Research ArticleCELL BIOLOGY

AMPK/ULK1-mediated phosphorylation of Parkin ACT domain mediates an early step in mitophagy

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Science Advances  07 Apr 2021:
Vol. 7, no. 15, eabg4544
DOI: 10.1126/sciadv.abg4544
  • Fig. 1 Parkin Ser108 is regulated by AMPK/ULK1 signaling.

    (A) Immunoblots of Parkin cotransfected with myc-tagged wild-type (WT) or kinase-inactive (KI) ULK1 in human embryonic kidney (HEK) 293T cells. (B) Immunoblots as in (A), but here, cell lysates were treated with lambda-phosphatase (PPase) as indicated. (C) Parkin Ser108 and Ser110 aligned with known ULK1 phosphorylation sites. Residues conforming to optimal ULK1 substrate motif highlighted. (D) ClustalW alignment of evolutionary conservation surrounding Parkin Ser108. (E) Immunoblots of indicated Parkin and ULK1 cDNAs cotransfected into HEK293T cells. Phosphorylation monitored by Parkin mobility shift. (F and G) As in (E) but monitored with Parkin phospho-ser108 antibody. (H) Immunoblots of cells transfected with Parkin and treated for 1 hour with vehicle (D), 20 μM CCCP (C), 10 μM 991, 10 μM 6965 ULK1 inhibitor (65), or 6965 combined with CCCP or 991 (65/C: 65/991). (I) Immunoblots of liver lysates from control or inducible knockout of AMPKa1/a2 (AMPK DKO) mice treated with vehicle or AMPK activator MK8722 for 2 hours. See Materials and Methods for details. (J) Immunoblots as in (I) but control or inducible KO of Ulk1/Ulk2. (K) Immunoblots of lysates from primary mouse hepatocytes from WT or AMPK liver DKO mice treated for 1 hour with vehicle or metformin.

  • Fig. 2 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.

  • Fig. 3 Parkin is phosphorylated at Ser108 in the cytosol and its timely translocation to mitochondria requires Ser108-Ser110.

    (A) Immunoblots of cytoplasmic and mitochondrial extracts isolated from YFP-Parkin-WT and YFP-Parkin-SA3 expressing HEK293T cells by cell membrane disruption and differential centrifugation, after treatment with 20 μM CCCP for the indicated time. Cytoplasmic proteins (30 μg) and mitochondria proteins (20 μg) per lane were analyzed. (B) Immunoblots of HEK293T cells transiently transfected with YFP-Parkin or YFP-Parkin-SA3 or YFP-Parkin-R104W then treated with 20 μM CCCP for the times indicated. MG132 (10 μM) was added to prevented proteasome-mediated degradation. (C) Quantification of Parkin mitochondrial translocation phenotype by immunofluorescence. Individual cells from the images in (D) were analyzed for Person correlation coefficient (PCC) analysis by ImageJ software. Data are shown in a violin plot. n = 19 for YFP-Parkin-WT; n = 24 for YFP-Parkin-SA3, **P < 0.01, ****P < 0.0001 by two-way analysis of variance (ANOVA). (D) Time-lapse live-cell images of U2OS cells stably expressing WT or SA3 YFP-Parkin treated with dimethyl sulfoxide (DMSO) or CCCP (20 μM) for 150 min. YFP-Parkin is shown as green, mitochondria were stained with MitoTracker (red). Clustered mitochondria with translocated YFP-Parkin are indicated with white arrowheads. Scale bars, 20 μm.

  • Fig. 4 Recruitment of Parkin and downstream mitophagy effectors to mitochondria is controlled by AMPK/ULK1 signaling following CCCP.

    (A) Immunoblots of Mito-tag purified mitochondria from HEK293T cells stably expressing WT or SA3 YFP-Parkin treated with 20 μM CCCP for indicated times. All cells also treated with 10 μM MG132 for 4 hours. (B) Representative immunocytochemistry of NDP52 (white) and Tom20 (red) in HeLa cells stably expressing WT or SA3 YFP-Parkin. Cells treated with DMSO or CCCP for 4 hours. Nuclei stained with 4′,6-diamidino-2-phenylindole (blue). NDP52 puncta number and puncta size were analyzed by ImageJ with 10 individual images for each condition. Data are shown as the means ± SEM. **P < 0.001; ****P < 0.0001 by two-way ANOVA. Scale bar, 20 μm. n.s., not significant. (C) Immunoblots of Mito-tag purified mitochondria from WT YFP-Parkin expressing cells treated with DMSO or 10 μM 6965 for 15 min before CCCP treatment for indicated times. Cells treated with MG132 as in (A). (D) Immunoblots of cell lysates from WT YFP-Parkin expressing cells treated with CCCP ± 15 min pretreated with 50 μM 991 or 10 μM 6965 as indicated. (E) Immunoblots of purified mitochondria from cells stably expressing WT or SA3 YFP-Parkin treated with CCCP ± 50 μM 991 for 1 hour. All cells also treated with 10 μM MG132 for 1 hour. (F) Immunoblots of supernatant (top) and purified mitochondria (bottom) from control or CRISPR-mediated AMPK KO cells treated with 20 μM CCCP for indicated times. Cells treated with MG132 as in (A).

  • Fig. 5 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.

Supplementary Materials

  • Supplementary Materials

    AMPK/ULK1-mediated phosphorylation of Parkin ACT domain mediates an early step in mitophagy

    Chien-Min Hung, Portia S. Lombardo, Nazma Malik, Sonja N. Brun, Kristina Hellberg, Jeanine L. Van Nostrand, Daniel Garcia, Joshua Baumgart, Ken Diffenderfer, John M. Asara, Reuben J. Shaw

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