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. 2011 Jul;7(7):737-47.
doi: 10.4161/auto.7.7.15491. Epub 2011 Jul 1.

ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding

Elaine A Dunlop  1 , David K Hunt, Hugo A Acosta-Jaquez, Diane C Fingar, Andrew R Tee
Affiliations

ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding

Elaine A Dunlop et al. Autophagy. 2011 Jul.

Abstract

Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.

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Figures

Figure 1
Figure 1
ULK1 induces phosphorylation of Raptor. (A) HEK293 cells transfected with Myc-mTOR, HA-Raptor, HA-mLST8 in addition to V5-tagged ULK1 (where indicated), were radiolabeled with [32P]-orthophosphate. After lysis, mTOR/Raptor/mLST8 was immunoprecipitated using anti-HA antibodies and phosphorylation of mTORC1 components assessed by autoradiography. (B) HA-Raptor and V5-ULK1 were co-expressed in HEK293 cells and Raptor was purified by HA-immunoprecipitation. One sample was treated with SAP, as indicated. Western blots were performed to determine the mobility of Raptor. (C) HEK293 cells were co-transfected with HA-Raptor and either wild-type (WT) or kinase dead (KD) V5-ULK1. Following HA-immunoprecipitation, samples were assessed for Raptor mobility by western blot. (D) As in (C) but cells were radiolabeled with [32P]-orthophosphate prior to lysis and immunoprecipitation. Phosphorylation of Raptor was determined by autoradiography.
Figure 2
Figure 2
ULK1 induces phosphorylation of Raptor on multiple sites in 293 cells. (A) A schematic showing the structure of Raptor, including the Raptor N-terminal conserved (RNC) domains, 3 HEAT repeats and 7 C-terminal WD40 repeats. The location of the phosphorylation sites examined in this study is shown in the central region of the protein. (B) HEK293 cells were transfected with HA-Raptor and either wild-type (WT) or kinase dead (KD) V5-ULK1. Following an HA immunoprecipitation, samples were assessed for phosphorylation of Raptor using site-specific antibodies. Total Raptor and ULK1 are shown as controls. (C) HEK293 cells were transfected with Myc-mTOR, HA-Raptor and either wild-type (WT) or kinase dead (KD) V5-ULK1 and treated with 1 µM Ku-0063794 for 3 h prior to lysis, where indicated. Immunoprecipitations and western blots were performed as in (B) with total mTOR, Raptor and ULK1 shown as controls.
Figure 3
Figure 3
ULK1 phosphorylates ATG13 and Raptor in vitro. (A) An in vitro ULK1 kinase assay was performed by immunoprecipitation of either wild-type (WT) or kinase dead (KD) V5-ULK1 followed by incubation with substrate (either purified GST-ATG13 or GST-Raptor) for 45 min in the presence of γ-[32P]-ATP. [32P]-radiolabel incorporation into substrates was determined by autoradiography. (B) The assay was performed as in (A) using GST-Raptor as substrate and cold ATP. Samples were assessed for Raptor phosphorylation using site-specific phospho-Raptor antibodies. Total Raptor and ULK1 are shown as controls.
Figure 4
Figure 4
ULK1 reduces S6K1 phosphorylation and activity. (A) HEK293 cells were transfected with HA-S6K1 with or without V5-ULK1, serum-starved overnight and stimulated with 100 nM insulin for 30 min prior to lysis where indicated. HA-S6K1 was immunoprecipitated and used in an in vitro kinase assay against recombinant rpS6 peptide. [32P]-radiolabel incorporation into rpS6 was determined by autoradiography. S6K1 phosphorylation was determined using phospho-Thr389 antibody. The densitometry of the phospho-rpS6 autorads from the three experiments is shown in the graph (mean ± SD). Statistical significance was analyzed using a student's t-test, *p < 0.01. (B) A S6K1 assay was performed as in (A) but in the presence or absence of Flag-Rheb expression instead of insulin stimulation. S6K1 activity was determined by Thr389 phosphorylation of S6K1 and [32P]-radiolabel incorporation into rpS6. The densitometry of the [32P]-radiolabeled rpS6 autorads from the three experiments is shown in the graph (mean ± SD). Statistical significance was analyzed using a student's t-test, *p < 0.05.
Figure 5
Figure 5
ULK1 inhibits phosphorylation of 4E-BP1 by mTORC1. (A) HEK293 cells were transfected with wild-type or kinase-dead ULK1, serum-starved overnight and then stimulated with 100 nM insulin for 30 min prior to lysis, where indicated. Phosphorylation of 4E-BP1 at Ser65 was analyzed by western blotting. Total 4E-BP1 and ULK1 are shown as controls. Densitometry of phospho-4E-BP1 from independent experiments is shown in the graph (mean ± SD). Statistical significance was analyzed using a student's t-test, *p < 0.01. (B) An in vitro mTORC1 kinase assay was performed by combining immunoprecipitated Myc-mTOR/HA-Raptor complexes, which were expressed with or without V5-ULK1, with recombinant GST-4E-BP1 in the presence or absence of active GST-Rheb (Q64L), as indicated. mTORC1 kinase activity was determined by western blotting using phospho-4E-BP1 (Thr37/46) antibody. The densitometry of the phospho-4E-BP1 blots from the three experiments is shown in the graph (mean ± SD). Statistical significance was analyzed using a student's t-test, *p < 0.05.
Figure 6
Figure 6
ULK1 causes loss of Raptor substrate binding without affecting mTORC1 association. (A) HA-Raptor was expressed in the presence or absence of V5-ULK1. Following HA-immunoprecipitation, western blotting was used to probe for the amount of endogenous mTOR and mLST8 co-immunoprecipitating with Raptor. (B) Raptor substrate binding was assessed using a Raptor overlay assay. 750 ng GST-4E-BP1 was resolved by SDS-PAGE and transferred to PVDF membrane. This was incubated with Myc-Raptor-containing lysate and the amount of Myc-Raptor bound to 4E-BP1 determined by western blotting with Myc antibodies. The graph shows the combined densitometry data of Raptor-4E-BP1 binding for three experiments (mean ± SD). Statistical significance was analyzed using a student's t-test, *p < 0.01. Anti-GST and anti-V5 antibodies were used to obtain total levels of GST-4E-BP1 and V5-ULK1 respectively.
Figure 7
Figure 7
Knockdown of endogenous ULK1 prevents Raptor phosphorylation and increases mTORC1 signaling. (A) Endogenous ULK1 expression was knocked down using shRNA and the phosphorylation of endogenous mTORC1 substrates was determined using phospho-specific antibodies (left part). Phosphorylation of Raptor was determined by [32P]-radiolabel incorporation into endogenous Raptor and using phospho-specific antibodies (right part). Scrambled (Scr) shRNA was used as a control. -serum = serum-free DMEM, -aa = amino acid- and serum-free Krebs Ringer Buffer. (B) V5-ULK2 and HA-Raptor were overexpressed in HEK293 cells. Following an HA-immunoprecipitation, phosphorylation of Raptor was determined using site-specific antibodies. Total Raptor levels are shown as a control. (C) Levels of endogenous phospho-AMPK (Thr172) and phospho-ACC (Ser79) were analyzed under normal growth conditions in HEK293 cells which had been transfected with V5-ULK1 or V5-ULK2 24 h prior to lysis. Total AMPK, ACC, HA-Raptor and V5-ULK1 are shown as controls. (D) A schematic diagram showing the proposed mechanism of ULK1-mediated inhibition of mTORC1. ULK1 inhibits signal transduction through mTORC1 (mTOR/Lst8/Raptor protein complex) by interaction with and phosphorylation of Raptor (ULK1 P-sites: Ser855, Ser859 and weakly at Ser792). ULK1 interaction with Raptor interferes with mTORC1 substrate recognition. AMPK, which binds to ULK1, also phosphorylates the inhibitory Ser792 site. Upon repression of autophagy, ULK1 dissociates from Raptor allowing Raptor to efficiently interact and phosphorylate mTORC1 substrates such as S6K1 on Thr389.
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