MARCH1 通过泛素化 TBK1 负调控 TBK1-mTOR 信号通路。

MARCH1 negatively regulates TBK1-mTOR signaling pathway by ubiquitinating TBK1.

机构信息

The Second Clinical Medical College , Binzhou Medical University, Yantai, Shandong, 264003, P.R. China.

Peninsular Cancer Research Center, Binzhou Medical University, Yantai, Shandong, 264003, P.R. China.

出版信息

BMC Cancer. 2024 Jul 26;24(1):902. doi: 10.1186/s12885-024-12667-y.

DOI:10.1186/s12885-024-12667-y

PMID:39061024

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11282859/

Abstract

BACKGROUND

TBK1 positively regulates the growth factor-mediated mTOR signaling pathway by phosphorylating mTOR. However, it remains unclear how the TBK1-mTOR signaling pathway is regulated. Considering that STING not only interacts with TBK1 but also with MARCH1, we speculated that MARCH1 might regulate the mTOR signaling pathway by targeting TBK1. The aim of this study was to determine whether MARCH1 regulates the mTOR signaling pathway by targeting TBK1.

METHODS

The co-immunoprecipitation (Co-IP) assay was used to verify the interaction between MARCH1 with STING or TBK1. The ubiquitination of STING or TBK1 was analyzed using denatured co-immunoprecipitation. The level of proteins detected in the co-immunoprecipitation or denatured co-immunoprecipitation samples were determined by Western blotting. Stable knocked-down cells were constructed by infecting lentivirus bearing the related shRNA sequences. Scratch wound healing and clonogenic cell survival assays were used to detect the migration and proliferation of breast cancer cells.

RESULTS

We showed that MARCH1 played an important role in growth factor-induced the TBK1- mTOR signaling pathway. MARCH1 overexpression attenuated the growth factor-induced activation of mTOR signaling pathway, whereas its deficiency resulted in the opposite effect. Mechanistically, MARCH1 interacted with and promoted the K63-linked ubiquitination of TBK1. This ubiquitination of TBK1 then attenuated its interaction with mTOR, thereby inhibiting the growth factor-induced mTOR signaling pathway. Importantly, faster proliferation induced by MARCH1 deficiency was weakened by mTOR, STING, or TBK1 inhibition.

CONCLUSION

MARCH1 suppressed growth factors mediated the mTOR signaling pathway by targeting the STING-TBK1-mTOR axis.

摘要

背景

TBK1 通过磷酸化 mTOR 正向调控生长因子介导的 mTOR 信号通路。然而，TBK1-mTOR 信号通路如何被调控仍不清楚。鉴于 STING 不仅与 TBK1 相互作用，还与 MARCH1 相互作用，我们推测 MARCH1 可能通过靶向 TBK1 来调节 mTOR 信号通路。本研究旨在确定 MARCH1 是否通过靶向 TBK1 来调节 mTOR 信号通路。

方法

采用免疫共沉淀（Co-IP）实验验证 MARCH1 与 STING 或 TBK1 的相互作用。采用变性 Co-IP 分析 STING 或 TBK1 的泛素化。通过 Western blot 检测 Co-IP 或变性 Co-IP 样本中的蛋白水平。通过感染携带相关 shRNA 序列的慢病毒构建稳定敲低细胞。划痕愈合和克隆形成细胞存活实验用于检测乳腺癌细胞的迁移和增殖。

结果

我们表明 MARCH1 在生长因子诱导的 TBK1-mTOR 信号通路中发挥重要作用。MARCH1 过表达减弱了生长因子诱导的 mTOR 信号通路的激活，而其缺失则产生相反的效果。机制上，MARCH1 与 TBK1 相互作用，并促进其 K63 连接的泛素化。这种 TBK1 的泛素化减弱了其与 mTOR 的相互作用，从而抑制了生长因子诱导的 mTOR 信号通路。重要的是，MARCH1 缺失引起的更快增殖被 mTOR、STING 或 TBK1 的抑制所削弱。