FIP200 controls the TBK1 activation threshold at SQSTM1/p62-positive condensates.

The protein kinase TBK1 is a central regulator of innate immune responses and autophagy, and ablation of either function has been linked to neuroinflammatory or degenerative diseases. Autophagy is an intracellular process that recycles old or damaged proteins and organelles. In recent years, the TBK1-dependent regulation of autophagy pathways has been characterized. However, the autophagy-dependent regulation of TBK1 activity awaits further clarification. Here, we observed that TBK1 is recruited to SQSTM1/p62-containing aggregates via the selective autophagy receptor TAX1BP1. In these aggregates, TBK1 phosphorylates SQSTM1/p62 at serine 403 and thus presumably regulates the efficient engulfment and clearance of these structures. We found that TBK1 activation is strongly increased if FIP200, a component of the autophagy-inducing ULK1 complex, is not present or cannot bind to TAX1BP1. Given our collective findings, we hypothesize that FIP200 ensures the inducible activation of TBK1 at SQSTM1/p62 condensates.

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

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.

The Autophagy-Initiating Kinase ULK1 Controls RIPK1-Mediated Cell Death.

Autophagy, apoptosis, and necroptosis are stress responses governing the ultimate fate of a cell. However, the crosstalk between these cellular stress responses is not entirely understood. Especially, it is not clear whether the autophagy-initiating kinase ULK1 and the cell-death-regulating kinase RIPK1 are involved in this potential crosstalk. Here, we identify RIPK1 as a substrate of ULK1. ULK1-dependent phosphorylation of RIPK1 reduces complex IIb/necrosome assembly and tumor necrosis factor (TNF)-induced cell death, whereas deprivation of ULK1 enhances TNF-induced cell death. We observe that ULK1 phosphorylates multiple sites of RIPK1, but it appears that especially phosphorylation of S357 within the intermediate domain of RIPK1 mediates this cell-death-inhibiting effect. We propose that ULK1 is a regulator of RIPK1-mediated cell death.

The mycotoxin phomoxanthone A disturbs the form and function of the inner mitochondrial membrane.

Mitochondria are cellular organelles with crucial functions in the generation and distribution of ATP, the buffering of cytosolic Ca2+ and the initiation of apoptosis. Compounds that interfere with these functions are termed mitochondrial toxins, many of which are derived from microbes, such as antimycin A, oligomycin A, and ionomycin. Here, we identify the mycotoxin phomoxanthone A (PXA), derived from the endophytic fungus Phomopsis longicolla, as a mitochondrial toxin. We show that PXA elicits a strong release of Ca2+ from the mitochondria but not from the ER. In addition, PXA depolarises the mitochondria similarly to protonophoric uncouplers such as CCCP, yet unlike these, it does not increase but rather inhibits cellular respiration and electron transport chain activity. The respiration-dependent mitochondrial network structure rapidly collapses into fragments upon PXA treatment. Surprisingly, this fragmentation is independent from the canonical mitochondrial fission and fusion mediators DRP1 and OPA1, and exclusively affects the inner mitochondrial membrane, leading to cristae disruption, release of pro-apoptotic proteins, and apoptosis. Taken together, our results suggest that PXA is a mitochondrial toxin with a novel mode of action that might prove a useful tool for the study of mitochondrial ion homoeostasis and membrane dynamics.

Targeting urothelial carcinoma cells by combining cisplatin with a specific inhibitor of the autophagy-inducing class III PtdIns3K complex.

BACKGROUND: Cisplatin-based regimens are routinely employed for the treatment of urothelial carcinoma. However, therapeutic success is hampered by the primary presence of or the development of cisplatin resistance. This chemoresistance is executed by multiple cellular pathways. In recent years, the cellular process of autophagy has been identified as a prosurvival pathway of cancer cells. On the one hand, autophagy enables cancer cells to survive conditions of low oxygen or nutrient supply, frequently found in tumors. On the other hand, autophagy supports chemoresistance of cancer cells. Here, we aimed at investigating the involvement of autophagy for cisplatin resistance in different urothelial carcinoma cell lines. MATERIALS & METHODS: We analyzed the expression levels of different autophagy-related proteins in cisplatin-sensitive and cisplatin-resistant urothelial carcinoma cell lines. Furthermore, we performed cell viability assays and caspase activity assays with cells treated with cisplatin, non-specific or specific autophagy inhibitors (chloroquine, 3-methyladenine, SAR405) or combinations thereof. RESULTS: We found that autophagy-related proteins are up-regulated in different cisplatin-resistant urothelial carcinoma cells compared to the sensitive parental cell lines. Furthermore, inhibition of autophagy, in general, or of the autophagy-inducing class III PtdIns3K complex, in particular, sensitized both sensitive and resistant urothelial carcinoma cells to cisplatin-induced cytotoxic effects. CONCLUSION: We propose that targeting the autophagic machinery might represent a suitable approach to complement or even increase cisplatin efficacy in order to overcome cisplatin resistance in urothelial carcinoma.

Systematic analysis of ATG13 domain requirements for autophagy induction.

Macroautophagy/autophagy is an evolutionarily conserved cellular process whose induction is regulated by the ULK1 protein kinase complex. The subunit ATG13 functions as an adaptor protein by recruiting ULK1, RB1CC1 and ATG101 to a core ULK1 complex. Furthermore, ATG13 directly binds both phospholipids and members of the Atg8 family. The central involvement of ATG13 in complex formation makes it an attractive target for autophagy regulation. Here, we analyzed known interactions of ATG13 with proteins and lipids for their potential modulation of ULK1 complex formation and autophagy induction. Targeting the ATG101-ATG13 interaction showed the strongest autophagy-inhibitory effect, whereas the inhibition of binding to ULK1 or RB1CC1 had only minor effects, emphasizing that mutations interfering with ULK1 complex assembly do not necessarily result in a blockade of autophagy. Furthermore, inhibition of ATG13 binding to phospholipids or Atg8 proteins had only mild effects on autophagy. Generally, the observed phenotypes were more severe when autophagy was induced by MTORC1/2 inhibition compared to amino acid starvation. Collectively, these data establish the interaction between ATG13 and ATG101 as a promising target in disease-settings where the inhibition of autophagy is desired.

Deubiquitinase inhibition by WP1130 leads to ULK1 aggregation and blockade of autophagy.

Autophagy represents an intracellular degradation process which is involved in both regular cell homeostasis and disease settings. In recent years, the molecular machinery governing this process has been elucidated. The ULK1 kinase complex consisting of the serine/threonine protein kinase ULK1 and the adapter proteins ATG13, RB1CC1, and ATG101, is centrally involved in the regulation of autophagy initiation. This complex is in turn regulated by the activity of different nutrient- or energy-sensing kinases, including MTOR, AMPK, and AKT. However, next to phosphorylation processes it has been suggested that ubiquitination of ULK1 positively influences ULK1 function. Here we report that the inhibition of deubiquitinases by the compound WP1130 leads to increased ULK1 ubiquitination, the transfer of ULK1 to aggresomes, and the inhibition of ULK1 activity. Additionally, WP1130 can block the autophagic flux. Thus, treatment with WP1130 might represent an efficient tool to inhibit the autophagy-initiating ULK1 complex and autophagy.

Expression of a ULK1/2 binding-deficient ATG13 variant can partially restore autophagic activity in ATG13-deficient cells.

Autophagy describes an intracellular process responsible for the lysosome-dependent degradation of cytosolic components. The ULK1/2 complex comprising the kinase ULK1/2 and the accessory proteins ATG13, RB1CC1, and ATG101 has been identified as a central player in the autophagy network, and it represents the main entry point for autophagy-regulating kinases such as MTOR and AMPK. It is generally accepted that the ULK1 complex is constitutively assembled independent of nutrient supply. Here we report the characterization of the ATG13 region required for the binding of ULK1/2. This binding site is established by an extremely short peptide motif at the C terminus of ATG13. This motif is mandatory for the recruitment of ULK1 into the autophagy-initiating high-molecular mass complex. Expression of a ULK1/2 binding-deficient ATG13 variant in ATG13-deficient cells resulted in diminished but not completely abolished autophagic activity. Collectively, we propose that autophagy can be executed by mechanisms that are dependent or independent of the ULK1/2-ATG13 interaction.