Park Sujin, Kim Chaewon, Heo Sukyeong, Kang Dongmin
Department of Life Science, Fluorescence Core Imaging Center and Bioimaging Data Curation Center, Ewha Womans University, Seoul 03760, Republic of Korea.
Department of Biomedical Engineering, Dongguk University, Seoul 10326, Republic of Korea.
Antioxidants (Basel). 2025 May 16;14(5):594. doi: 10.3390/antiox14050594.
Receptor-mediated endocytosis (RME) is a commonly recognized receptor internalization process of receptor degradation or recycling. However, recent studies have supported that RME is closely related to signal propagation and amplification from the plasma membrane to the cytosol. Few studies have elucidated the role of HO, a mild oxidant among reactive oxygen species (ROS) in RME and second messenger of signal propagation. In the present study, we investigated the regulatory function of HO in early endosomes during signaling throughout receptor-mediated endocytosis. In mammalian cells with a physiological amount of HO generated during epidermal growth factor (EGF) activation, fluorescence imaging showed that the levels of two activating phosphorylations on Ser and Thr of Akt were transiently increased in the plasma membrane, but the predominant p-Akt on Ser appeared in early endosomes. To examine the role of endosomal HO molecules as signaling mediators of Akt activation in endosomes, we modulated endosomal HO through the ectopic expression of an endosomal-targeting catalase (Cat-Endo). The forced removal of endosomal HO inhibited the Akt phosphorylation on Ser but not on Thr. The levels of mSIN and rictor, two components of mTORC2 that work as a kinase in Akt phosphorylation on Ser, were also selectively diminished in the early endosomes of Cat-Endo-expressing cells. We also observed a decrease in the endosomal level of the adaptor protein containing the PH domain, the PTB domain, and the Leucine zipper motif 1 (APPL1) protein, which is an effector of Rab5 and key player in the assembly of signaling complexes regulating the Akt pathway in Cat-Endo-expressing cells compared with those in normal cells. Therefore, the HO-dependent recruitment of the APPL1 adaptor protein into endosomes was required for full Akt activation. We proposed that endosomal HO is a promoter of Akt signaling.
受体介导的内吞作用（RME）是一种普遍认可的受体内化过程，用于受体降解或再循环。然而，最近的研究表明，RME与从质膜到细胞质溶胶的信号传播和放大密切相关。很少有研究阐明羟基自由基（HO）作为活性氧（ROS）中的一种温和氧化剂在RME和信号传播的第二信使中的作用。在本研究中，我们研究了HO在整个受体介导的内吞作用信号传导过程中对早期内体的调节功能。在表皮生长因子（EGF）激活过程中产生生理量HO的哺乳动物细胞中，荧光成像显示，Akt的丝氨酸和苏氨酸上的两种激活磷酸化水平在质膜中短暂升高，但丝氨酸上的主要p-Akt出现在早期内体中。为了研究内体HO分子作为内体中Akt激活的信号介质的作用，我们通过内体靶向过氧化氢酶（Cat-Endo）的异位表达来调节内体HO。内体HO的强制去除抑制了丝氨酸而非苏氨酸上的Akt磷酸化。mTORC2的两个成分mSIN和rictor作为Akt丝氨酸磷酸化的激酶，在表达Cat-Endo的细胞的早期内体中也选择性降低。我们还观察到，与正常细胞相比，在表达Cat-Endo的细胞中，含有PH结构域、PTB结构域和亮氨酸拉链基序1（APPL1）蛋白的衔接蛋白的内体水平降低，APPL1蛋白是Rab5的效应器，也是调节Akt途径的信号复合物组装中的关键参与者。因此，APPL1衔接蛋白依赖HO募集到内体中是Akt完全激活所必需的。我们提出内体HO是Akt信号传导的促进剂。
