Fig 1: In vitro kinase assays show BNIP3 and BNIP3L are phosphorylated by ULK1 on S17 and S35 respectively. (a) Primary amino acid sequence alignment of putative ULK1 phosphorylation sites in BNIP3 and BNIP3L aligned to ULK1 phosphorylation sites in validated ULK1 substrates (ATG101, ATG9, ATG14, BECLIN1, ATG4B, FUNDC1, VPS34). (b) Cartoon illustrating key domains in BNIP3. (c) Cartoon illustrating key domains in BNIP3L. (d) In vitro kinase assay testing the ability of recombinant ULK1 kinase to phosphorylate recombinant BNIP3 (lanes 3–5) or BNIP3 mutated to S17A (lanes 6–8) in the presence or absence of ULK-101 to inhibit ULK1 kinase activity (lanes 2, 5, 8) and using ULK1 autophosphorylation as a control for ULK1 activity (lower panel). Fold change in phosphorylation for the mutant is shown relative to WT. (e) In vitro kinase assay testing the ability of recombinant ULK1 kinase to phosphorylate recombinant BNIP3L (lanes 3–5) or BNIP3L mutated to S35A (lanes 6–8) in the presence or absence of ULK-101 to inhibit ULK1 kinase activity (lanes 2, 5, 8) and using ULK1 autophosphorylation as a control for ULK1 activity (lower panel). Fold change in phosphorylation for the mutant is shown relative to WT. (f) In vitro kinase assay testing the ability of recombinant ULK1 kinase to phosphorylate different mutant forms of BNIP3. Fold changes in phosphorylation for each mutant is shown relative to WT.
Fig 2: ULK1-dependent phosphorylation of Parkin is required for maximal Parkin activity.(A) Immunoblots of HEK293T cells transfected with YFP-Parkin-C431S or YFP-Parkin-C431S/SA3. Twenty-four hours after transfection, cells were treated with CCCP (20 μM), valinomycin (5 μM), AO (2.5 μM antimycin A + 5 μM oligomycin) or vehicle (DMSO) for 2 hours. Red arrow indicates higher molecular weight Parkin-ubiquitin thioester species. (B) YFP-Parkin-C431S–expressing HEK293T cells treated as in (A) ± SBI-0206965 (10 μM) for 2 hours. (C) Functional analysis of Parkin-mediated mitophagy activity in HEK293T cells stably bearing mito-Keima reporter. The graph indicates the mitophagy-positive cell population quantified by flow cytometry. Data are shown as the means ± SEM of two independent experiments. *P < 0.05 when compared to the control cells at 2 or 4 hours, respectively, by two-way ANOVA. (D) Immunoblot analysis of endogenous Parkin in hESC-derived induced neurons treated with AO for indicated times or 60 min with 10 or 50 μM 6965 pretreatment. (E) Model for Parkin activation. CCCP treatment causes rapid increases in AMP and mtROS, which activate AMPK to phosphorylate and activate ULK1 within minutes. ULK1, in turn, phosphorylates Parkin maximally at Ser108 in the cytoplasm within 2 min of CCCP treatment. Meanwhile, PINK1 is slowly becoming stabilized on the mitochondrial outer membrane following CCCP, with detection of its phosphorylation of ubiquitin and Parkin Ser65 appearing around 30 min and becoming maximal at 60 min or later.
Fig 3: Mutation of S17 in BNIP3 modulates its LC3B interaction and mitophagy. (a) Pulldown of GFP-LC3 stably expressed in HEK-293 T cells with transiently expressed HA-BNIP3 (WT) and different HA-BNIP3 mutants (W18A, S17A, S17E) or empty vector (EV) control, in the presence or absence of 100 nM bafilomycin A1. Inputs to the pulldown are shown on the left and the result of the pulldown on the right. Fold changes in protein levels of BNIP3 dimer and BNIp3 monomer are shown relative to WT. (b) Pulldown of GFP-LC3 with HA-BNIP3, as described in (a), in the presence (lanes 6–9, 16–19) or absence (lanes 1–5, 10–15) of exogenous FLAG-ULK1. Fold change in protein levels of BNIP3 dimer and BNIp3 monomer are shown. Fold changes in protein levels of BNIP3 dimer and BNIp3 monomer are shown relative to WT. (c–f) Immunofluorescent staining for TOMM20 (green, mitochondria), LC3B (magenta, autophagosomes), HA-BNIP3 (red) and DAPI (blue) in U2OS cells transiently expressing HA-BNIP3 (c), HA-BNIP3W18A (d), HA-BNIP3S17A (e) or HA-BNIP3S17E (f). Cells expressing exogenous HA-BNIP3 are asterisked (*) and LC3B/TOMM20 overlap is detected as white puncta (green and magenta overlap). (g) Quantification using Image J of LC3B/TOMM20 overlap per cell for at least at least 15 cells per field for each of the different forms of BNIP3 compared to cells not expressing BNIP3. Data were statistically analyzed as described in “Materials and methods” section. All data are shown as the mean ± s.e.m. Values of p ≤ 0.05 are considered significant. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.
Fig 4: The BH3 domain promotes BNIP3 turnover in a manner inhibited by ULK1. (a) Western blot for different mutant forms of HA-BNIP3 (WT, S17A, S17E, DBH3, DPEST, G180A, DTMD) expressed in HEK-293 T cells in the presence (lanes 8–14) or absence (lanes 1–7) of exogenous FLAG-ULK1. Fold change in protein levels of BNIP3 dimer and BNIP3 monomer are shown relative to WT. (b) Western blot for HA-BNIP3 and different mutant forms (WT, S17A, S17E, DBH3, DBH3/S17A, DBH3/S17E) of HA-BNIP3 expressed in HEK-293 T cells in the presence (lanes 7–12) or absence (lanes 1–6) of exogenous FLAG-ULK1. Fold change in protein levels of BNIP3 dimer and BNIP3 monomer are shown relative to WT. (c) Pulldown of GFP-LC3 stably expressed in HEK-293 T cells with transiently expressed HA-BNIP3 (WT) and different HA-BNIP3 mutants (DBH3, S17A/DBH3, S17E/DBH3) in the presence or absence of 100 nM bafilomycin A1. Inputs to the pulldown are shown on the left and the result of the pulldown on the right. Fold change in protein levels of BNIP3 dimer and BNIP3 monomer are shown relative to WT. (d) Pulldown of FLAG-ULK1 stably expressed in HEK-293 T cells with transiently expressed HA-BNIP3 (WT) and different CTD mutant forms of FLAG-ULK1 (full-length, D829–1051, D1038–1051). Fold change in levels of BNIP3 dimer and BNIP3 monomer are shown relative to Full-length. (e) Immunofluorescent staining for TOMM20 (green, mitochondria), LC3B (magenta, autophagosomes), HA-BNIP3 (red) and DAPI (blue) in U2OS cells transiently expressing HA-BNIP3 (top panels) or HA-BNIP3DBH3 (bottom panels).
Fig 5: Parkin Ser108phosphorylation is rapidly induced following AO and CCCP and precedes activation of PINK1 and TBK1.(A) Schematic of overall Parkin domain structure with inset of ClustalW alignment of residues 59 to 115 of human Parkin. The evolutionary conservation of the PINK1 site Ser65 and ULK1 site Ser108 in Parkin across vertebrates is shown in red. Ser108 is one of only two fully conserved serines in the recently described ACT element (7) involved in Parkin activation (residues 101 to 110). At right, structural model of Ser108 in ACT element shown in red, Arg104 shown in green is mutated in a familial Parkinson’s patient (R104W). (B) Immunoblots of HEK293T cells stably expressing YFP-Parkin was subjected to AO (2.5 μM antimycin A and 5 μM oligomycin) treatment for the times indicated. (C) Immunoblots of HEK293T cells stably expressing YFP-Parkin were subjected to 10 μM CCCP treatment for the times indicated.
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