Laboratory of Veterinary Hygiene, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi, 753-8515, Japan.
Laboratory of Veterinary Hygiene, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi, 753-8515, Japan.
Exp Cell Res. 2022 Sep 1;418(1):113246. doi: 10.1016/j.yexcr.2022.113246. Epub 2022 Jun 10.
Mechanistic target of rapamycin complex 1 (mTORC1) phosphorylates and inhibits eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). This leads to the release of eIF4E from 4E-BP1 and the initiation of eIF4E-dependent mRNA translation. In this study, we examined the expression of a 4E-BP1-based reporter (mTORC1 activity reporter; TORCAR) with various localization signal tags to clarify the relationship between the localization of 4E-BP1 and its phosphorylation. Phosphorylation of 4E-BP1 at threonine 37/46 and serine 65 was efficient at lysosomes and the plasma membrane, whereas it was significantly decreased in the nucleus. In addition, the localization of endogenous eIF4E shifted from the cytoplasm to the nucleus only when nuclear-localized TORCAR was expressed. Nuclear-localized TORCAR decreased cyclin D1 protein levels and altered cell cycle distribution. These data provide an experimental tool to manipulate the localization of endogenous eIF4E without affecting mTORC1 and highlight the important role of nuclear-cytoplasmic shuttling of eIF4E.
雷帕霉素靶蛋白复合物 1（mTORC1）磷酸化并抑制真核起始因子 4E（eIF4E）结合蛋白 1（4E-BP1）。这导致 eIF4E 从 4E-BP1 中释放出来，并启动 eIF4E 依赖性 mRNA 翻译。在这项研究中，我们研究了带有各种定位信号标签的基于 4E-BP1 的报告基因（mTORC1 活性报告基因；TORCAR）的表达，以阐明 4E-BP1 的定位与其磷酸化之间的关系。4E-BP1 在苏氨酸 37/46 和丝氨酸 65 处的磷酸化在溶酶体和质膜中效率很高，而在核中则显著降低。此外，只有表达核定位的 TORCAR 时，内源性 eIF4E 的定位才从细胞质转移到细胞核。核定位的 TORCAR 降低了细胞周期蛋白 D1 蛋白水平并改变了细胞周期分布。这些数据提供了一种实验工具，可以在不影响 mTORC1 的情况下操纵内源性 eIF4E 的定位，并强调了 eIF4E 的核质穿梭的重要作用。
