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 1

FBXO2 promotes amino acid–induced mTORC1 activation.

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FBXO2 promotes amino acid–induced mTORC1 activation. (A and B) HEK293T c...
(A and B) HEK293T cells stably expressing shRNAs against FBXO2 (shFBXO2) (A) or FBXO2-knockout cells (B) were deprived of amino acids and serum for 2 hours, then stimulated with amino acids for 10 minutes. Whole cell lysates (WCLs) were analyzed by immunoblotting with the indicated antibodies. AAs, amino acids. (C) HEK293T cells stably expressing shFBXO2 were transduced with lentiviruses expressing Flag-FBXO2-rsm (resistant to shFBXO2). WCLs of the cells treated as in (A and B) were analyzed by immunoblots. (D and E) HEK293T cells stably expressing the indicated shRNAs were deprived of amino acids and serum for 2 hours, then stimulated with amino acids for 10 minutes, followed by immunofluorescence analysis with the indicated antibodies. Representative images were shown (D), and colocalization of mTOR with LAMP2 was quantified (E). Scale bar, 10 μm. Insets were digitally zoomed. (F and G) HEK293T cells stably expressing EGFP-tagged TFEB (EGFP-TFEB) were transduced with lentiviruses expressing the indicated shRNAs for 72 hours. The transduced cells were deprived of amino acids and serum for 2 hours, then stimulated with amino acids for 2 hours, followed by nucleus staining with DAPI. Representative images of EGFP-TFEB localization were shown in F, and the quantitative results of EGFP-TFEB localization were presented in G. Scale bar, 10 μm. Data are presented as means ± SEM; n = 9 independent fields per condition; ****P < 0.0001, 1-way ANOVA followed by Tukey’s multiple comparisons test (E and G). Data are representative of at least 2 independent experiments (A–C).