GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction

Douglas S. Grunwald, Neil Michael Otto, Ji-Man Park, Daihyun Song, Do-Hyung Kim

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

ULK1 (unc-51 like autophagy activating kinase 1) is the key mediator of MTORC1 signaling to macroautophagy/autophagy. ULK1 functions as a protein complex by interacting with ATG13, RB1CC1/FIP200, and ATG101. How the ULK1 complex is regulated to trigger autophagy induction remains unclear. In this study, we have determined roles of Atg8-family proteins (ATG8s) in regulating ULK1 activity and autophagy. Using human cells depleted of each subfamily of ATG8, we found that the GABARAP subfamily positively regulates ULK1 activity and phagophore and autophagosome formation in response to starvation. In contrast, the LC3 subfamily negatively regulates ULK1 activity and phagophore formation. By reconstituting ATG8-depleted cells with individual ATG8 members, we identified GABARAP and GABARAPL1 as positive and LC3B and LC3C as negative regulators of ULK1 activity. To address the role of ATG8 binding to ULK1, we mutated the LIR of endogenous ULK1 to disrupt the ATG8-ULK1 interaction by genome editing. The mutation drastically reduced the activity of ULK1, autophagic degradation of SQSTM1, and phagophore formation in response to starvation. The mutation also suppressed the formation and turnover of autophagosomes in response to starvation. Similar to the mutation of the ULK1 LIR, disruption of the ATG13-ATG8 interaction suppressed ULK1 activity and autophagosome formation. In contrast, RB1CC1 did not show any specific binding to ATG8s, and mutation of its LIR did not affect ULK1 activity. Together, this study demonstrates differential binding and opposite regulation of the ULK1 complex by GABARAPs and LC3s, and an important role of the ULK1- and ATG13-ATG8 interactions in autophagy induction. Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; ATG8: autophagy related 8; ATG13: autophagy related 13; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; ATG101: autophagy related 101; BAFA1: bafilomycin A1; BECN1: beclin 1; Cas9: CRISPR associated protein 9; CRISPR: clustered regularly interspaced short palindromic repeats; EBSS: earle’s balanced salt solution; DAPI: 4ʹ-6-diamidino-2-phenylindole; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor-associated protein like 1; GABARAPL2: GABA type A receptor-associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; gRNA: guide RNA; KI: kinase inactive mutant; KO: knockout; LC3A: microtubule associated protein 1 light chain 3 alpha; LC3B: microtubule associated protein 1 light chain 3 beta; LC3C: microtubule associated protein 1 light chain 3 gamma; LIR: LC3-interacting region; MTORC1: mechanistic target of rapamycin kinase complex 1; PBS: phosphate buffered saline; PCR: polymerase chain reaction; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; qPCR: quantitative PCR; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RPS6KB1: ribosomal protein S6 kinase B1; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TALEN: transcription activator-like effector nuclease; TUBA: tubulin alpha; ULK1: unc-51 like autophagy activating kinase 1; WB: western blotting; WIPI2: WD repeat domain phosphoinositide interacting 2; WT: wild type.

Original languageEnglish (US)
JournalAutophagy
DOIs
StatePublished - Jan 1 2019

Fingerprint

Autophagy
Microtubule-Associated Proteins
GABA-A Receptors
Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Starvation
Mutation
Proteins
Light
CRISPR-Associated Proteins
Polymerase Chain Reaction
Phosphotransferases
Ribosomal Protein S6 Kinases
Glyceraldehyde-3-Phosphate Dehydrogenases
Tubulin
Phosphatidylinositols
Salts
Fluorescence
Western Blotting
Phosphates

Keywords

  • ATG8
  • GABARAP
  • LC3
  • LIR
  • ULK1

PubMed: MeSH publication types

  • Journal Article

Cite this

GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction. / Grunwald, Douglas S.; Otto, Neil Michael; Park, Ji-Man; Song, Daihyun; Kim, Do-Hyung.

In: Autophagy, 01.01.2019.

Research output: Contribution to journalArticle

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title = "GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction",
abstract = "ULK1 (unc-51 like autophagy activating kinase 1) is the key mediator of MTORC1 signaling to macroautophagy/autophagy. ULK1 functions as a protein complex by interacting with ATG13, RB1CC1/FIP200, and ATG101. How the ULK1 complex is regulated to trigger autophagy induction remains unclear. In this study, we have determined roles of Atg8-family proteins (ATG8s) in regulating ULK1 activity and autophagy. Using human cells depleted of each subfamily of ATG8, we found that the GABARAP subfamily positively regulates ULK1 activity and phagophore and autophagosome formation in response to starvation. In contrast, the LC3 subfamily negatively regulates ULK1 activity and phagophore formation. By reconstituting ATG8-depleted cells with individual ATG8 members, we identified GABARAP and GABARAPL1 as positive and LC3B and LC3C as negative regulators of ULK1 activity. To address the role of ATG8 binding to ULK1, we mutated the LIR of endogenous ULK1 to disrupt the ATG8-ULK1 interaction by genome editing. The mutation drastically reduced the activity of ULK1, autophagic degradation of SQSTM1, and phagophore formation in response to starvation. The mutation also suppressed the formation and turnover of autophagosomes in response to starvation. Similar to the mutation of the ULK1 LIR, disruption of the ATG13-ATG8 interaction suppressed ULK1 activity and autophagosome formation. In contrast, RB1CC1 did not show any specific binding to ATG8s, and mutation of its LIR did not affect ULK1 activity. Together, this study demonstrates differential binding and opposite regulation of the ULK1 complex by GABARAPs and LC3s, and an important role of the ULK1- and ATG13-ATG8 interactions in autophagy induction. Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; ATG8: autophagy related 8; ATG13: autophagy related 13; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; ATG101: autophagy related 101; BAFA1: bafilomycin A1; BECN1: beclin 1; Cas9: CRISPR associated protein 9; CRISPR: clustered regularly interspaced short palindromic repeats; EBSS: earle’s balanced salt solution; DAPI: 4ʹ-6-diamidino-2-phenylindole; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor-associated protein like 1; GABARAPL2: GABA type A receptor-associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; gRNA: guide RNA; KI: kinase inactive mutant; KO: knockout; LC3A: microtubule associated protein 1 light chain 3 alpha; LC3B: microtubule associated protein 1 light chain 3 beta; LC3C: microtubule associated protein 1 light chain 3 gamma; LIR: LC3-interacting region; MTORC1: mechanistic target of rapamycin kinase complex 1; PBS: phosphate buffered saline; PCR: polymerase chain reaction; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; qPCR: quantitative PCR; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RPS6KB1: ribosomal protein S6 kinase B1; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TALEN: transcription activator-like effector nuclease; TUBA: tubulin alpha; ULK1: unc-51 like autophagy activating kinase 1; WB: western blotting; WIPI2: WD repeat domain phosphoinositide interacting 2; WT: wild type.",
keywords = "ATG8, GABARAP, LC3, LIR, ULK1",
author = "Grunwald, {Douglas S.} and Otto, {Neil Michael} and Ji-Man Park and Daihyun Song and Do-Hyung Kim",
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T1 - GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction

AU - Grunwald, Douglas S.

AU - Otto, Neil Michael

AU - Park, Ji-Man

AU - Song, Daihyun

AU - Kim, Do-Hyung

PY - 2019/1/1

Y1 - 2019/1/1

N2 - ULK1 (unc-51 like autophagy activating kinase 1) is the key mediator of MTORC1 signaling to macroautophagy/autophagy. ULK1 functions as a protein complex by interacting with ATG13, RB1CC1/FIP200, and ATG101. How the ULK1 complex is regulated to trigger autophagy induction remains unclear. In this study, we have determined roles of Atg8-family proteins (ATG8s) in regulating ULK1 activity and autophagy. Using human cells depleted of each subfamily of ATG8, we found that the GABARAP subfamily positively regulates ULK1 activity and phagophore and autophagosome formation in response to starvation. In contrast, the LC3 subfamily negatively regulates ULK1 activity and phagophore formation. By reconstituting ATG8-depleted cells with individual ATG8 members, we identified GABARAP and GABARAPL1 as positive and LC3B and LC3C as negative regulators of ULK1 activity. To address the role of ATG8 binding to ULK1, we mutated the LIR of endogenous ULK1 to disrupt the ATG8-ULK1 interaction by genome editing. The mutation drastically reduced the activity of ULK1, autophagic degradation of SQSTM1, and phagophore formation in response to starvation. The mutation also suppressed the formation and turnover of autophagosomes in response to starvation. Similar to the mutation of the ULK1 LIR, disruption of the ATG13-ATG8 interaction suppressed ULK1 activity and autophagosome formation. In contrast, RB1CC1 did not show any specific binding to ATG8s, and mutation of its LIR did not affect ULK1 activity. Together, this study demonstrates differential binding and opposite regulation of the ULK1 complex by GABARAPs and LC3s, and an important role of the ULK1- and ATG13-ATG8 interactions in autophagy induction. Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; ATG8: autophagy related 8; ATG13: autophagy related 13; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; ATG101: autophagy related 101; BAFA1: bafilomycin A1; BECN1: beclin 1; Cas9: CRISPR associated protein 9; CRISPR: clustered regularly interspaced short palindromic repeats; EBSS: earle’s balanced salt solution; DAPI: 4ʹ-6-diamidino-2-phenylindole; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor-associated protein like 1; GABARAPL2: GABA type A receptor-associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; gRNA: guide RNA; KI: kinase inactive mutant; KO: knockout; LC3A: microtubule associated protein 1 light chain 3 alpha; LC3B: microtubule associated protein 1 light chain 3 beta; LC3C: microtubule associated protein 1 light chain 3 gamma; LIR: LC3-interacting region; MTORC1: mechanistic target of rapamycin kinase complex 1; PBS: phosphate buffered saline; PCR: polymerase chain reaction; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; qPCR: quantitative PCR; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RPS6KB1: ribosomal protein S6 kinase B1; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TALEN: transcription activator-like effector nuclease; TUBA: tubulin alpha; ULK1: unc-51 like autophagy activating kinase 1; WB: western blotting; WIPI2: WD repeat domain phosphoinositide interacting 2; WT: wild type.

AB - ULK1 (unc-51 like autophagy activating kinase 1) is the key mediator of MTORC1 signaling to macroautophagy/autophagy. ULK1 functions as a protein complex by interacting with ATG13, RB1CC1/FIP200, and ATG101. How the ULK1 complex is regulated to trigger autophagy induction remains unclear. In this study, we have determined roles of Atg8-family proteins (ATG8s) in regulating ULK1 activity and autophagy. Using human cells depleted of each subfamily of ATG8, we found that the GABARAP subfamily positively regulates ULK1 activity and phagophore and autophagosome formation in response to starvation. In contrast, the LC3 subfamily negatively regulates ULK1 activity and phagophore formation. By reconstituting ATG8-depleted cells with individual ATG8 members, we identified GABARAP and GABARAPL1 as positive and LC3B and LC3C as negative regulators of ULK1 activity. To address the role of ATG8 binding to ULK1, we mutated the LIR of endogenous ULK1 to disrupt the ATG8-ULK1 interaction by genome editing. The mutation drastically reduced the activity of ULK1, autophagic degradation of SQSTM1, and phagophore formation in response to starvation. The mutation also suppressed the formation and turnover of autophagosomes in response to starvation. Similar to the mutation of the ULK1 LIR, disruption of the ATG13-ATG8 interaction suppressed ULK1 activity and autophagosome formation. In contrast, RB1CC1 did not show any specific binding to ATG8s, and mutation of its LIR did not affect ULK1 activity. Together, this study demonstrates differential binding and opposite regulation of the ULK1 complex by GABARAPs and LC3s, and an important role of the ULK1- and ATG13-ATG8 interactions in autophagy induction. Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; ATG8: autophagy related 8; ATG13: autophagy related 13; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; ATG101: autophagy related 101; BAFA1: bafilomycin A1; BECN1: beclin 1; Cas9: CRISPR associated protein 9; CRISPR: clustered regularly interspaced short palindromic repeats; EBSS: earle’s balanced salt solution; DAPI: 4ʹ-6-diamidino-2-phenylindole; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor-associated protein like 1; GABARAPL2: GABA type A receptor-associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; gRNA: guide RNA; KI: kinase inactive mutant; KO: knockout; LC3A: microtubule associated protein 1 light chain 3 alpha; LC3B: microtubule associated protein 1 light chain 3 beta; LC3C: microtubule associated protein 1 light chain 3 gamma; LIR: LC3-interacting region; MTORC1: mechanistic target of rapamycin kinase complex 1; PBS: phosphate buffered saline; PCR: polymerase chain reaction; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; qPCR: quantitative PCR; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RPS6KB1: ribosomal protein S6 kinase B1; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TALEN: transcription activator-like effector nuclease; TUBA: tubulin alpha; ULK1: unc-51 like autophagy activating kinase 1; WB: western blotting; WIPI2: WD repeat domain phosphoinositide interacting 2; WT: wild type.

KW - ATG8

KW - GABARAP

KW - LC3

KW - LIR

KW - ULK1

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