Functional characterization of lysosomal interaction of Akt with VRK2

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.

The FBXW2–MSX2–SOX2 axis regulates stem cell property and drug resistance of cancer cells

“…VRK2 was first identified in 1997 as a putative serine/threonine kinase with structural similarity to vaccinia virus B1R kinase (40), and was recently characterized as a candidate gene for psychiatric and neurological disorders (41). Involvement of VRK2 in human cancer is much less studied, with few reports on its interactions with p53 (42), Bcl-xL (43), and Akt (44) and its inhibition of MAPK (45). How VRK2 expression is regulated under physiological or stressed conditions is completely unknown.…”

“…The Fbxw2 Δ/Δ mice were generated via crossing Fbxw2 fl/fl mice with EIIa-Cre transgenic mice and were viable. Mouse embryonic fibroblasts were generated from embryonic day (E)13.5 embryos of these mice as described (44) and cultured in DMEM with 15% FBS, 2 mM L-glutamine, and 0.1 mM MEM nonessential amino acids. Fbxw2 fl/fl MEFs were…”

Central cellular signaling pathways involved with the regulation of lipid metabolism in the liver: a review

“…Cellular regulation Metabolic effects References AKT -Growth regulation, metabolism control -Glucose metabolism: ↑ glucose transporters on cell surface → glycolysis -↓ FOXO1: ↓ gluconeogenesis -↑ PI3K/AKT/mTOR: biosynthesis of macromolecules -Insulin resistance: FOXO1 activation → ↓ AKT → hyperglycemia and phosphorylation of IRS-1, ↓ GLUT-4 translocation, ↓ glucose transported into cells -Caloric abundance: ↑ mTOR → ↑ lipid droplets Hirata et al, 2018;Józwiak, Forma, Brys, & Krzeslak, 2014;Manning & Toker, 2017;Semenkovich, Goldberg, & Goldberg, 2016;Welty, Alfaddagh, & Elajami, 2016;Zhang et al, 2018 AMPK -↑ demand for energy or caloric restriction: ↓ ATP → ↑ AMP/ATP -SIRT1 acetylation: simulates a caloric depleted state → AMPK activation → ↓ de novo lipogenesis and ↑ insulin sensitivity, lipolysis, and mitochondrial fatty acid (FA) oxidation -TSH → AMPK: ↓ HMG-CoAR and ↓ cholesterol synthesis -↓ insulin sensitivity: ↓ AMPK, defective autophagy and ROS generation -Starvation: ↑ autophagy, to ↑ nutrients available to cells → ↑ ULK1 and mTORC1 -AMPK improves NASH induced by highfat-diet: suppression of several key lipogenic factors, such as SREBP-1 (↓ triglyceride synthesis), HMG-CoAR, cholesterol synthesis; and ↑ ACC:…”

<b><i>VRK2</i></b>, a Candidate Gene for Psychiatric and Neurological Disorders

“…Vrk2 could phosphorylate dysbindin, which will consequently enhance its ubiquitination and decrease protein stability [113]. Moreover, Hirata et al[131] found that Vrk2 interacts with Akt in the lysosomes, and the lysosomal Vrk2-Akt complex controls cellular proliferation and mitochondrial outer-membrane stabilization in the process of autophagy. Intriguingly, AKT1 is also one of the best candidate genes for SCZ [132-137].…”