TNF-induced necroptosis initiates early autophagy events via RIPK3-dependent AMPK activation, but inhibits late autophagy

Autophagy. 2021 Dec;17(12):3992-4009. doi: 10.1080/15548627.2021.1899667. Epub 2021 Mar 28.

Abstract

Macroautophagy/autophagy and necroptosis represent two opposing cellular s tress responses. Whereas autophagy primarily fulfills a cyto-protective function, necroptosis is a form of regulated cell death induced via death receptors. Here, we aimed at investigating the molecular crosstalk between these two pathways. We observed that RIPK3 directly associates with AMPK and phosphorylates its catalytic subunit PRKAA1/2 at T183/T172. Activated AMPK then phosphorylates the autophagy-regulating proteins ULK1 and BECN1. However, the lysosomal degradation of autophagosomes is blocked by TNF-induced necroptosis. Specifically, we observed dysregulated SNARE complexes upon TNF treatment; e.g., reduced levels of full-length STX17. In summary, we identified RIPK3 as an AMPK-activating kinase and thus a direct link between autophagy- and necroptosis-regulating kinases.Abbreviations: ACACA/ACC: acetyl-CoA carboxylase alpha; AMPK: AMP-activated protein kinase; ATG: autophagy-related; BECN1: beclin 1; GFP: green fluorescent protein; EBSS: Earle's balanced salt solution; Hs: Homo sapiens; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLKL: mixed lineage kinase domain like pseudokinase; Mm: Mus musculus; MTOR: mechanistic target of rapamycin kinase; MVB: multivesicular body; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PLA: proximity ligation assay; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKAA2: protein kinase AMP-activated catalytic subunit alpha 2; PRKAB2: protein kinase AMP-activated non-catalytic subunit beta 2; PRKAG1: protein kinase AMP-activated non-catalytic subunit gamma 1; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; STX7: syntaxin 7; STX17: syntaxin 17; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; WT: wild-type.

Keywords: AMPK; RIPK3; STX17; autophagy; necroptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases* / metabolism
  • Animals
  • Autophagy* / physiology
  • Class III Phosphatidylinositol 3-Kinases / metabolism
  • Fibroblasts / metabolism
  • Mice
  • Necroptosis*
  • Receptor-Interacting Protein Serine-Threonine Kinases* / metabolism

Substances

  • Class III Phosphatidylinositol 3-Kinases
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ripk3 protein, mouse
  • AMP-Activated Protein Kinases

Grants and funding

This work was supported by the Deutsche Forschungsgemeinschaft [STO 864/4-1]; Deutsche Forschungsgemeinschaft [STO 864/5-1]; Deutsche Forschungsgemeinschaft [STO 864/6-1]; Deutsche Forschungsgemeinschaft [GRK 2158]; Düsseldorf School of Oncology (funded by the Comprehensive Cancer Center Düsseldorf/Deutsche Krebshilfe and the Medical Faculty of the Heinrich Heine University Düsseldorf).