异常调控细胞生长和代谢的机械靶向雷帕霉素(mTOR)途径在多种癌症类型中受到异常调控。mTOR复合物2(mTORC2)由核心成分mTOR、Rictor、mSin1和mLST8组成，主要响应生长信号。然而，mTORC2组装和活性之间的协调关系仍不清楚。Keap1作为细胞中氧化应激的主要传感器，是Cullin 3-RING E3泛素连接酶(KRL3)的底物适配体，促进了NF-E2相关因子2(NRF2)的蛋白酶体降解，后者是一种保护细胞免受氧化和电子亲合应激的转录因子。在本研究中，我们证明Keap1通过一个保守的ETGE基序与mLST8结合。CRL3Keap1泛素连接酶复合物促进了mLST8的非降解性泛素化，从而降低了mTORC2复合物的完整性和mTORC2-AKT激活。然而，这种效应可以被氧化/电子亲合应激和生长因子信号诱导的活性氧自由基(ROS)阻断。癌症源性Keap1或mLST8突变会破坏Keap1-mLST8相互作用，使mLST8得以逃脱Keap1介导的泛素化，从而增强mTORC2-AKT的活化，促进细胞恶性化和改变细胞代谢。我们的研究结果揭示了Keap1/mLST8突变驱动的肿瘤发生机制，通过促进mTORC2-AKT的活化，独立于经典的NRF2途径。版权所有 © 2023 The Authors. Published by Elsevier B.V. All rights reserved.

The mechanistic target of the rapamycin (mTOR) pathway, which participates in the regulation of cellular growth and metabolism, is aberrantly regulated in various cancer types. The mTOR complex 2 (mTORC2), which consists of the core components mTOR, Rictor, mSin1, and mLST8, primarily responds to growth signals. However, the coordination between mTORC2 assembly and activity remains poorly understood. Keap1, a major sensor of oxidative stress in cells, functions as a substrate adaptor for Cullin 3-RING E3 ubiquitin ligase (CRL3) to promote proteasomal degradation of NF-E2-related factor 2 (NRF2), which is a transcription factor that protects cells against oxidative and electrophilic stress. In the present study, we demonstrate that Keap1 binds to mLST8 via a conserved ETGE motif. The CRL3Keap1 ubiquitin ligase complex promotes non-degradative ubiquitination of mLST8, thus reducing mTORC2 complex integrity and mTORC2-AKT activation. However, this effect can be prevented by oxidative/electrophilic stresses and growth factor signaling-induced reactive oxygen species (ROS) burst. Cancer-derived Keap1 or mLST8 mutations disrupt the Keap1-mLST8 interaction and allow mLST8 to evade Keap1-mediated ubiquitination, thereby enhancing mTORC2-AKT activation and promoting cell malignancy and remodeling cell metabolism. Our findings provide new insights into the molecular mechanisms of Keap1/mLST8 mutation-driven tumorigenesis by promoting mTORC2-AKT activation, which is independent of the canonical NRF2 pathway.Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.