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Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity

Haller, Martina, Hock, Andreas K, Giampazolias, Evangelos, Oberst, Andrew, Green, Douglas R, Debnath, Jayanta, Ryan, Kevin M, Vousden, Karen H, Tait, Stephen W G·2014·Autophagy
DOI 10.4161/15548627.2014.981914PMC PMC4502749
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Figure 1. Free ATG12 is rapidly degraded in a proteasome-dependent manner (A) Western blot detection of ATG12 in U2OS cells. (B) Endogenous ATG12 expression in U2OS cells following CHX treatment. (C) U2OS expressing empty vector or ATG12 were treated with CHX for various times and probed for ATG12 expression. (D) U2OS cells were treated were treated for 8 h as indicated with MG132 and/or CHX and probed for ATG12 expression. (E) U2OS were treated for 8 h with MG132 or lactacystin and examined for ATG12 expression. (F) U2OS expressing ATG12 were treated for 8 h with MG132 and/or CHX as indicated and cell lysates were probed for ATG12 expression. ACT or TOMM20 were used as a loading control.
Figure 1. Free ATG12 is rapidly degraded in a proteasome-dependent manner (A) Western blot detection of ATG12 in U2OS cells. (B) Endogenous ATG12 expression in U2OS cells following CHX treatment. (C) U2OS expressing empty vector or ATG12 were treated with CHX for various times and probed for ATG12 expression. (D) U2OS cells were treated were treated for 8 h as indicated with MG132 and/or CHX and probed for ATG12 expression. (E) U2OS were treated for 8 h with MG132 or lactacystin and examined for ATG12 expression. (F) U2OS expressing ATG12 were treated for 8 h with MG132 and/or CHX as indicated and cell lysates were probed for ATG12 expression. ACT or TOMM20 were used as a loading control.CC BY-NC · PubMed Central
Figure 2. Proteasomal degradation of free ATG12 protein occurs independent of autophagy (A) E1A and Ras-transformed WT or Atg7 knockout MEF were treated for 8 h with MG132 and cell lysates were probed for ATG12 expression. (B) Atg3 or (C) Atg5 knockout MEFs were treated with MG132 for 4 h and 8 h and analyzed for ATG12 expression. (D) U2OS cells expressing ATG12G140A were treated for 8 h with MG132 and/or CHX as indicated and lysates were examined for ATG12 expression. In all immunoblots, ACT was used as a loading control.
Figure 2. Proteasomal degradation of free ATG12 protein occurs independent of autophagy (A) E1A and Ras-transformed WT or Atg7 knockout MEF were treated for 8 h with MG132 and cell lysates were probed for ATG12 expression. (B) Atg3 or (C) Atg5 knockout MEFs were treated with MG132 for 4 h and 8 h and analyzed for ATG12 expression. (D) U2OS cells expressing ATG12G140A were treated for 8 h with MG132 and/or CHX as indicated and lysates were examined for ATG12 expression. In all immunoblots, ACT was used as a loading control.CC BY-NC · PubMed Central
Figure 3. Direct ubiquitination of free ATG12 regulates its proteasomal degradation (A) 293T cells expressing vector or ATG12 and His-ubiquitin were treated with MG132 as indicated. Cell lysates were subject to His-tag affinity isolation and immunoblotted for ATG12 and ubiquitin expression (B) Atg12 knockout MEFs, as well as MEFs stably expressing Atg12, were treated for 4 h with chloroquine, then examined for ATG12 and LC3B expression. (C) 293T cells expressing ATG12 or ATG12[K-] together with His-ubiquitin, were treated as indicated, subject to His-tag affinity isolation and probed for ATG12 and ubiquitin expression. (D) U2OS cells expressing ATG12 or ATG12[K-] and treated for 6 h with CHX and analyzed for ATG12 and TOMM20 expression, densitometric analysis was performed using ImageJ software, normalizing to TOMM20 levels. (E) U2OS cells expressing ATG12[K-] were treated with MG132 and/or CHX for 8 h as indicated and examined for ATG12 expression. ACT or TOMM20 were used as loading controls.
Figure 3. Direct ubiquitination of free ATG12 regulates its proteasomal degradation (A) 293T cells expressing vector or ATG12 and His-ubiquitin were treated with MG132 as indicated. Cell lysates were subject to His-tag affinity isolation and immunoblotted for ATG12 and ubiquitin expression (B) Atg12 knockout MEFs, as well as MEFs stably expressing Atg12, were treated for 4 h with chloroquine, then examined for ATG12 and LC3B expression. (C) 293T cells expressing ATG12 or ATG12[K-] together with His-ubiquitin, were treated as indicated, subject to His-tag affinity isolation and probed for ATG12 and ubiquitin expression. (D) U2OS cells expressing ATG12 or ATG12[K-] and treated for 6 h with CHX and analyzed for ATG12 and TOMM20 expression, densitometric analysis was performed using ImageJ software, normalizing to TOMM20 levels. (E) U2OS cells expressing ATG12[K-] were treated with MG132 and/or CHX for 8 h as indicated and examined for ATG12 expression. ACT or TOMM20 were used as loading controls.CC BY-NC · PubMed Central
Figure 4. Free ATG12 promotes proteasome inhibitor-mediated cell death (A) U2OS cells stably expressing empty vector or BCL2L1 were treated with MG132 and cell viability was determined by SYTOX Green staining using an Incucyte Imager; data represents the mean +/− standard error of the mean (SEM) of 3 experiments at a representative time-point (24 h). (B) U2OS cells were assessed for ATG12 expression 2 d post-transfection with control or ATG12 siRNA. Cell viability of control or ATG12 siRNA-transfected U2OS cells, treated with MG132 (C) HBSS (D) or Act D (E) was determined by SYTOX Green staining using an Incucyte Imager; representative time-points shown (24 h MG132, 30 h HBSS, 24 h Act D). Graphs represent the mean +/− SEM of 4 experiments. (F) U2OS, transfected with control or ATG12 siRNA, or stably expressing vector or BCL2L1 were starved in HBSS. After 48 h of starvation, cells were washed, cultivated for 7 d in DMEM and colonies were stained with methylene blue. (G) U2OS cells were treated with HBSS (24 h), MG132 (16 h) or Act D (16 h) and examined for ATG12 expression. In all immunoblots, ACT was used as a loading control.
Figure 4. Free ATG12 promotes proteasome inhibitor-mediated cell death (A) U2OS cells stably expressing empty vector or BCL2L1 were treated with MG132 and cell viability was determined by SYTOX Green staining using an Incucyte Imager; data represents the mean +/− standard error of the mean (SEM) of 3 experiments at a representative time-point (24 h). (B) U2OS cells were assessed for ATG12 expression 2 d post-transfection with control or ATG12 siRNA. Cell viability of control or ATG12 siRNA-transfected U2OS cells, treated with MG132 (C) HBSS (D) or Act D (E) was determined by SYTOX Green staining using an Incucyte Imager; representative time-points shown (24 h MG132, 30 h HBSS, 24 h Act D). Graphs represent the mean +/− SEM of 4 experiments. (F) U2OS, transfected with control or ATG12 siRNA, or stably expressing vector or BCL2L1 were starved in HBSS. After 48 h of starvation, cells were washed, cultivated for 7 d in DMEM and colonies were stained with methylene blue. (G) U2OS cells were treated with HBSS (24 h), MG132 (16 h) or Act D (16 h) and examined for ATG12 expression. In all immunoblots, ACT was used as a loading control.CC BY-NC · PubMed Central
Figure 5. Free ATG12 promotes cell death independent of autophagy (A) U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4BC74A were treated for 4 h with chloroquine and cell lysates were blotted for RFP and LC3B. U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4BC74A were transfected with control or ATG12 siRNA. (B) Following MG132 treatment (24 h) or (C) HBSS starvation (48 h), cell viability was determined by SYTOX Green exclusion in an Incuyte Imager. Graphs show the mean +/− SEM of 3 (B) or 5 (C) experiments at representative time-points (24h MG132, 48 h HBSS). Western blots show cell lysates of U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4BC74A, transfected with control or ATG12 siRNA, treated for 8 h with MG132 (B) or HBSS (C) and probed for RFP, LC3B, and ATG12. In all immunoblots, ACT or TOMM20 were used as a loading control.
Figure 5. Free ATG12 promotes cell death independent of autophagy (A) U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4BC74A were treated for 4 h with chloroquine and cell lysates were blotted for RFP and LC3B. U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4BC74A were transfected with control or ATG12 siRNA. (B) Following MG132 treatment (24 h) or (C) HBSS starvation (48 h), cell viability was determined by SYTOX Green exclusion in an Incuyte Imager. Graphs show the mean +/− SEM of 3 (B) or 5 (C) experiments at representative time-points (24h MG132, 48 h HBSS). Western blots show cell lysates of U2OS cells stably expressing vector, Strawberry or Strawberry-ATG4BC74A, transfected with control or ATG12 siRNA, treated for 8 h with MG132 (B) or HBSS (C) and probed for RFP, LC3B, and ATG12. In all immunoblots, ACT or TOMM20 were used as a loading control.CC BY-NC · PubMed Central

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Metadata

Authors
Haller, M., Hock, A. K., Giampazolias, E., Oberst, A., Green, D. R., Debnath, J., Ryan, K. M., Vousden, K. H., & Tait, S. W. G.
Venue
Autophagy
Publisher
Informa UK Limited
Published
2014-12-02
Volume
10
Issue
12
Pages
2269-2278
ISSN
  • 1554-8627
  • 1554-8635
DOI
10.4161/15548627.2014.981914
PMC
PMC4502749
Cited by
58
References
2
Language
en

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