Division of Cancer Biology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan.
Chemistry Department, Faculty of Science, Tanta University, Tanta, Egypt.
Oncogene. 2018 Oct;37(40):5367-5386. doi: 10.1038/s41388-018-0330-0. Epub 2018 Jun 5.
Serine-threonine kinase Akt (also known as PKB, protein kinase B), a core intracellular mediator of cell survival, is involved in various human cancers and has been suggested to play an important role in the regulation of autophagy in mammalian cells. Nonetheless, the physiological function of Akt in the lysosomes is currently unknown. We have reported previously that PtdIns(3)P-dependent lysosomal accumulation of the Akt-Phafin2 complex is a critical step for autophagy induction. Here, to characterize the molecular function of activated Akt in the lysosomes in the process of autophagy, we searched for the molecules that interact with the Akt complex at the lysosomes after induction of autophagy. By time-of-flight-mass spectrometry (TOF/MS) analysis, kinases of the VRK family, a unique serine-threonine family of kinases in the human kinome, were identified. VRK2 interacts with Akt1 and Akt2, but not with Akt3; the C terminus of Akt and the N terminus of VRK2 facilitate the interaction of Akt and VRK2 in mammalian cells. The kinase-dead form of VRK2A (KD VRK2A) failed to interact with Akt in coimmunoprecipitation assays. Bimolecular fluorescence complementation (BiFC) experiments showed that, in the lysosomes, Akt interacted with VRK2A but not with VRK2B or KD VRK2A. Immunofluorescent assays revealed that VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction. WT VRK2A, but not KD VRK2A or VRK2B, facilitated accumulation of phosphorylated Akt in the lysosomes. Downregulation of VRK2 abrogated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB; these events coincided to inhibition of autophagy induction. The VRK2-Akt complex is required for control of lysosomal size, acidification, bacterial degradation, and for viral replication. Moreover, lysosomal VRK2-Akt controls cellular proliferation and mitochondrial outer-membrane stabilization. Given the roles of autophagy in the pathogenesis of human cancer, the current study provides a novel insight into the oncogenic activity of VRK2-Akt complexes in the lysosomes via modulation of autophagy.
丝氨酸-苏氨酸激酶 Akt（也称为 PKB，蛋白激酶 B）是细胞存活的核心细胞内介质，涉及多种人类癌症，并被认为在哺乳动物细胞自噬的调节中发挥重要作用。尽管如此，Akt 在溶酶体中的生理功能目前尚不清楚。我们之前曾报道过，PtdIns(3)P 依赖性溶酶体积累 Akt-Phafin2 复合物是自噬诱导的关键步骤。在这里，为了描述 Akt 在自噬过程中溶酶体中的激活分子的功能，我们在诱导自噬后，在溶酶体上搜索与 Akt 复合物相互作用的分子。通过飞行时间质谱（TOF/MS）分析，鉴定出 VRK 家族的激酶，VRK 家族是人类激酶组中独特的丝氨酸-苏氨酸激酶家族。VRK2 与 Akt1 和 Akt2 相互作用，但不与 Akt3 相互作用；Akt 的 C 端和 VRK2 的 N 端促进了哺乳动物细胞中 Akt 和 VRK2 的相互作用。激酶失活形式的 VRK2A（KD VRK2A）在免疫沉淀测定中不能与 Akt 相互作用。双分子荧光互补（BiFC）实验表明，在溶酶体中，Akt 与 VRK2A 相互作用，但不与 VRK2B 或 KD VRK2A 相互作用。免疫荧光测定显示，自噬诱导后，VRK2 和磷酸化 Akt 积累在溶酶体中。WT VRK2A，但不是 KD VRK2A 或 VRK2B，促进了磷酸化 Akt 在溶酶体中的积累。VRK2 的下调消除了磷酸化 Akt 的溶酶体积累，并损害了 TFEB 的核定位；这些事件与自噬诱导的抑制同时发生。VRK2-Akt 复合物是控制溶酶体大小、酸化、细菌降解和病毒复制所必需的。此外，溶酶体 VRK2-Akt 控制细胞增殖和线粒体外膜稳定。鉴于自噬在人类癌症发病机制中的作用，本研究通过调节自噬，为 VRK2-Akt 复合物在溶酶体中通过调节自噬发挥致癌活性提供了新的见解。
