Abstract
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.
Highlights
Serine–threonine kinase Akt, a major downstream effector of the phosphatidylinositol-3 kinase (PI3K) pathway, regulates diverse cellular processes, including antiapoptotic processes, proliferation, the cell cycle, cytoskeletal organization, vesicle trafficking, and glucose transport [1,2,3,4]
VRK2A interacts with Akt in the lysosomes
Colocalization of Bimolecular fluorescence complementation (BiFC) (WT VRK2 and WT Akt, green signal) and p-Ser473 Akt was seen only in the cells transfected with the combination of WT VRK2A and WT Akt in the lysosomes (LAMP2, red signal), with the relative intensities of these signals (Fig. 3h–l) and quantification presented as a bar graph (Fig. 3m). These results suggested that the kinase activities of both VRK2A and Akt are important for formation of the Akt–VRK2 complex and for lysosomal accumulation of activated Akt
Summary
Serine–threonine kinase Akt, a major downstream effector of the phosphatidylinositol-3 kinase (PI3K) pathway, regulates diverse cellular processes, including antiapoptotic processes, proliferation, the cell cycle, cytoskeletal organization, vesicle trafficking, and glucose transport [1,2,3,4]. Genetic and functional alterations of the Akt signaling pathways underlie the pathogenesis of a wide variety of human oncological diseases, glucose intolerance, viral infections, and autoimmune diseases [3,4,5]. Autophagy is an evolutionarily conserved mechanism in diverse life forms ranging from yeast to mammalian cells; it facilitates degradation and recycling of cellular components during cellular stress, such as nutrient starvation [6,7,8]. Autophagy is thought to underlie various processes in oncological diseases thereby modulating initiation and/or maintenance of cancers [14,15,16,17,18,19,20]
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