TPT1 (tumor protein, translationally-controlled 1) negatively regulates autophagy through the BECN1 interactome and an MTORC1-mediated pathway

Abstract

TPT1/TCTP (tumor protein, translationally-controlled 1) is highly expressed in tumor cells, known to participate in various cellular activities including protein synthesis, growth and cell survival. In addition, TPT1 was identified as a direct target of the tumor suppressor TP53/p53 although little is known about the mechanism underlying the anti-survival function of TPT1. Here, we describe a role of TPT1 in the regulation of the MTORC1 pathway through modulating the molecular machinery of macroautophagy/autophagy. TPT1 inhibition induced cellular autophagy via the MTORC1 and AMPK pathways, which are inhibited and activated, respectively, during treatment with the MTOR inhibitor rapamycin. We also found that the depletion of TPT1 potentiated rapamycin-induced autophagy by synergizing with MTORC1 inhibition. We further demonstrated that TPT1 knockdown altered the BECN1 interactome, a representative MTOR-independent pathway, to stimulate autophagosome formation, via downregulating BCL2 expression through activating MAPK8/JNK1, and thereby enhancing BECN1-phosphatidylinositol 3-kinase (PtdIns3K)-UVRAG complex formation. Furthermore, reduced TPT1 promoted autophagic flux by modulating not only early steps of autophagy but also autophagosome maturation. Consistent with in vitro findings, in vivo organ analysis using Tpt1 heterozygote knockout mice showed that autophagy is enhanced because of haploinsufficient TPT1 expression. Overall, our study demonstrated the novel role of TPT1 as a negative regulator of autophagy that may have potential use in manipulating various diseases associated with autophagic dysfunction.

Keywords: BCL2; BECN1; MTORC1; TP53 target gene; TPT1/TCTP; autophagy.

Figure 1.

TPT1 negatively regulates basal autophagy. (A) GFP-LC3 puncta were analyzed in HeLa GFP-LC3 cells, transiently transfected with TPT1 shRNA or control shRNA. Representative images were taken at x 600 magnification. Cells were stained with DAPI for the nucleus (blue). Scale bars: 20 μm. The number of GFP-LC3 dots per cell in each case was quantified. Data are presented as means ± S. E. M. (n = 3). P** < 0.0.1 (B) The lysates from either control shRNA or TPT1 shRNA transiently transfected HeLa GFP-LC3 cells were immunblotted with the indicated antibodies. ACTB served as a loading control. (C) GFP-LC3 puncta were analyzed in control shRNA and TPT1 shRNA stably transfected HeLa GFP-LC3 cells. The number of GFP-LC3 dots per cell in each case was quantified. Scale bars: 20 μm. Data are presented as means ± S. E. M. (n = 3). P** < 0.01. (D) Cell lysates from shRNA stably transduced HeLa GFP-LC3 cells were harvested at the indicated times after renewing the cell culture media and then subjected to immunoblotting analysis. ACTB served as a loading control. (E) Tpt1^+/+ and Tpt1^+/− MEFs were stained with an antibody to endogenous LC3 (green). Scale bars: 20 μm. The number of LC3 puncta per cell in each condition was quantified. Data are presented as means ± S. E. M. (n = 3). P** < 0.01. (F) Cell lysates from Tpt1^+/+ and Tpt1^+/− MEFs were immunoblotted. ACTB served as a loading control.

Figure 2.

Reduction in TPT1 enhances the on-rate of autophagy. (A and B) HeLa GFP-LC3 cells, stably transduced with shRNA for control (shCont) or TPT1 (shTPT1) were cultured in the presence or absence of 100 nM of bafilomycin A₁ (Baf A1) for 8 h. (A) GFP-LC3 puncta were analyzed. Representative images were taken at x 600 magnification. The number of GFP-LC3 dots per cell in each condition was quantified. Scale bars: 20 μm. Data are presented as means ± S. E. M. (n = 3). P** < 0.01, P*** < 0.001. (B) Cell lysates were immunoblotted with the indicated antibodies. ACTB served as a loading control. The experiments were repeated at least 6 times. The levels of GFP-LC3-II relative to ACTB were quantified by densitometry analysis. (C and D) Stable HeLa GFP-LC3-shCont and shTPT1 cells were cultured in the presence or absence of 50 µM of chloroquine (CQ) for 8 h. (C) GFP-LC3 puncta were analyzed. Representative images were taken at x 600 magnification. Scale bars: 20 μm. The number of GFP-LC3 dots per cell in each condition was quantified. Data are presented as means ± S. E. M. (n = 3). P** < 0.01, P*** < 0.001. (D) Cell lysates were immunoblotted with the indicated antibodies. ACTB served as a loading control. The experiments were repeated at least 6 times. The levels of GFP-LC3-II relative to ACTB were quantified by densitometry analysis.

Figure 3.

Reduction of TPT1 stimulates autophagosome maturation. (A and B) HeLa cells stably transduced with either control shRNA or TPT1 shRNA were then transfected with mRFP-GFP-LC3 for 24 h. (A) Representative images were taken at x 800 magnification. Scale bars: 20 μm. (B) The number of yellow puncta and the number of mRFP-LC3-positive puncta (red) in the merged images were counted and the total number of puncta per cell was calculated. Data are presented as means ± S. E. M. (n = 3). P** < 0.01, P*** < 0.001. (C) HeLa cells stably transduced with either control shRNA or TPT1 shRNA were cotransfected with RFP-LC3 and GFP-LAMP1 for 24 h, in the presence or absence of 100 nM of Baf A1 for 8 h. The colocalization of LC3 and LAMP1 was analyzed. Representative fluorescence images are shown together with the profiles of colocalization. Scale bars: 20 μm.

Figure 4.

Downregulation of TPT1 alters the BECN1 interactome to promote autophagy. (A) HeLa GFP-LC3 cells were transiently transfected with shRNA for control and TPT1. After 24 h, cell lysates were immunoblotted with the indicated antibodies. ACTB served as a loading control. (B) HeLa GFP-LC3 cells, stably transduced with shRNA for control (shCont) or TPT1 (shTPT1), were harvested at the indicated times after changing the cell culture medium and then immunoblotted. ACTB served as a loading control. (C and D) Stable HeLa GFP-LC3-shCont and shTPT1 cells were harvested 2 h after changing the cell culture medium and then immunoprecipitated with BCL2 antibodies (C) and BECN1 antibodies (D) and then blotted with the indicated antibodies. ACTB served as a loading control.

Figure 5.

Knockdown of TPT1 potentiates rapamycin-induced autophagy. HeLa GFP-LC3 cells, stably transduced with shRNA for control (shCont) or TPT1 (shTPT1) were treated either with DMSO or 200 nM rapamycin for 2 h. (A) Analysis of GFP-LC3 puncta. Representative images were taken at x 600 magnification. Scale bars: 20 μm. The number of GFP-LC3 dots per cell in each condition was quantified. Data are presented as means ± S. E. M. (n = 3). P** < 0.01, P*** < 0.001. (B) Immunoblotting of cell lysates with the indicated antibodies. The experiments were repeated at least 6 times. ACTB served as a loading control. The levels of GFP-LC3-II relative to ACTB were quantified by densitometry analysis. (C and D) Tpt1^+/+ and Tpt1^+/− MEFs were treated with 200 nM rapamycin for the indicated times. (C) LC3 puncta were immunostained and observed by confocal microscopy. Representative images were taken at x 600 magnification. Scale bars: 20 μm. The number of LC3 dots per cell in each case was quantified. Data are presented as means ± S. E. M. (n = 3). P* < 0.05, P** < 0.01, P*** < 0.001. (D) Cell lysates from Tpt1^+/+ or Tpt1^+/- MEFs were immunoblotted with the indicated antibodies. The experiments were repeated twice. ACTB served as a loading control. The levels of LC3-II relative to ACTB were quantified by densitometer analysis.

Figure 6.

Downregulation of TPT1 modulates the MTORC1 and AMPK pathways. (A) HeLa GFP-LC3 cells were transiently transfected with shRNA for control or TPT1. After 24 h, cell lysates were immunoblotted with the indicated antibodies. (B) HeLa GFP-LC3 cells, stably transduced with shRNA for control (shCont) or TPT1 (shTPT1) were harvested at the indicated times after changing cell culture media and then immunoblotted. The levels of p-PRKAA/AMPKα and p-ACACA/ACC relative to each total forms were quantified by densitometry analysis. (C and D) Stable HeLa GFP-LC3-shCont and shTPT1 cells were treated with 200 nM rapamycin for 2 h and harvested for immunoblotting. Treatment with DMSO was used as a control. ACTB was used as a loading control. Quantification of total MTOR derived from western blotting is depicted in (D). Data are presented as means ± S. E. M. (n = 3). P* < 0.05. (E) Livers were dissected from 3 representative sets of wild (Tpt1^+/+) and Tpt1 heterozygote knockout mice (Tpt1^+/−). Autophagy-related molecules were immunoprobed with the indicated antibodies. ACTB served as a loading control.

Figure 7.

Haploinsufficient TPT1 expression in vivo leads to enhanced autophagy. Organs were dissected from 3 representative sets of wild-type (Tpt1^+/+) and Tpt1 heterozygote knockout mice (Tpt1^+/−). Autophagy-related molecules were immunoprobed with the indicated antibodies in livers (A) and kidneys (B). ACTB served as a loading control. Quantification of LC3-II, SQSTM1 and TPT1 derived from western blotting depicted in (C) livers and (D) kidneys. Data are presented as means ± S. E. M. (n = 6). P** < 0.01, P*** < 0.001. (E) Basal autophagic flux in Tpt1^+/+ and Tpt1^+/− mice was analyzed using the leupeptin assay. Mice were injected with PBS or 40 mg/kg leupeptin and killed 4 h later. Autophagy-related molecules were immunoprobed with the indicated antibodies in livers. ACTB served as a loading control. Data are presented as means ± S. E. M. (n = 3). P** < 0.01, P*** < 0.001.