Journal: Autophagy

Article Title: Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates

doi: 10.1080/15548627.2024.2394306

Figure Lengend Snippet: IκB kinases induce TAX1BP1 phosphorylation. (A) 293T cells were co-transfected with Flag-TAX1BP1 together with the indicated kinase plasmids and 24 h later lysed, and the cell extracts were immunoblotted with the indicated antibodies. (B) 293T cells were transfected with the indicated plasmids and lysates were incubated with λ-phosphatase for 30 min prior to immunoblotting with anti-Flag. (C) 293T cells were transfected with Flag-TAX1BP1 and either Flag-TBK1, Flag-IKBKE/IKKi or kinase dead mutants Flag-TBK1 K38A or Flag-IKBKE/IKKi K38A , respectively. Lysates were subjected to immunoblotting with anti-Flag antibody. (D) in vitro kinase assays. Purified GST-tagged TAX1BP1 (300 ng) was incubated with 50 ng of purified recombinant GST-tagged IKBKE/IKKi or hexahistidine-tagged TBK1 in the presence or absence of ATP. The reaction mixtures were subjected to immunoblotting with antibodies to TAX1BP1, IKBKE/IKKi and TBK1. (E) DLD-1 cells were transfected with the indicated amount of plasmids for 24 h and then treated with BafA1 (20 nM) for 18 h. Cells were then lysed and subjected to immunoblotting analysis with the indicated antibodies. (F) Control and ATG3 KO DLD-1 cells were transfected with the indicated amount of plasmids for 24 h and then lysed and subjected to immunoblotting with the indicated antibodies. (G) Colloidal blue staining of in vitro kinase reaction mixtures containing TAX1BP1 with or without IKBKE/IKKi. Individual bands within the red rectangle were cut and gelextracted for mass spectrometry (MS) analysis. (H) Schematic diagram of TAX1BP1 domains. The indicated ten and thirteen predicted phosphorylation sites were substituted with alanine, generating the TAX1BP1 10A and 13A mutants, respectively. SKICH, the SKIP carboxy homology domain; LIR, LC3-interacting region; CC, coiled-coil domain; ZF, zinc finger domain.

Article Snippet: Flag-IKBKE/IKKi K38A , A gift from Tom Maniatis (Columbia University) , Addgene 26202.

Techniques: Phospho-proteomics, Transfection, Incubation, Western Blot, In Vitro, Purification, Recombinant, Control, Staining, Mass Spectrometry

Journal: Autophagy

Article Title: Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates

doi: 10.1080/15548627.2024.2394306

Figure Lengend Snippet: Plasmids used in the study.

Article Snippet: Flag-IKBKE/IKKi K38A , A gift from Tom Maniatis (Columbia University) , Addgene 26202.

Techniques: Mutagenesis, Cloning, Negative Control

Journal: Autophagy

Article Title: Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates

doi: 10.1080/15548627.2024.2394306

Figure Lengend Snippet: Oligonucleotides used in the study.

Article Snippet: Flag-IKBKE/IKKi K38A , A gift from Tom Maniatis (Columbia University) , Addgene 26202.

Techniques: Mutagenesis

Journal: Nature Communications

Article Title: ALLO-1- and IKKE-1-dependent positive feedback mechanism promotes the initiation of paternal mitochondrial autophagy

doi: 10.1038/s41467-024-45863-2

Figure Lengend Snippet: a , b EPG-7 is necessary for the accumulation of ALLO-1b around paternal mitochondria (mt). GFP-ALLO-1b (green) and HSP-6-mCherry (paternal mt; magenta) were observed in the wild-type (WT) or epg-7 mutant zygotes. In b , the intensity of GFP-ALLO-1b was quantified and normalized based on the fluorescence of paternal mitochondria. n = 154 (wild type, WT) and n = 108 ( epg-7(tm2508) ) paternal mitochondria or their clusters from 9 or 10 zygotes, respectively, were analyzed. All zygotes were imaged between the pronuclear expansion to the pseudo-cleavage stage. c – e Accumulation of ALLO-1b depends on the FIP200-interacting region (FIR) motif. GFP-ALLO-1b WT or GFP-ALLO-1b D11AE12A (FIR mut) (green) and HSP-6-mCherry (paternal mt; magenta) were observed in zygotes. In d , the intensity of GFP co-localized with paternal mitochondria was quantified. n = 207 (GFP-ALLO-1b WT) and n = 140 (GFP-ALLO-1b FIR mut) paternal mitochondria or their clusters from 10 zygotes for each genotype were analyzed. In e , the intensity of cytoplasmic GFP was quantified outside the vicinity of the paternal organelles to compare GFP expression levels between transgenes ( n = 12 or 18 zygotes for WT or FIRmut, respectively). All zygotes were imaged between the pronuclear expansion to the pseudo-cleavage stage. Volcano plot of tandem mass tag (TMT)-based quantitative phosphoproteomics identifying the decrease in phosphorylation of EPG-7 S949 in ikke-1 ( f ) and allo-1 ( g ) mutants. Red arrows indicate phosphorylated S949-containing peptides of EPG-7. Magenta dots in f indicate the peptides of ALLO-1 (see Supplementary Table ). The x -axis shows the ratio of wild type to the mutants in the amount of detected peptides. p values in the volcano plots were calculated by the two-tailed Student’s t -test. h Model of ALLO-1 accumulation via a positive feedback loop by IKKE-1. P, phosphorylation. Scale bars, 5 μm. p values were calculated using the two-tailed Mann–Whitney U test ( b , d , e ). Error bars represent the mean ± standard deviation (SD). Source data are provided as a Source Data file.

Article Snippet: The deletion mutants, allo-1 ( tm4756 ), ikke-1(tm4102) , and epg-7 ( tm2508 ), were provided by S. Mitani of the Japanese National Bioresource Project for the Experimental Animal “Nematode C. elegans .” Each mutant was backcrossed with the wild-type N2 at least twice.

Techniques: Mutagenesis, Fluorescence, Expressing, Phospho-proteomics, Two Tailed Test, MANN-WHITNEY, Standard Deviation