Research ArticleCell Biology

Regulated proteolysis of p62/SQSTM1 enables differential control of autophagy and nutrient sensing

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Sci. Signal.  04 Dec 2018:
Vol. 11, Issue 559, eaat6903
DOI: 10.1126/scisignal.aat6903
  • Fig. 1 Caspase-8 cleaves p62 to generate p62TRM.

    (A) Immunoblots of healthy donor–derived skin fibroblast (C12 cells) left untreated (UT) or treated with PAM2CSK4 (5 μg/ml) or poly(I:C) (10 μg/ml) for 20 hours. (B and C) Representative two-channel images (B) and immunoblots (C) of C12 cells left untreated (UT) or treated with poly(I:C) (10 μg/ml, 20 hours), staurosporine (STS; 1 μM, 6 hours), or actinomycin D (ActD; 2 μM, 20 hours). In (B), cells were treated in the presence of PI (red). Scale bars, 100 μm. (D) Immunoblots from C12 cells transfected with nontargeting (CTRL) or the indicated small interfering RNA (siRNA) for 72 hours and left untreated or treated with poly(I:C). (E) Immunoblots from HeLa cells transfected with nontargeting (CTRL) or caspase-8 siRNA for 72 hours and then left untreated or treated with poly(I:C) as indicated. (F) Top: Schematic of GST-p62297–440 showing the region around the caspase-8 cleavage site (Asp329) and expected sizes of fragments after proteolysis. Bottom: Coomassie-stained gel of assay with increasing amounts of caspase-8 and WT or D329A GST-p62297–440; IETD, caspase-8 inhibitor Ac-IETD-fmk; *, caspase-8 p20 from self-processing; ter, terminus. (G) Immunoblots from C12 cells transfected with indicated Flagp62 variants and treated with poly(I:C). Data represent n = 2 (D) or n = 3 (A to C and E to G) biologically independent experiments.

  • Fig. 2 RIPK1 is required for caspase-8–driven generation of p62TRM.

    (A) Schematic (left) of TLR3 signaling and immunoblots (right) from skin fibroblasts of individuals with dominant loss of function in the indicated genes that were untreated (−) or treated (+) with poly(I:C) as indicated. (B) Immunoblots from C12 cells stably expressing nontargeting control (CTRL) or RIPK1-targeting miR treated with poly(I:C). (C) Immunoblots from C12 cells treated with poly(I:C) in the presence of the indicated inhibitors or vehicle [dimethyl sulfoxide (DMSO)]. (D) Immunoblots from HeLa cells left untreated or treated with poly(I:C) in the presence of DMSO or the indicated inhibitors. (E) Immunoblots from HeLa cells stably expressing CTRL or ATG7 miR left untreated or treated with poly(I:C). Blots on the right show ATG7 expression. IETD, Ac-IETD-fmk (caspase-8 inhibitor); NEC1, necrostatin-1 (RIPK1 inhibitor). Data represent n = 2 (D) or n = 3 (A to C and E) biologically independent experiments.

  • Fig. 3 p62TRM cannot execute autophagy-related functions.

    (A) Top: Schematic representation of p62 and p62TRM. Bottom: Immunoblots (IBs) from co-IP experiments in HEK293E cells expressing nontargeting (CTRL) or p62-targeting miR. Cells were transiently transfected with MycLC3B and Flagp62TRM. (B) Immunoblots from HEK293E cells showing p62 turnover upon treatment with Torin 1 (250 nM) in a Baf-A1 (100 nM)–sensitive manner. Indicated Flag-tagged p62 variants were transiently transfected in HEK293E cells, and endogenous p62 was stably silenced with a p62 3′ untranslated region (3′UTR)–specific miR (HEKp62miR cells). (C) Immunoblots from HEK293E cells transiently transfected with either Flagp62 or Flagp62TRM and left untreated or treated with Torin 1 (250 nM) without or with Baf-A1 (100 nM) for 4 hours. (D and E) Representative images and quantification from immunofluorescence analyses of Δp62 HeLa cells stably expressing the indicated Flag-tagged p62 variants showing trafficking defects of p62TRM. In (D), cells were treated with poly(I:C) for 20 hours, and Flagp62 variants and ubiquitin (Ub) were stained. In (E), cells were infected with L. monocytogenes ΔactA for 3 hours, and Flagp62 variants and bacteria were stained. Regions in white box are shown in insets. Scale bars, 10 μm. Plots show means ± SEM. n = 3 independent experiments. nd, not detected. ***P < 0.005 by paired Student’s t test. (F) Impaired restriction of L. monocytogenes ΔactA by p62TRM. Plots show fold replication between 2 and 10 hours after infection of indicated Δp62 HeLa cells (matched means; n = 6 independent experiments). ns, not significant; ***P ≤ 0.005 by paired Student’s t test. Data in (A) to (E) represent n = 3 biologically independent experiments.

  • Fig. 4 p62TRM promotes leucine sensing by mTORC1.

    (A and B) Immunoblots from C12 cells (A) or HeLa cells (B) starved of either leucine (Leu), arginine (Arg), or glucose (Glc) and then reactivated with these substances for 30 min. P, phosphorylated. (C) IL-6 enzyme-linked immunosorbent assay (ELISA) from Δp62 HeLa cells expressing the indicated p62 variants 20 hours after plating. Means ± SEM are plotted; Δp62 compared to +WT (n = 33 independent experiments), +TRM (n = 17 independent experiments), +iso2 (n = 14 independent experiments), or yellow fluorescent protein (YFP) (n = 9 independent experiments). (D) Immunoblots from Δp62 or Δp62 HeLa cells expressing the indicated p62 variants that were leucine-starved for 4 hours and left untreated or reactivated with leucine for 30 min. Intervening irrelevant lanes were removed, and pictures from the same blot developed with antibodies to p62 or β-actin (below) are shown. (E) Immunoblots from C12 cells transfected with nontargeting (CTRL) or caspase-8 siRNA for 72 hours and then left for 1 hour in serum-free RPMI with or without (w/o) leucine. (F) Immunoblots from C12 cells transfected with the indicated siRNA for 72 hours, then starved for either leucine or glucose, and left untreated (−) or treated with leucine or glucose, respectively, for 30 min. ***P ≤ 0.005, ****P < 0.0001 by paired Student’s t test. Data in (A), (B), and (D) to (F) represent n = 3 biologically independent experiments.

  • Fig. 5 Natural D329H and D329G mutations in p62 abolish caspase-8–mediated processing and mTORC1 activation.

    (A) Immunoblots from C12 cells transfected with the indicated Flag-tagged p62 variants and left untreated or treated with poly(I:C). (B) Coomassie-stained gel from assay with recombinant GST-p62297–440 variants and caspase-8. Also see schematic in Fig. 1F. (C) Representative images from immunofluorescence staining of Flagp62 variants and L. monocytogenes ΔactA in Δp62 HeLa cells stably expressing the indicated Flag-p62 proteins 3 hours after infection and quantification of p62-positive bacteria on the right (means ± SEM; n = 3 independent experiments). (D) Fold replication of L. monocytogenes ΔactA between 2 and 10 hours after infection in indicated ΔHeLa cells. Matched means are shown (n = 5 independent experiments). (E) ELISA quantification of IL-6 secreted by Δp62 HeLa cells expressing the indicated p62 variants. Means ± SEM are plotted; Δp62 compared to +WT (n = 33 independent experiments), +D329H (n = 16 independent experiments), or +D329G (n = 27 independent experiments). (F) Immunoblots from indicated Δp62 HeLa cells leucine-starved for 4 hours and then left untreated or reactivated with leucine (Leu) for 30 min. ***P ≤ 0.005 by one-way analysis of variance (ANOVA) (C) or paired Student’s t test (D and E). Data represent n = 3 (A and B) or n = 4 (F) biologically independent experiments.

  • Fig. 6 Synthetic generation of p62TRM by TEV protease–mediated cleavage bypasses the requirement for caspase-8.

    (A) Schematic of the strategy for synthetic generation of p62TRM. The Asp329 region in p62 was replaced with a TEV recognition sequence to generate the Flagp62TEV plasmid for stable constitutive expression in Δp62 HeLa cells. Doxycycline (DOX) was used to control expression of TEV-T2A-GFP (green fluorescent protein); T2A is a self-cleaving peptide sequence. (B) Immunoblots showing doxycycline-inducible expression of GFP and cleavage of p62TEV into p62TRM in Δp62/Flagp62TEV+TEV-T2A-GFP cells. (C) Representative immunoblots from Δp62/p62TEV+TEV-T2A-GFP cells either leucine-starved for 4 hours or glucose-starved for 6 hours, followed by reactivation with Leu or Glc for 30 min as indicated. Cells were either untreated (w/o) or treated with doxycycline for 24 hours and recovered without doxycycline for 18 hours before starvation/reactivation treatments. (D) Δp62/p62TEV/TEV-T2A-GFP cells were transfected with nontargeting (CTRL) or caspase-8 siRNA for 72 hours and treated with doxycycline before leucine starvation/reactivation as in (C). Graph below shows ratio of P-p70-S6K1/p70-S6K1 after leucine reactivation relative to CTRL siRNA-transfected cells. Means ± SEM (n = 3 independent experiments) are plotted. ns, not significant, by paired Student’s t test. Data in (B) and (C) represent n = 3 biologically independent experiments.

  • Fig. 7 RIPK1-dependent p62TRM generation and leucine sensing through mTORC1.

    (A) Immunoblots from IP experiments in HEK293p62miR cells transfected with Flagp62 variants and YFP or MycRIPK1 as indicated. (B) ELISA quantification of IL-6 secreted by Δp62 HeLa cells stably reconstituted with the indicated Flagp62 variants. +D329G was compared to +WT (n = 22 independent experiments), +ΔZZ/UBA (n = 17 independent experiments), or +D329GΔUBA (n = 13 independent experiments). (C) Immunoblots from Δp62 HeLa cells expressing the indicated p62 constructs leucine-starved for 4 hours and then left untreated or treated with leucine for 30 min. (D) IL-6 ELISA from C12 cells stably expressing nontargeting (CTRL) or RIPK1 miR 20 hours after plating. n = 9 independent experiments. (E) Immunoblots from C12 cells transfected with nontargeting (CTRL) or RIPK1 siRNA for 72 hours and then treated with serum-free RPMI containing the indicated concentrations of leucine (Leu) for 1 hour. (F) Immunoblots from C12 cells stably expressing nontargeting (CTRL) or RIPK1 miRNA starved for either leucine or glucose for 1 hour and then untreated (−) or treated with leucine or glucose, respectively, for 30 min. (G) Model showing RIPK1–caspase-8–driven cleavage (showed by green scissor) that generates p62TRM. Full-length p62 and D329H/G variants are competent in autophagy and antimicrobial xenophagy. p62TRM generated from p62 (or p62iso2; not shown) can regulate mTORC1. Natural D329H/G mutants cannot be processed by caspase-8 or activate mTORC1. RIPK1–caspase-8 contributes to regulation of mTORC1 through p62TRM production. Means ± SEM are plotted in graphs. ***P < 0.005, ****P < 0.0001 by paired Student’s t tests. Data represent n = 3 (A, C, and E) or n = 4 (F) biologically independent experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/559/eaat6903/DC1

    Fig. S1. TLR3 induces cell death–independent, caspase-8–dependent processing of p62 at Asp329.

    Fig. S2. Caspase-8–dependent processing of p62 at Asp329.

    Fig. S3. Role of RIPK1 in p62TRM production and the instability of the p62330–440 fragment.

    Fig. S4. Characterization of p62TRM.

    Fig. S5. Characterization of p62TRM, p62D329, p62D329H, and other variants.

    Fig. S6. Synthetic cleavage of p62 using TEV.

    Fig. S7. Role of RIPK1 and caspase-8 in mTORC1 regulation.

    Fig. S8. Role of p62 variants and RIPK1 in regulating mTORC1.

    Table S1. List of antibodies.

    Table S2. List of key reagents and resources.

    Table S3. siRNA sequences used in the study.

  • This PDF file includes:

    • Fig. S1. TLR3 induces cell death–independent, caspase-8–dependent processing of p62 at Asp329.
    • Fig. S2. Caspase-8–dependent processing of p62 at Asp329.
    • Fig. S3. Role of RIPK1 in p62TRM production and the instability of the p62330–440 fragment.
    • Fig. S4. Characterization of p62TRM.
    • Fig. S5. Characterization of p62TRM, p62D329, p62D329H, and other variants.
    • Fig. S6. Synthetic cleavage of p62 using TEV.
    • Fig. S7. Role of RIPK1 and caspase-8 in mTORC1 regulation.
    • Fig. S8. Role of p62 variants and RIPK1 in regulating mTORC1.
    • Table S1. List of antibodies.
    • Table S2. List of key reagents and resources.
    • Table S3. siRNA sequences used in the study.

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