TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction

Figures & data

Figure 1. Cytoplasmic TP53INP2 stimulates autophagy. (a) Domain architecture of TP53INP2, TP53INP2[NLSΔ] and TP53INP2[LIRΔ]. LIR, LC3-interacting region; NLS, nuclear localization signal; NoLS, nucleolar localization signal. (b) The intracellular distribution of GFP-tagged TP53INP2, TP53INP2[NLSΔ] or TP53INP2[LIRΔ] in HeLa cells. (c) Confocal images of HEK293 cells stably expressing GFP-LC3B with RFP-tagged TP53INP2, TP53INP2[NLSΔ] or TP53INP2[LIRΔ] transfection. (d) Quantification of GFP-LC3B puncta in (c). The data are presented as mean ± SEM, n = 30 cells. (e) Western blot analysis of LC3B-II production and GFP-LC3B cleavage in HEK293 cells stably expressing GFP-LC3B and transfected with the indicated plasmids. (f) Confocal images of HEK293 cells stably expressing mCherry-LC3B and transfected with plasmids expressing GFP-[3NES]-TP53INP2[NLSΔ] or GFP-[3NLS]-TP53INP2[NLSΔ]. (g) Western blot analysis of intracellular LC3B in HEK293 cells that were starved or transfected with the GFP-tagged indicated plasmids. (h) LDH sequestration assay of HEK293 cells treated as in (g) in the presence of chloroquine. The data are presented as mean ± SEM of triplicates. ***, P < 0.001. Scale bars: 10 µm.

Figure 2. Association of TP53INP2 with early autophagic structures. (a-e) Colocalization of RFP-TP53INP2 with ULK1-GFP (a), GFP-ATG14 (b), GFP-BECN1 (c), GFP-ZFYVE1 (d) or WIPI2-GFP (e) in starved MEFs. (f and g) Localization of RFP-TP53INP2 or RFP-TP53INP2[NLSΔ] in starved BECN1-KO (f) or 3-MA-treated (g) HEK293 cells. The cells were imaged by confocal microscope 24 h after cotransfection of the plasmids. (h) Formation of puncta in HEK293 cells stably expressing ULK1-GFP, GFP-BECN1, GFP-ZFYVE1 or GFP-LC3B. The cells were infected with lentivirus expressing non-silencing control shRNA or TP53INP2 shRNA for 72 h, with or without cell starvation for 2 h. (I) Quantification of the puncta in (h). The data are presented as mean ± SEM, n = 30 cells. ***, P < 0.001. Scale bars: 10 µm.

Figure 3. LC3 mediates the association of TP53INP2 with autophagic membranes. (a) Colocalization analysis of RFP-TP53INP2 and GFP-ATG14 in starved WT, atg5^−/- or atg7^−/- MEFs. (b) Colocalization analysis of RFP-TP53INP2 and Flag-ATG14 in starved HEK293 cells coexpressing GFP-LC3B, GFP-[3NLS]-LC3B or GFP-LC3B^G120A. (c) HEK293 cells stably expressing GFP-LC3B were cotransfected with Flag-ATG14 and RFP-TP53INP2[NLSΔ], or with Flag-ATG14 and RFP-TP53INP2^W35,I38A[NLSΔ]. Cells were then immunostained with anti-Flag and imaged with confocal microscopy. (d) GFP-LC3B punctum formation in HEK293 cells stably expressing GFP-LC3B with or without cell starvation. The cells were transiently transfected with RFP-TP53INP2[NLSΔ] or RFP-TP53INP2^W35,I38A[NLSΔ]. (e) Quantification of GFP-LC3B puncta in (d). The data are presented as mean ± SEM, n = 30 cells. ***, P < 0.001. Scale bars: 10 µm.

Figure 4. TP53INP2 forms a complex with LC3B and ATG7. (a) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-TP53INP2, GFP-TP53INP2[NLSΔ] or GFP-TP53INP2[LIRΔ] from HEK293 cells. TP53INP2 proteins were immunoprecipitated by anti-GFP. The coprecipitated ATG7, ATG3 or ATG12–ATG5 was detected by western blot using anti-ATG3, anti-ATG7 or anti-ATG5 respectively. (b) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-tagged TP53INP2[NLSΔ], TP53INP2^W35,I38A[NLSΔ] or TP53INP2[LIRΔ]. GFP-tagged TP53INP2 mutants were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7, anti-ATG3 or anti-ATG5. (c) In vitro TP53INP2-ATG7 binding assay. Purified GST-TP53INP2 or GST-TP53INP2^W35,I38A was incubated with purified LC3B[G120] and ATG7. After affinity-isolating GST-TP53INP2 or GST-TP53INP2^W35,I38A with glutathione-sepharose 4B beads, the bound LC3B[G120] and ATG7 were analyzed by western blot. (d) HEK293T cells were cotransfected with Flag-LC3B, TP53INP2-MYC and HA-ATG7. The cells were lysed 48 h after transfection and Flag-LC3B was immunoprecipitated with anti-Flag. After incubation of the Flag-LC3B precipitates with Flag peptide, the eluate was used for immunoprecipitation with either anti-MYC or anti-HA. The immunoprecipitates were then analyzed by western blot by anti-Flag, anti-MYC and anti-HA respectively. (e) Coimmunoprecipitation of ATG7 with each of the indicated GFP-tagged truncated TP53INP2 mutants in HEK293 cells. TP53INP2 proteins were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7. (f) Purified GST-tagged TP53INP2[NLSΔ], TP53INP2^W35,I38A[NLSΔ], TP53INP2^W35,I38A[Δ1-28],[NLSΔ] or SQSTM1 was incubated with purified ATG7, then the GST-tagged proteins were affinity-isolated by glutathione-sepharose 4B beads and bound ATG7 was detected by western blot using anti-ATG7.

Figure 5. TP53INP2 facilitates LC3B-ATG7 interaction. (a) Coprecipitation of endogenous ATG7 with exogenous Flag-LC3B in TP53INP2-MYC cotransfected HEK293 cells with or without cell starvation. Flag-LC3B was immunoprecipitated using anti-Flag, then ATG7 and TP53INP2-MYC were detected by anti-ATG7 and anti-MYC respectively. (b) Coprecipitation of ATG7 with Flag-LC3B from HEK293 cells transiently expressing RFP-tagged TP53INP2 or each of the indicated TP53INP2 mutants. Flag-LC3B was immunoprecipitated using anti-Flag. (c) HEK293 cells stably expressing non-silencing shRNA or TP53INP2 shRNA were transfected with Flag-LC3B^K49,51R and starved. The cells were then fractionated by differential centrifugation. Flag-LC3B^K49,51R was immunoprecipitated from the cell cytosol using anti-Flag and the coprecipitated ATG7 was detected by western blot. (d) In vitro affinity-isolation assay of LC3B[G120]-ATG7 interaction. Purified GST-LC3B[G120] was incubated with cell lysate from HEK293 cells expressing the indicated RFP-tagged TP53INP2 mutants. After affinity-isolating GST-LC3B[G120] using glutathione-sepharose 4B beads, GST-LC3B[G120]-bound ATG7 was analyzed by western blot. (e) Confocal images of HEK293 cells stably expressing GFP-LC3B and transfected with plasmids expressing each of the indicated RFP-tagged TP53INP2 truncated mutants. (f) Quantification of GFP-LC3B puncta in (e). The data are presented as mean ± SEM, n = 30 cells. ***, P < 0.001. Scale bars: 10 µm.