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

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.