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. 2011 Mar 22;108(12):4788-93.
doi: 10.1073/pnas.1100844108. Epub 2011 Mar 7.

Nutrient starvation elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its subsequent dissociation from AMPK

Libin Shang  1 , She Chen, Fenghe Du, Shen Li, Liping Zhao, Xiaodong Wang
Affiliations

Nutrient starvation elicits an acute autophagic response mediated by Ulk1 dephosphorylation and its subsequent dissociation from AMPK

Libin Shang et al. Proc Natl Acad Sci U S A. .

Abstract

Macroautophagy (herein referred to as autophagy) is an evolutionarily conserved self-digestive process cells adapt to starvation and other stress responses. Upon starvation, autophagy is induced, providing cells with needed nutrient supplies. We report here that Unc-51-like kinase 1 (Ulk1), a key initiator for mammalian autophagy, undergoes dramatic dephosphorylation upon starvation, particularly at serine 638 and serine 758. Phosphorylations of Ulk1 are mediated by mammalian target-of-rapamycin (mTOR) kinase and adenosine monophosphate activated protein kinase (AMPK). AMPK interacts with Ulk1 in a nutrient-dependent manner. Proper phosphorylations on Ulk1 are crucial for Ulk1/AMPK association, as a single serine-to-alanine mutation (S758A) at Ulk1 impairs this interaction. Compared to the wild-type ULK1, this Ulk1-S758A mutant initiates starvation-induced autophagy faster at an early time point, but does not alter the maximum capacity of autophagy when starvation prolongs. This study therefore revealed previously unnoticed acute autophagy response to environmental changes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Phosphorylations of Ulk1 at serine 638 and serine 758 were regulated differently. (A) Dynamics of Ulk1 phosphorylation. U2OS cells were starved in total starvation medium (HBSS solution containing 1% rich medium), and then replenished with rich medium (DMEM plus 10% dialyzed FBS) for durations as indicated. Cell extracts were analyzed by Western blotting to visualize phosphorylations of Ulk1 at serine 638 and serine 758. (B) U2OS cells were treated for 2 h as indicated. For serum withdrawal, cells were cultured in DMEM without FBS; for DMEM withdrawal, cells were cultured using 10% dialyzed FBS in HBSS. Phosphorylations of Ulk1 at Ser638 and Ser758 were analyzed using phospho-specific antibodies against these two sites. (C) Calcium is crucial for phosphorylation of Ulk1 at serine 638, but not serine 758. U2OS cells are treated for 2 h as indicated. Lane 3: calcium was added into total starvation medium to reach the same calcium concentration as in regular DMEM (0.2 g/L); Lane 4: regular DMEM medium (containing 10% dialyzed FBS) with single drop-out of calcium. See also Fig. S1.
Fig. 2.
Fig. 2.
Phosphorylations of Ulk1 at serine 638 and serine 758 were mediated differently by mTOR and AMPK. (A) Rapamycin treatment induced dephosphorylation of Ulk1 at both serine 638 and serine 758. U2OS cells were cultured in rich medium and rapamycin was added (100 nM). Cells were then collected at given time points after rapamycin treatment. (B) Knockdown of mTOR induced dephosphorylation of Ukl1 at both serine 638 and serine 758. U2OS cells were transfected with either control siRNA (Luc) or mTOR siRNA oligos. 72 h after transfection, cells were collected and extracts analyzed by Western blotting. (C) Knockdown of AMPK induced dephosphorylation of Ulk1 at serine 638, but not serine 758. U2OS stable cell lines that inducibly knock down AMPKα (α1 and α2) or AMPKβ (β1 and β2) upon addition of doxycycline (Dox) were generated. 72 h after addition of Dox, phosphorylations of Ulk1 at Ser638 and Ser758 were visualized by Western blotting. (D) AMPK activity correlates with phosphorylation of Ulk1 at serine 638 but not serine 758 in response to various nutrient conditions. U2OS cells were treated for 2 h as indicated and cell extracts were analyzed by Western blotting.
Fig. 3.
Fig. 3.
Ulk1 interacts with AMPK in response to nutrient availability. (A) Only Ulk1, but not mAtg13, associates with AMPK when cells are fed. In HeLa cells, Flag-HA-Ulk1, and Flag-HA-Atg13 stable lines were generated. Cells were treated for 2 h with either rich medium or total starvation medium. Flag-HA tandem IP assays were performed using cell extracts prepared from above treatments. Proteins in the final eluates were separated with 4–12% SDS-PAGE gel and were visualized by silver staining. Arrows indicates major coimmunoprecipitated proteins identified by MS. (B) Dynamics of Ulk1/AMPK interaction in response to nutrient availability. Cells were starved and repleted with rich medium for durations as indicated. Flag IP assays were then performed. Ulk1/AMPK association patterns were analyzed by Western blotting. (C) Phosphorylation of Ulk1 at serine 758 was required for Ulk1/AMPK association. HEK 293T cells were transfected with either wild-type Flag-Ulk1 or Flag-Ulk1 mutants as indicated, and treated for 2 h in rich or total starvation medium. Cell extracts were immunoprecipitated with anti-Flag beads, and the eluates were analyzed by Western blotting. (D) Phosphorylation of Ulk1 at serine 638 was not required for Ulk1/AMPK association. Experiments were performed the same as in (C) with either wild-type Ulk1 or indicated mutants. See also Fig. S2.
Fig. 4.
Fig. 4.
Functional outputs of Ulk1 phosphorylations at serine 638 and serine 758. (A) LLPD assays showed Ulk1-S758A initiates starvation-induced autophagy faster compared to wild-type Ulk1 (*P = 0.003 in one-tailed Student’s t-test with equal variances, n = 3). Error bar represents SD. U2OS stable cell line that inducibly knock down mAtg13 upon addition of Dox was generated. Absence of mAtg13 also led to absence of Ulk1. 72 h after addition of Dox, cells were transfected with either wild-type Ulk1 or Ulk1-S758A construct, and cultured for another 48 h in rich medium containing no 3H-labeled but excessive cold leucine. Cells were than cultured in either rich or total starvation medium for 10 min. Percentages of 3H-labeled leucine released from cells into medium were calculated. (B) In either fed condition or 10 min after starvation, cells expressing Ulk1-S758A exhibited higher hVps34 activity compared to the wild type. Using same cells and same transfections as in (A), kinase activity of hVps34 was assayed by quantifying conversion from PI to PI3P in given time as described in Materials and Methods. Error bar represents SD. (C) Upper: LC3II formation in U2OS cells expressing either Ulk1-S758A mutant or wild-type Ulk1, in fed condition (0 min) and 30 or 120 min after starvation; lower: Quantitative analysis of the ratio of LC3II to LC3I (n = 3). (D) In Ulk1-S638A background, serine 758 could not be properly rephosphorylated 60 min after medium replenishment, while in wild-type Ulk1 background this process took less than 15 min. U2OS stable cell line that inducibly knock down Ulk1 upon addition of Dox was generated. 72 h after addition of Dox, cells were transfected with either wild-type Ulk1 or Ulk1-S638A construct, and cultured for another 48 h in rich medium to allow expression. Cells were then treated as indicated. (E) In S638A background, Ulk1/AMPK reassociation is slowed down. Using same cell line and transfections as in (D), cells were starved for 1 h and recovered for 20 min. Extracts were immunoprecipitated with anti-Flag beads, and the eluates were analyzed by Western blotting. See also Fig. S4.
Fig. 5.
Fig. 5.
Model of Ulk1 phosphorylation in response to nutrients. When cells are fed, Ulk1 is hyper-phosphorylated at serine 638 and serine 758. Upon starvation, serine 638 is firstly dephosphorylated; then followed by dephosphorylation at serine 758. Dephosphorylation of Ulk1 leads to dissociation of Ulk1/AMPK. When cells are replenished with rich medium, mTOR is activated; it phosphorylates serine 638 and serine 758. The phosphorylation of Ulk1 at serine 758 then leads to reassociation between Ulk1 and AMPK. When in close proximity, AMPK functions to maintain phosphorylation Ulk1 at serine 638.
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