g, h HEK293 cells (g) and value? ?0.05) (Supplementary Fig.?S9a). enzyme ovarian tumor domain-containing protein 5 (OTUD5) as a novel positive regulator of the mTOR complex (mTORC) 1 and 2 signaling pathways. We exhibited that OTUD5 stabilized -transducin repeat-containing protein 1 (TrCP1) proteins via its deubiquitinase (DUB) activity, leading to the degradation of Disheveled, Egl-10, and pleckstrin domain-containing mTOR-interacting protein (DEPTOR), which is an inhibitory protein of mTORC1 and 2. We also showed that mTOR directly phosphorylated OTUD5 and activated its DUB activity. RNA sequencing analysis revealed that OTUD5 regulates the downstream gene expression of mTOR. Additionally, OTUD5 depletion elicited several mTOR-related phenotypes such as decreased cell size and increased autophagy in mammalian cells as well as the suppression of a (10?g) were incubated with or without mTOR wild type or KD mutant precipitates in mTOR kinase reaction buffer (without [-32P] ATP) at 30?C for 2?h as described in the In Vitro mTOR Kinase Assay subsection of the Materials and methods section. The reactants were separated via SDS-PAGE. The gel was stained with Coomassie blue so that the OTUD5 bands could be visualized for gel excision. The phosphopeptides from the OTUD5 bands were analyzed Mogroside IV by BioCon (Seoul, Republic of Korea) using liquid chromatography-mass spectrometry. Flag-OTUD5 purification from mammalian cells HEK293T cells were transfected with pcDNA3.1-Flag-OTUD5-wild type, -S177A, and -3SA mutant constructs. After 72?h incubation, the cells were lysed and the Flag-OTUD5 proteins were immunoprecipitated with anti-DYKDDDDK magnetic beads. The immune complexes were incubated at 4?C for 6?h with Flag elution buffer (25?mM HEPES, pH 7.5, 50?mM NaCl, 1?mM EDTA, 10% glycerol, 250?g 3??Flag peptide [Sigma Aldrich], phosphatase inhibitor cocktail [Roche]) without protease inhibitor to elute Mogroside IV the bound proteins from the beads. Then, the Mouse monoclonal antibody to RanBP9. This gene encodes a protein that binds RAN, a small GTP binding protein belonging to the RASsuperfamily that is essential for the translocation of RNA and proteins through the nuclear porecomplex. The protein encoded by this gene has also been shown to interact with several otherproteins, including met proto-oncogene, homeodomain interacting protein kinase 2, androgenreceptor, and cyclin-dependent kinase 11 protein concentration was determined by the Bradford method. In vitro DUB assay Various types of Flag-OTUD5 proteins (100?nM) were reacted with 1?M Ub-AMC (Boston Biochem, Cambridge, MA, USA) in DUB buffer (50?mM Tris-HCl, pH 8.0, 100?mM NaCl, 1% glycerol, 5?mM DTT) at room temperature. The samples fluorescence was read at an excitation wavelength of 345?nm and an emission wavelength of 445?nm every 10?min for 2?h in VICTORTM X Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). RNA sequencing Total RNA was extracted from for 3?min, the cells were washed once with 1% FBS/PBS, and the pellets were resuspended in 75% ethanol/PBS and incubated at 4?C for 12?h for fixation. The fixed cells were centrifuged at 200??g for 3?min and washed once with 1% FBS/PBS. Thereafter, they were incubated with 1% FBS/PBS made up of 0.1% Triton X-100 and 250?g/ml RNase A at 37?C for 30?min. After incubation, the cells were stained with 20?g propidium iodide and analyzed by fluorescence-activated cell sorting. Travel stocks used Following fly stocks were obtained from Vienna Resource Center (Vienna, Austria): (v27588), (v27589), and (v109912). (BL9688) was obtained from Bloomington Stock Center (Bloomington, IN, USA). was kindly provided by Prof. J. Chung (Seoul National University, Republic of Korea). Immunofluorescence microscopy HeLa cells were seeded in -Slide 8-well chamber slides (#80826, Ibidi, Gr?felfing, Germany) at a density of 5??104 cells/well. The following day, the cells were washed with warm PBS and then fixed in 4% paraformaldehyde in PBS for 20?min at room heat. After a wash-out with PBS, the cells were mounted in Fluoroshield Mounting Medium with DAPI (ab104139, Abcam). The fluorescence signals were measured using a ZEISS LSM 880 laser scanning microscope. Proliferation assay A cell proliferation assay was performed using a CellTiter-Glo? Luminescent Cell Viability Assay Kit (Promega, Madison, WI, USA) according to the manufacturers protocol. Statistical analysis All experiments were performed in replicate or triplicate and representative results were presented where data were expressed as mean??SD mentioned in physique legends. Variance between groups statistically compared was comparable. All statistical analyses were carried out using Microsoft Excel program. Two-tailed students test was used to compare the difference between groups. Results OTUD5 is usually a positive regulator of mTORC1 and mTORC2 signaling pathways To identify DUBs that were involved in mTORC1 and mTORC2 signaling pathways, we examined the Mogroside IV activity of mTOR signaling using HEK293 stable cell lines that expressed short hairpin RNAs (shRNAs) targeting.