indicated. B, RNAi depletion of ATG13. Agarose gel depicting RT-PCR of ATG13 and GAPDH mRNA isolated from control-depleted and two ATG13-depleted cell clones (13-4 and 13-5) is shown. C, loss of ULK1 complex proteins inhibits autophagy. MEF cells, depleted of the indicated proteins and expressing GFP-LC3, were incubated in complete medium or amino acid-free medium for 1 h, followed by lysis, SDS-PAGE, and immunoblot with anti-GFP to detect GFP-LC3. D, same cells described in C were grown on glass coverslips and following incubation in complete medium or amino acid-free medium for 1 h, were fixed and GFP-LC3 visualized under the fluorescence microscope. E, ULK1, ATG13, and FIP200 colocalize upon autophagy stimulation. U2OS cells, grown on glass coverslips, were transfected with mCherry-ULK1 and GFP-ATG13 (top panels) or GFP-ULK1 and mCherry-FIP200 (bottom panels). 24-h post-transfection, cells were washed and incubated in amino acid-free medium for 1 h, followed by fixing and visualization under the fluorescence microscope. Arrows indicate examples of co-localization between mCherry-ULK1 and GFP-ATG13 (top panels) or GFP-ULK1 and mCherry-FIP200 (bottom panels).
FIGURE 2.
FIGURE 2.
ULK1, ATG13, and FIP200 form a large protein complex. A, ULK1 interacts with ATG13 and FIP200. Equimolar amounts of the indicated recombinant proteins (U, ULK1; F, FIP200; 13, ATG13) were incubated together at room temperature for 1 h before precipitation of ULK1 with anti-ULK1 IgG. Following washes, bound proteins were subjected to immunoblotting with the indicated antibodies. B, ATG13 interacts with ULK1 and FIP200. Binding assay was carried out as in A, but T7-tagged ATG13 was precipitated using anti-T7 antibody followed by washing and analysis of bound proteins with immunoblotting. C, ULK1, ATG13, and FIP200 form a large protein complex. The indicated proteins were incubated alone or in combination followed by gel filtration over a Superose 6 column. The indicated fractions were subjected to immunoblotting with the relevant antibodies. The gel filtration position of the molecular weight standards (MW) is shown at the top.
FIGURE 3.
FIGURE 3.
Localization of ULK1 to the isolation membrane requires both ATG13 and FIP200. A, ULK1 localizes to isolation membranes upon autophagy stimulation. MEF cells stably expressing GFP-ATG5 alone (top panels) or GFP-ATG5 plus FLAG-S-tagged ULK1 (bottom panels) were grown on glass coverslips and following incubation in amino acid-free medium for 1 h, were fixed and processed for immunofluorescence. Costaining for endogenous ULK1 (red) is shown in the top panels along with GFP fluorescence (green), while staining with anti-S tag (for FLAG-S-ULK1) along with GFP fluorescence is shown in the bottom panels. B, localization of ULK1 is disrupted in ATG13- and FIP200-depleted cells. Left panels show 1-h amino acid-starved MEF cells, with the indicated protein depleted by RNAi, stained with anti-ULK1 (green) and DAPI (blue, to mark the nuclei). Right panels show the same depleted MEF cells expressing FLAG-S-tagged ULK1, treated as in the left panels, but stained with anti-S tag (red) to detect the tagged ULK1 as well as DAPI (blue).
FIGURE 4.
FIGURE 4.
Stimulation of ULK1 kinase activity by ATG13 and ULK1. A, ATG13 stimulates ULK1 kinase activity. 10 nm recombinant ULK1 was incubated with the indicated amounts of recombinant FLAG-ATG13 in the presence of 0.3 mg/ml MBP and [γ-32P]ATP as described under “Experimental Procedures.” Reactions were stopped by the addition of SDS sample buffer followed by SDS-PAGE and transfer to nitrocellulose. The upper panel shows the autoradiograph of 32P-labeled MBP with the total amount of MBP shown by Ponceau-S staining of the membrane below. The quantities of ULK1 and FLAG-ATG13 in each reaction are indicated by immunoblot in the lower panels. B, ATG13 and FIP200 act additively to stimulate ULK1 kinase activity. 10 nm ULK1, 200 nm FLAG-ATG13, or 200 nm hisFIP200 were incubated alone or in combination, as indicated, along with MBP and [γ-32P]ATP and processed as in A. C, ULK1 kinase activity is impaired in ATG13- and FIP200-depleted cells. MEF cells, either control-, ATG13-, or FIP200-depleted were incubated in complete medium or amino acid-free medium for 1 h, followed by lysis and immunoblotting of FIP200 (top panel) and ULK1 (bottom panel). Protein loading of the duplicate samples is shown by Ponceau-S staining in the lower panel.
FIGURE 5.
FIGURE 5.
ULK1 and ATG13 are subjected to mTOR-mediated phosphorylation. A, ULK1 remains partially phosphorylated following autophagy induction. MEF cells were incubated in amino acid-free medium (starve) for 1 h followed by lysis. Lysates were then treated with phosphatase, or mock-treated, followed by immunoblot to analyze the mobility of ULK1. B, ATG13 is dephosphorylated upon autophagy induction. Lysates derived from MEF cells stably expressing GFP-ATG13 were either treated or mock-treated with phosphatase then subject to SDS-PAGE and immunoblotting anti-GFP. C, ULK1 and ATG13 are dephosphorylated upon autophagy induction. MEF cells expressing GFP-ATG13 were incubated in complete medium (control), medium containing 500 nm rapamycin (rapamycin) or amino acid-free medium (starve), followed by lysis and immunoblot with anti-ULK1 (top panel) or anti-GFP (bottom panel). Samples were loaded in duplicate. D, mTOR overexpression increases ATG13 phosphorylation. 293T cells were transfected with Myc-tagged ATG13, either alone or in combination with Myc-tagged wild type (WT) or kinase-dead (dead) mTOR as indicated. 24-h post-transfection cells were incubated in complete medium, medium containing 500 nm rapamycin, or amino acid-free medium (starve) for 1.5 h. Cells were then lysed, loaded in duplicate, and subjected to SDS-PAGE and immunoblot with anti-Myc.
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