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 6

FBXO2-mediated KPTN ubiquitination drives HCC progression via mTORC1 activation.

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FBXO2-mediated KPTN ubiquitination drives HCC progression via mTORC1 act...
(A) KPTN-knockout HuH-7 cells were transduced with lentiviruses expressing WT KPTN or KPTN-4KR mutant, deprived of amino acids and serum for 2 hours, and stimulated with amino acids for 20 minutes. WCLs were analyzed by immunoblotting with the indicated antibodies. Data are representative of at least 2 independent experiments. (B–D) The transduced cells (A) were seeded in 24-well pates for cell proliferation (B) or in 6-well pates to be examined for colony formation in soft agar (C and D). Cell numbers were counted for 4 consecutive days (B). Representative pictures at 4× objective were shown (C), and colonies with diameter > 10 μm were quantified (D). Scale bar, 100 μm. Data are presented as means ± SEM; n = 3 biologically independent repeats; *P < 0.05, ***P < 0.001, ****P < 0.0001, 1-way ANOVA followed by Tukey’s multiple comparisons test. (E–G) The transduced cells in A were subcutaneously injected into nude mice for xenograft growth. The tumor volumes at the indicated days postinoculation (E) and the tumor weights of the last time point (F) were measured, and the tumors of the last time point were photographed (G). Data are presented as means ± SEM, n = 8; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 1-way ANOVA followed by Tukey’s multiple comparisons test. (H) The xenograft tumors (G) were analyzed by immunoblotting with the indicated antibodies. (I) The relative levels of p-S6K1 (H) were quantified. Data are presented as means ± SEM, n = 8; ****P < 0.0001, 1-way ANOVA followed by Tukey’s multiple comparisons test. (J) A model depicting how FBXO2-mediated KPTN ubiquitination promotes amino acid–dependent mTORC1 signaling and tumor growth.