Suppression of autophagy during mitosis via CUL4-RING ubiquitin ligases-mediated WIPI2 polyubiquitination and proteasomal degradation

Autophagy. 2019 Nov;15(11):1917-1934. doi: 10.1080/15548627.2019.1596484. Epub 2019 Mar 30.

Abstract

Macroautophagy/autophagy is a cellular process in which cytosolic contents are degraded by lysosome in response to various stress conditions. Apart from its role in the maintenance of cellular homeostasis, autophagy also involves in regulation of cell cycle progression under nutrient-deprivation conditions. However, whether and how autophagy is regulated by the cell cycle especially during mitosis remains largely undefined. Here we show that WIPI2/ATG18B (WD repeat domain, phosphoinositide interacting 2), an autophagy-related (ATG) protein that plays a critical role in autophagosome biogenesis, is a direct substrate of CUL4-RING ubiquitin ligases (CRL4s). Upon mitosis induction, CRL4s are activated via neddylation, and recruit WIPI2 via DDB1 (damage specific DNA binding protein 1), leading to polyubiquitination and proteasomal degradation of WIPI2 and suppression of autophagy. The WIPI2 protein level and autophagy during mitosis could be rescued by knockdown of CRL4s or treatment with MLN4924/Pevonedistat, a selective inhibitor of CRLs, via suppression of NAE1 (NEDD8 activating enzyme E1 subunit 1). Moreover, restoration of WIPI2 rescues autophagy during mitosis and leads to mitotic slippage and cell senescence. Our study thus discovers a novel function of CRL4s in autophagy by targeting WIPI2 for polyubiquitination and proteasomal degradation during mitosis. Abbreviations: ACTB, actin beta; ATG, autophagy-related; AMPK, AMP-activated protein kinase; AURKB/ARK2, aurora kinase B; BafA1, bafilomycin A1; CCNB1, cyclin B1; CDK1, cyclin dependent kinase 1; CHX, cycloheximide; CQ, chloroquine; CRL4s, CUL4-RING ubiquitin ligases; DDB1, damage specific DNA binding protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione S-transferase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; STK11/LKB1,serine/threonine kinase 11; MTORC1/MTOR complex 1, mechanistic target of rapamycin kinase complex 1; NAE1, NEDD8 activating enzyme E1 subunit 1; NOC, nocodazole; RING, really interesting new gene; RBX1, ring-box 1; SA-GLB1/β-gal, senescence-associated galactosidase beta 1; TSC2, TSC complex subunit 2; TUBA, tubulin alpha; WIPI2, WD repeat domain, phosphoinositide interacting 2.

Keywords: Autophagy; CRL4s; MLN4924; WIPI2; mitosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy / genetics*
  • Cellular Senescence / genetics
  • Cyclopentanes / pharmacology
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Leupeptins / pharmacology
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mitosis / genetics*
  • NEDD8 Protein / genetics
  • NEDD8 Protein / metabolism
  • Phosphate-Binding Proteins / genetics
  • Phosphate-Binding Proteins / metabolism*
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Binding / genetics
  • Pyrimidines / pharmacology
  • Signal Transduction / genetics
  • Ubiquitin / metabolism*
  • Ubiquitin-Activating Enzymes / antagonists & inhibitors
  • Ubiquitin-Activating Enzymes / genetics
  • Ubiquitin-Activating Enzymes / metabolism
  • Ubiquitin-Protein Ligases / antagonists & inhibitors
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination / genetics*

Substances

  • Cyclopentanes
  • DDB1 protein, human
  • DNA-Binding Proteins
  • Leupeptins
  • Membrane Proteins
  • NEDD8 Protein
  • NEDD8 protein, human
  • Phosphate-Binding Proteins
  • Pyrimidines
  • Ubiquitin
  • WIPI2 protein, human
  • CULL-RING ligase, human
  • Ubiquitin-Protein Ligases
  • Proteasome Endopeptidase Complex
  • Ubiquitin-Activating Enzymes
  • NAE protein, human
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde
  • pevonedistat