VWCE modulates amino acid-dependent mTOR signaling and coordinates with KICSTOR to recruit GATOR1 to the lysosomes

Abstract

The mechanistic target of rapamycin complex 1 (mTORC1) is a crucial regulator of cell growth. It senses nutrient signals and adjusts cellular metabolism accordingly. Deregulation of mTORC1 has been associated with metabolic diseases, cancer, and aging. Amino acid signals are transduced to mTORC1 through sensor proteins and two protein complexes named GATOR1 and GATOR2. In this study, we identify VWCE (von Willebrand factor C and EGF domains) as a negative regulator of amino acid-dependent mTORC1 signaling. Knockdown of VWCE promotes mTORC1 activity even in the absence of amino acids. VWCE interacts with the KICSTOR complex to facilitate the recruitment of GATOR1 to the lysosomes. Bioinformatic analysis reveals that expression of VWCE is reduced in prostate cancer. More importantly, overexpression of VWCE inhibits the development of prostate cancer. Therefore, VWCE may serve as a potential therapeutic target for the treatment of prostate cancers.

Fig. 1. VWCE regulates amino acid-dependent mTORC1 activity.

a A schematic diagram depicting amino acid (a.a.) deprivation and restimulation. b Knockdown of VWCE impairs the suppression of mTORC1 activity upon amino acid deprivation. Cells transfected with non-targeting siRNA (siCtrl) or siRNA targeting VWCE (siVWCE) were starved of amino acids for 50 min (-), or starved for 50 min and restimulated with amino acids for 10 min (+). Cell lysates were analyzed by immunoblotting. c The knockdown efficiency of VWCE in (b) is tested by RT-qPCR (N = 3). Data are mean ± s.d. ***p < 0.001 (unpaired two-sided Student’s t-test). d Overexpression of VWCE restores the ability of VWCE-deficient cells to sense amino acid deprivation. The cells were transfected with the indicated siRNAs and then transfected with the indicated plasmids after 6 h. After another 6 h, the cell culture medium was replaced with fresh medium, and the cells were allowed to grow for 60 h before harvesting. e mTOR (red) constitutively localizes on lysosomes in VWCE-knockdown cells, even in the absence of amino acids. The cells were treated by amino acid starvation or starvation plus restimulation before preparation for immunostaining. LAMP2 (green) was used as a lysosome marker. f Quantification of co-localization between mTOR and LAMP2 in (e) (N = 30). Data are mean ± s.d. ****p < 0.0001 (unpaired two-sided Student’s t-test). The immunoblotting assays were independently replicated three times with consistent results. Source data are provided as Source data files.

Fig. 2. VWCE interacts with KICSTOR and localizes on the lysosomes.

a VWCE acts downstream of GATOR2 to regulate mTORC1. Cells transfected with siCtrl, siWDR59, siVWCE, or siWDR59 plus siVWCE were treated by amino acid starvation (-) or starvation then restimulation (+) and lysed for immunoblotting. b GATOR1 overexpression inhibits mTORC1 activity to a lesser extent in VWCE-deficient cells. Cells transfected with the indicated siRNAs and plasmids were harvested for immunoprecipitation and immunoblotting. MetAP2 serves as a control. c VWCE acts upstream of Rag GTPases to regulate mTORC1. The siRNA-transfected cells were then transfected with dominant-negative Rag heterodimers (RagB^T54N+RagC^Q120L). d The interaction network of VWCE, KICSTOR components, GATOR1 components and GATOR2 components from BioPlex. e VWCE interacts with all 4 subunits of the KICSTOR complex (KPTN, ITFG2, C12orf66, SZT2). Cells stably expressing FLAG-tagged VWCE were transfected with the indicated HA-tagged cDNAs. Cell lysates and immunoprecipitates were analyzed by immunoblotting after the indicated amino acid treatments. f VWCE interacts with KPTN in an amino acid-independent manner. Cells transiently expressing the indicated cDNAs were lysed after the indicated amino acid treatments. Immunoprecipitates and lysates were analyzed by immunoblotting. g Amino acid-insensitive localization of VWCE on lysosomes. Lysosomes were immunopurified from HEK293T cells stably expressing 3×HA-tagged TMEM192 and FLAG-tagged VWCE. Purified lysosomes (Lyso-IP) and whole cell lysates (WCL) were analyzed by immunoblotting (g, h). Organelle-specific marker proteins are indicated. h Lysosomal localization of VWCE is independent of the KICSTOR complex. The experiments were independently replicated three times with consistent results. Source data are provided as a Source data file.

Fig. 3. Knockdown of VWCE suppresses the lysosomal localization of GATOR1.

a, b Knockdown of VWCE impairs the GATOR1-KICSTOR (a) and GATOR1-GATOR2 (b) interactions. Cells were transfected with the indicated siRNAs and cDNAs. The immunoprecipitates and lysates were analyzed by immunoblotting. c The relative levels of immunoprecipitated MIOS/HA-DEPDC5 or KPTN/HA-DEPDC5 in (b) are quantified across three independent replicates. Data are mean ± s.d. *p < 0.05 (unpaired two-sided Student’s t-test). d Knockdown of VWCE affects the lysosomal localization of GATOR1. Cells stably expressing 3×HA-tagged TMEM192 were transfected with siCtrl or siVWCE. Immunopurified lysosomes and cell lysates were analyzed by immunoblotting. e, g Cells stably expressing EGFP-NPRL2 (e) or expressing endogenous 3×HA-tagged DEPDC5 (g) were transfected with the indicated siRNAs. Immunostaining experiments were performed. LAMP2 was used as a lysosomal marker. f, h Quantification of the co-localization between EGFP-NPRL2 and LAMP2 in (d) (N = 20) or 3×HA-DEPDC5 and LAMP2 in (g) (N = 20). Data are mean ± s.d. **p < 0.01; ****p < 0.0001 (unpaired two-sided Student’s t-test). i A proposed model of how VWCE regulates amino acid-dependent mTORC1 signaling. The immunoblotting assays were independently replicated three times with consistent results. Source data are provided as Source data files.

Fig. 4. VWCE inhibits cancer development through mTORC1 signaling.

a Differential expression of VWCE in normal versus tumor tissues, as derived from the TCGA database. Sample sizes for normal (N) and tumor (T) tissues are indicated in the figure. *p < 0.05; **p < 0.01; ****P < 0.0001 (unpaired two-sided Student’s t-test). The full names corresponding to the cancer types abbreviations are provided in Supplementary Table 2. b VWCE is highly expressed in liver cancer cell lines, but not in prostate cancer cell lines. The expression levels of VWCE in the indicated cancer cell lines were obtained from the CCLE database. c, d The expression levels of VWCE in the cancer cell lines were measured by RT-qPCR (c) and immunoblotting (d). Data are mean ± s.d. in (c) (N = 3). e Overexpression of VWCE inhibits amino acid-dependent mTORC1 activation in the PC3 cells. The cells stably expressing vector or FLAG-tagged VWCE were treated as indicated and lysed for immunoblotting. f The relative levels of p-S6K1/Actin in (e) are quantified across three independent replicates. Data are mean ± s.d. *P < 0.05 (unpaired two-sided Student’s t-test). g Stable expression of VWCE inhibits the lysosomal localization of mTOR in PC3 cells. The cells stably expressing vector or VWCE were treated as indicated and immunostained with anti-mTOR (red) and anti-LAMP2 (green) antibodies. h Quantification of co-localization between mTOR and LAMP2 in (g) (N = 20). Data are mean ± s.d. *p < 0.05; ****p < 0.0001 (unpaired two-sided Student’s t-test). i Stable expression of VWCE inhibits anchorage-independent cell growth of PC3 cells. A total of 10,000 PC3 cells were seeded into each well of a 6-well cell culture plate. The number of colonies were quantified (N = 6). Data are mean ± s.d. ****p < 0.0001 (unpaired two-sided Student’s t-test). j–l Overexpression of VWCE inhibits subcutaneous xenograft tumor growth of PC3 cells. The PC3 cells stably expressing vector or VWCE were subcutaneously injected into nude mice for xenograft growth. Tumor images (j), tumor volumes (k), and tumor weights (l) are shown. Tumor volumes and tumor weights are quantified (N = 10). Data are mean ± s.d., ***p < 0.001; ****p < 0.0001 (unpaired two-sided Student’s t-test). Source data are provided as Source data files.