FBXO2-mediated KPTN ubiquitination promotes amino acid–dependent mTORC1 signaling and tumor growth
Jianfang Gao, Jina Qing, Xianglong Li, Yuxuan Luo, Lingwen Huang, Hongxia Li, Huan Zhang, Jiao Zhang, Pei Xiao, Jinsong Li, Tingting Li, Shanping He
Jianfang Gao, Jina Qing, Xianglong Li, Yuxuan Luo, Lingwen Huang, Hongxia Li, Huan Zhang, Jiao Zhang, Pei Xiao, Jinsong Li, Tingting Li, Shanping He
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Research Article Cell biology Oncology

FBXO2-mediated KPTN ubiquitination promotes amino acid–dependent mTORC1 signaling and tumor growth

Abstract

Mechanistic target of rapamycin complex 1 (mTORC1) is a master controller of cell growth, and its dysregulation is associated with cancer. KICSTOR, a complex comprising KPTN, ITFG2, C12orf66, and SZT2, functions as a critical negative regulator of amino acid–induced mTORC1 activation. However, the regulatory mechanisms governing KICSTOR remain largely unclear. In this study, we identify F-box only protein 2 (FBXO2) as a key modulator of amino acid–dependent mTORC1 signaling. Mechanistically, FBXO2 colocalizes and directly interacts with KPTN via its F-box–associated domain, promoting K48- and K63-linked polyubiquitination of KPTN at lysine residues 49, 67, 262, and 265. FBXO2-mediated KPTN ubiquitination disrupted its interaction with ITFG2 and SZT2, while enhancing its interaction with C12orf66, thereby impairing the ability of KICSTOR to recruit the GATOR1 complex — comprising DEPDC5, NPRL2, and NPRL3 — to the lysosomal surface. Notably, FBXO2 protein levels were substantially upregulated in patients with liver cancer, and FBXO2-mediated KPTN ubiquitination facilitated the progression of hepatocellular carcinoma (HCC). These results reveal a key regulatory mechanism of mTORC1 signaling and highlight FBXO2 and KPTN ubiquitination as therapeutic targets for HCC treatment.

Authors

Jianfang Gao, Jina Qing, Xianglong Li, Yuxuan Luo, Lingwen Huang, Hongxia Li, Huan Zhang, Jiao Zhang, Pei Xiao, Jinsong Li, Tingting Li, Shanping He

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Figure 3

FBXO2 activates mTORC1 by promoting KPTN ubiquitination.

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FBXO2 activates mTORC1 by promoting KPTN ubiquitination. (A and B) HEK29...
(A and B) HEK293T cells stably expressing Flag-KPTN were transduced with lentiviruses expressing the indicated shRNAs (A) or transfected with plasmids expressing the indicated genes (B), then treated with MG132 (20 μM) for 12 hours at 60 hours posttransduction or 36 hours posttransfection. (C and D) HEK293T cells stably expressing Flag-KPTN (C) or HEK293T cells (D) were transfected with plasmids expressing the indicated genes, then treated with MG132 (20 μM) for 12 hours at 60 hours posttransfection. WT, wild-type; Ub-K48, with the other 6 lysine residues of ubiquitin (Ub) mutated to arginine except lysine 48 (K48); Ub-K63, with the other 6 lysine residues of Ub mutated to arginine except lysine 63 (K63); KPTN-4KR, KPTN-K49/67/262/265R. WCLs (A–D) were denatured and then immunoprecipitated with anti-Flag magnetic beads, followed by immunoblotting with the indicated antibodies. (E and F) KPTN-knockout HEK293T cells stably expressing EGFP-TFEB were transduced with lentiviruses expressing Flag-tagged KPTN or KPTN-4KR for 24 hours, deprived of amino acids and serum for 2 hours, and stimulated with amino acids for 2 hours, followed by nucleus staining with DAPI. Representative images of EGFP-TFEB localization were shown (E), and the quantitative results of EGFP-TFEB localization were presented (F). Scale bar, 10 μm. Data are presented as means ± SEM; n = 9 independent fields per condition; ***P < 0.001, ****P < 0.0001, 1-way ANOVA followed by Tukey’s multiple comparisons test. (G) WT and KPTN-knockout HEK293T cells were transduced with lentiviruses expressing Flag-tagged KPTN or KPTN-4KR for 24 hours, deprived of amino acids and serum for 2 hours, and stimulated with amino acids for 10 minutes. WCLs were analyzed by immunoblotting with the indicated antibodies. Data are representative of at least 2 independent experiments (A–D and G). dIP, denaturing immunoprecipitation.