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. 2016 Dec 9;11(1):76.
doi: 10.1186/s13024-016-0141-0.

ULK1-mediated phosphorylation of ATG14 promotes autophagy and is impaired in Huntington's disease models

Mitchell S Wold  1 , Junghyun Lim  1   2 , Véronik Lachance  1 , Zhiqiang Deng  1   3 , Zhenyu Yue  4   5
Affiliations

ULK1-mediated phosphorylation of ATG14 promotes autophagy and is impaired in Huntington's disease models

Mitchell S Wold et al. Mol Neurodegener. .

Abstract

Background: Autophagy is a bulk degradation pathway for long-lived proteins, protein aggregates, and damaged organelles. ULK1 protein kinase and Vps34 lipid kinase are two key autophagy regulators that are critical for autophagosome biogenesis. However, it isn't fully understood how ULK1 regulates Vps34, especially in the context of disease. Polyglutamine expansion in huntingtin (Htt) causes aberrant accumulation of the aggregated protein and disrupts various cellular pathways including autophagy, a lysosomal degradation pathway, underlying the pathogenesis of Huntington's disease (HD). Although autophagic clearance of Htt aggregates is under investigation as therapeutic strategy for HD, the precise mechanism of autophagy impairment remains poorly understood. Moreover, in-vivo assays of autophagy have been particularly challenging due to lack of reliable and robust molecular biomarkers.

Method: We generated anti-phosphorylated ATG14 antibody to determine ATG14-mediated autophagy regulation; we employed Huntington's disease (HD) genetic cell models and animal models as well as autophagy reporter animal model to understand autophagy signaling and regulation in vivo. We applied biochemical analysis and molecular biology approaches to dissect the alteration of autophagy kinase activity and regulation.

Results: Here, we demonstrate that ULK1 phosphorylates ATG14 at serine 29 in an mTOR-dependent manner. This phosphorylation critically regulates ATG14-Vps34 lipid kinase activity to control autophagy level. We also show that ATG14-associated Vps34 activity and ULK1-mediated phosphorylation of ATG14 and Beclin 1 are compromised in the Q175 mouse model of Huntington's disease. Finally, we show that ATG14 phosphorylation is decreased during general proteotoxic stress caused by proteasomal inhibition. This reduction of the specific phosphorylation of ATG14 and Beclin 1 is mediated, in part, by p62-induced sequestration of ULK1 to an insoluble cellular fraction. We show that increased ULK1 levels and phosphor-mimetic mutant ATG14 facilitate the clearance of polyQ mutant in cells.

Conclusion: Our study identifies a new regulatory mechanism for ATG14-Vps34 kinase activity by ULK1, which can be used as valuable molecular markers for in-vivo autophagic activity as well as potential therapeutic target for the clearance of polyglutamine disease protein.

Keywords: ATG14; Autophagy; Huntington’s; ULK1; Vps34.

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Figures

Fig. 1
Fig. 1
ULK1 phosphorylates ATG14 at serine 29. a Representation of ATG14 structure with the location of serine 29. 4C-ER – cysteine repeats needed for ER localization. CCD – coiled-coil domains. BATS – BARKOR autophagosome targeting sequence. b Co-immunoprecipitation of ATG14 and ULK1. Myc-ULK1 and ATG14-GFP were overexpressed in HEK cells and pulled down using antibodies against GFP. c Autoradiogram from in-vitro kinase assay with ULK1 and ATG14 a.a. 20–73. Myc-ULK1 WT, KI, or empty vector was overexpressed in HEK cells and immunopurified with antibodies against myc. ATG14 a.a. 20–73 WT, S29A, or S61A were purified from e. coli. MBP (myelin basic protein) was used as a positive control. d Similar experiment as (c), except using an antibody against phosphorylated serine 29. e ATG14-GFP and myc-ULK1 WT or KI were co-transfected in HEK cells. Half of the IP was treated with an alkaline phosphatase at 37 °C for 30 min
Fig. 2
Fig. 2
ATG14 is phosphorylated in an mTOR-dependent fashion by ULK1 upon autophagy induction. a Phosphorylation of ATG14 in HCT116 cells after glucose, serum, or amino acid withdrawal, medium starvation with HBSS, or Torin 1 treatment for 6 h. b ATG14 phosphorylation levels were normalized to total ATG14 levels and compared to the control condition. **p < 0.01 *p < 0.05 n.s. not significant (n = 3). Data are represented as mean +/− SEM. c ATG14 phosphorylation after 15, 30, 60, and 120 min of Torin 1 treatment in HCT116 cells. d ATG14 phosphorylation levels were normalized to total ATG14 levels and compared to the control condition. A one-way ANOVA with Bonferroni’s posttest was performed. F(4, 10) = 12.58, p = 0.0006 ***p < 0.001 **p < 0.01 *p < 0.05 (n = 3). Data are represented as mean +/− SEM. e ATG14 phosphorylation in Beclin WT, KO or Beclin rescued MEF cells treated with Torin 1 or vehicle (DMSO) for 60 min. Arrow indicates pATG14 band. f Quantification of ATG14 phosphorylation normalized to total ATG14. **p < 0.01 (n = 3). Data are represented as mean +/− SEM. g MEF WT and ULK1/2 DKO cells were treated with Torin 1 for 60 min. ATG14 was immunopurified to enhance the phospho-signal. h Validation of phospho-ATG14 (S29) antibody specificity. ATG14 phosphorylation signal disappears in ATG14 KO HCT116 cells after medium starvation or Torin 1 treatment for 60 min
Fig. 3
Fig. 3
ATG14 phosphorylation promotes Vps34 lipid kinase activity. a Vps34 lipid kinase assay. Empty vector or FLAG-ATG14 S29 WT, A, or E mutants were overexpressed in HEK cells and pulled down using antibodies against FLAG. b Quantification of PI(3)P levels were normalized to levels of Vps34 in the IP. Values were then normalized to the WT condition. **p < 0.01 (n = 5). Data are represented as mean +/− SEM. c Co-immunoprecipitation of ATG14 and Vps34 complex members, Beclin 1, Vps34, Vps15 and NRBF2. Empty vector or FLAG-ATG14 S29 WT, A, or E mutants were overexpressed in HEK cells and pulled down using antibodies against FLAG. d Torin 1 treatment does not alter binding of Vps34 complex members. MEF cells were treated with Torin 1 for 60 min. Cell lysates were subject to IP using antibody against ATG14. e Phosphorylation levels of Beclin 1 and ATG14 from HCT116 cells treated with Torin 1 for 15, 30, 60, and 120 min. f Vps34 lipid kinase assay for Beclin 1 and ATG14 phosphorylation mutation comparison. Beclin 1-AsRed, FLAG-ATG14, and dual myc-Vps34 his-Vps15 plasmid were overexpressed and purified from HEK cells using antibodies against FLAG. g Quantification of PI(3)P levels were normalized to levels of Vps34 in the IP. Values were then normalized to the WT condition. Two-way ANOVA analysis with Bonferroni posttest was used for the effect of the nature of the mutation and which protein was being mutated. Main effect of Beclin 1 or ATG14 mutation F(1, 18) = 4.08, p = 0.0587; Main effect of type of mutation F(1, 18) = 0.71, p = 0.5043; Interaction F(2, 18) = 3.80, p = 0.0421. **p < 0.01 (n = 5). Data are represented as mean +/− SEM
Fig. 4
Fig. 4
ATG14-associated Vps34 activity is reduced in Q175 brains. a ATG14 phosphorylation in Q175 brains. ATG14 was immunopurified using antibodies against ATG14. Two representative lanes shown for each experiment. b Quantification of ATG14 phosphorylation. **p < 0.01 (n = 5). Data are represented as mean +/− SEM. c In-vitro lipid kinase assay of Vps34 complex pulled down with antibodies against ATG14 from the brains of Q175 mice. Thin layer chromatography was used to separate 32P-PI(3)P. d Quantification of PI(3)P levels were normalized to levels of Vps34 in the IP. ***p < 0.001 (n = 4). Data are represented as mean +/− SEM. e Beclin 1 S14 phosphorylation in Q175 mice. f Quantification of Beclin 1 phosphorylation. **p < 0.01 (n = 5). Data are represented as mean +/− SEM. g ATG14 phosphorylation in mCFP-103Q expressing HeLa cells. Doxycycline was removed for 7 days to induce protein expression. IP was performed against ATG14. h Quantification of ATG14 phosphorylation. Values were normalized to control. *p < 0.05 (n = 4). Data are represented as mean +/− SEM. I Immunofluorecent imaging of autophagosome reporter GFP-LC3 distribution in striatal nuclei of Q175;GFP-LC3 and control WT;GFP-LC3 mice. Mice at 6, 10 and 15 months are examined. mHtt nuclear aggregate staining in red and GFP-LC3 in green. Scale bar 10 μm
Fig. 5
Fig. 5
Proteotoxic stress causes a decrease in ATG14 phosphorylation and a redistribution of ULK1 into insoluble fraction. a ATG14 phosphorylation after proteasome inhibition. Phosphorylation of p62 is at S409. MEF cells were treated with MG132 for 16 h then separated into Triton X-100 soluble and insoluble fractions. b Quantification of ATG14 phosphorylation. ***p < 0.001 (n = 3). Data are represented as mean +/− SEM. c ULK1 accumulates in the Triton X-100 insoluble fraction in the brains of Q175 mice. d Quantification of ULK1 in the insoluble fraction. *p < 0.05 (n = 3). Data are represented as mean +/− SEM. e ATG14 phosphorylation after proteasome inhibition. P62 WT or KO MEF cells were treated with MG132 for 16 h then separated into Triton X-100 soluble and insoluble fractions. f Quantification of ATG14 phosphorylation in the soluble fraction. Two-way ANOVA was performed. Main effect of MG132 F(1, 8) = 92.60, p < 0.0001; Main effect of p62 KO F(1, 8) = 0.56, p = 0.4748; Interaction F(1, 8) = 7.82, p = 0.0233. (n = 3). Data are represented as mean +/− SEM. g Quantification of ULK1 in the insoluble fraction. Main effect of MG132 F(1, 8) = 252.94, p < 0.0001; Main effect of p62 KO F(1, 8) = 87.94, p < 0.0001; Interaction F(1, 8) = 81.33, p < 0.0001 (n = 3). Data are represented as mean +/− SEM
Fig. 6
Fig. 6
ULK1 activity regulates the degradation of polyQ proteins. a FLAG-ATG14 S29 WT, SA, or SE, Beclin 1-AsRed, and dual myc-Vps34 his-Vps15 plasmid were overexpressed in mCFP-65Q expressing HeLa cells then treated with rapamycin overnight. Cell lysates were separated into soluble and insoluble fractions. PolyQ was measured in the insoluble fraction. b Quantification of polyQ degradation in the insoluble fraction. Degradation was calculated as the ratio of control sample vs. rapamycin sample **p < 0.01 (n = 4). Data are represented as mean +/− SEM. c Myc empty vector, myc-ULK1 WT or KI were overexpressed in mCFP-65Q expressing HeLa cells and cells were lysed 48 h after transfection. Cell lysates were separated into soluble and insoluble fractions. PolyQ was measured in the insoluble fraction. d Quantification of polyQ levels in the insoluble fraction. Values were normalized to empty vector condition. *p < 0.05 n.s. not significant (n = 4). Data are represented as mean +/− SEM
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References

    1. Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet. 2004;36:585–95. doi: 10.1038/ng1362. - DOI - PubMed
    1. Tellez-Nagel I, Johnson AB, Terry RD. Studies on brain biopsies of patients with Huntington’s chorea. J Neuropathol Exp Neurol. 1974;33:308–32. doi: 10.1097/00005072-197404000-00008. - DOI - PubMed
    1. Heng MY, Duong DK, Albin RL, Tallaksen-Greene SJ, Hunter JM, Lesort MJ, et al. Early autophagic response in a novel knock-in model of Huntington disease. Hum Mol Genet. 2010;19:3702–20. doi: 10.1093/hmg/ddq285. - DOI - PMC - PubMed
    1. Kegel KB, Kim M, Sapp E, McIntyre C, Castaño JG, Aronin N, et al. Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation, and autophagy. J Neurosci. 2000;20:7268–78. - PMC - PubMed
    1. Cortes CJ, La Spada AR. The many faces of autophagy dysfunction in Huntington’s disease: from mechanism to therapy. Drug Discov Today. 2014;19:963–71. doi: 10.1016/j.drudis.2014.02.014. - DOI - PMC - PubMed

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