Fig 2: Phosphorylation of ubiquitin-specific protease-14 (USP14) by Akt activates USP14 DUB activity.(A) Akt activates USP14 DUB activity in vitro. USP14 protein (1 µg) was incubated with or without active Akt (1 µg) in kinase assay buffer in a total volume of 50 µL for 1 hr at 30°C, then the reaction mixtures were subjected to Ub-AMC assay. RFU, relative fluorescence units. (B, C) Akt activates USP14 in cells. USP14 was immunoprecipitated from HEK293T cells coexpressed with activated Akt (B) or treated with 10 µM MK2206 for 4 hr (C) and then eluted with HA-peptide following Ub-AMC hydrolysis assay. (D) Activation of USP14 by stimulating cells with IGF-1. HEK293T cells were serum-starved and pretreated with or without Akt inhibitor MK2206 (1 µM) for 30 min before stimulation with IGF-1 (100 ng/mL) for 30 min. USP14 was then immunoprecipitated and eluted with HA-peptide. The activity of USP14 was determined using Ub-AMC hydrolysis assay. (E) USP14 activation by Akt is blocked by S432A mutation. Ub-AMC hydrolysis assay of wildde type USP14 or S432A mutant in the presence or absence of active Akt. (F) Ub-AMC hydrolysis assay of bacterially expressed and purified wild type USP14 or S432E mutant. (G) Lineweaver–Burk analysis of USP14 S432E, obtained by measuring the initial rates at varying Ub-AMC concentrations (see Figure 3—figure supplement 2E for reference). (H) The activity of phospho-mimetic USP14 mutant can be further stimulated by the presence of proteasome. Ub-AMC hydrolysis assay of wild type USP14 or S432E mutant in the presence or absence of Ub-VS-treated human proteasome (VS-proteasome (see Lee et al., 2010); 1 nM). Ptsm, 26S proteasome.DOI: http://dx.doi.org/10.7554/eLife.10510.007

Fig 3: Purification of USP14 interacting proteins and identification of potential USP14 substrates. a The immunoprecipitation of Flag-USP14 interacting protein was visualized by Coomassie blue staining. b Venn diagram showing the number of downregulated proteins identified in response to USP14 KD (green), USP14 interaction candidates identified in Co-IP process (blue), upregulated ubiquitin sites identified in response to USP14 KD (red) and overlapping proteins in the datasets. The chart below shows potential substrates of the overlapped proteins

Fig 4: USP14 suppresses Vps34 activity by interacting with Beclin 1. (A) USP14 deficiency enhances Vps34 activity. H4-RFP-FYVE cells were transfected with control siRNA or siRNA pools targeting Usp14. Seventy-two hours after transfection, the cells were imaged using an ArrayScan HCS 4.0 reader. Representative cells are shown. The average RFP-FYVE spot intensity in 1000 cells from each indicated sample was determined. The data are displayed as the means ± SEM of the spot intensity per cell. USP14 knockdown efficiency is shown at the right. (B) The Beclin 1-associated Vps34 lipid kinase assay. H4 cells (2 × 107 cells) were lysed after being transfected with either a control siRNA or siRNA pools targeting Usp14 for 72 h, and the cell lysates were incubated with anti-Beclin 1 antibody to precipitate the Beclin 1/Vps34 complex. The immunoprecipitates were separated into two parts: one for input detection and the other for kinase assays. The production of PtdIns3P (PI3P) was detected by protein–lipid blot assay. Lipids were extracted and applied onto a Hybond C-extra membrane. The commercial PtdIns3P was spotted as indicated for control. The levels of PtdIns3P were detected using the GST-PX-p40 domain protein, which binds to PtdIns3P and anti-GST antibody. Ratios of PtdIns3P levels/Vps34 were calculated and are shown. PI3P (0.2 pM) was used as a positive control. (C) USP14 associates with Vps34 complexes. The expression vectors of HA-tagged USP14 and Flag-tagged Beclin 1, Vps34, Atg14L, or UVRAG were cotransfected into HEK293T cells, respectively, and immunoprecipitations (IP) were performed at 24 h after transfection. (D) H4 cells (2 × 107 cells) were lysed, the lysates were analyzed by immunoprecipitation with anti-IgG control or anti-USP14, and the immunocomplexes were analyzed by Western blot analysis. (E) H4 cells with Beclin 1 stable knockdown were virally infected with expression vectors for either control or Flag-tagged Beclin 1 for 12 h and then treated with 50 µM IU1 for the indicated periods of times. The cells were harvested for Western blot analysis. The ratios of LC3-II/GAPDH were calculated and are shown at the right. (F) Mapping the interacting regions of Beclin 1 with USP14. The expression vectors of Flag-tagged Beclin 1 truncations encoding the indicated regions were cotransfected with that of full-length USP14 into HEK293T cells and analyzed by immunoprecipitation and Western blotting.

Fig 5: Identification of FASN as a direct target of USP14. a The lysate from mouse primary hepatocytes (MPH) were incubated with either anti-USP14 antibody or normal rabbit IgG, and the immunocomplexes were probed with the indicated antibodies. b Relative mRNA levels of FASN in MPH overexpressing USP14 or GFP for 48 h. c Representative protein levels of SREBP-1c, SCD-1 and ACC1 in MPH were shown and with the quantification plot based on scanning densitometry analysis (n = 4 for each group). The two lanes are two representative biological replicates. Each lane represents the tissue protein sample from one mouse. ***p < 0.001 versus indicated groups (Student’s t-test). d MPH were transfected with shRNA targeting USP14 or negative control (NC) for 48 h. The protein levels of FASN, USP14 and ß-actin were detected by western blot with the indicated antibodies and relative mRNA levels of FASN were determined by quantitative PCR. e MPH with or without USP14 knockdown were treated with 25 µg/mL cycloheximide (CHX) for the indicated time. The whole cell lysate was detected by western blot with the indicated antibodies. f MPH with or without USP14 overexpression were treated with 25 µg/mL cycloheximide (CHX) for the indicated time. The whole cell lysate was detected by western blot. Data are presented as mean ± SEM