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 2

FBXO2 directly binds to KPTN via its F-box–associated domain.

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FBXO2 directly binds to KPTN via its F-box–associated domain. (A and B) ...
(A and B) HEK293T cells stably expressing Flag-tagged RagA-Q66L (Flag-RagA-Q66L) (A) or NPRL2-knockout cells (B) were transduced with lentiviruses expressing either shCTL or shFBXO2 for 72 hours, deprived of amino acids and serum for 2 hours, and then stimulated with amino acids for 10 minutes. WCLs were analyzed by immunoblotting with the indicated antibodies. (C) Wild-type (WT), KPTN-knockout, or NPRL2-knockout HEK293T cells were transduced with lentiviruses expressing Flag-FBXO2. (D and E) HEK293T cells stably expressing Flag-FBXO2 (D) or KPTN-knockout HEK293T cells (E) were transfected with plasmids containing the indicated genes. WCLs (C–E) were immunoprecipitated with anti-Flag (C) or anti-HA (D and E) magnetic beads, followed by immunoblotting with the indicated antibodies. (F) HEK293T cells were cotransfected with plasmids expressing mCherry-C160–tagged FBXO2 (mCherry-C160-FBXO2) and mCherry-N159–tagged SKP1, KPTN, or Raptor, followed by nucleus staining with DAPI. Representative images of mCherry were shown. Scale bar, 100 μm. (G) HeLa cells stably expressing Flag-FBXO2 were subjected to immunofluorescence assays with the indicated antibodies. Scale bar, 10 μm. (H) Schematic diagram of the domains of FBXO2. FBA, F-box–associated. (I) WCLs of HEK293T cells transfected with plasmids expressing HA-tagged FBXO2 or its mutant FBXO2-Y279A/W280A were incubated with purified GST-tagged KPTN (GST-KPTN) or GST proteins from bacteria, followed by in vitro GST pulldown assays. GST, glutathione-S-transferase. (J) Diagram of FBXO2 interacting with KPTN rather than other components of KICSTOR or GATOR1. (K) WT or KPTN-knockout HEK293T cells were transduced with lentiviruses expressing either shCTL or shFBXO2 for 72 hours, deprived of amino acids and serum for 2 hours, and then 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–E, I, and K).