Fig 2: JNK1-mediated phosphorylation stabilizes ASCT2 by triggering ZDHHC14 degradation.a The lysates of HEK293T cells with ZDHHC14 and indicated JNK members expression were subjected to IP with Flag magnetic beads, and then their interactions were analyzed by western blot. b Representative immunoblotting bands for GST precipitation showing ZDHHC14-JNK1 binding by treating purified GST or GST-ZDHHC14 protein with purified 6His-JNK1 protein in vitro. c NCI-H1299 (left) and NCI-H1975 (right) cells were transfected with stable empty vector or knockdown of JNK1 expression, then cultured in the medium with or without glutamine for 12 h, the expression of ASCT2, ZDHHC14, JNK1, and p-JNK was measured by western blot. d NCI-H1299 (left) and NCI-H1975 (right) cells were treated with DMSO, 10 μM JNK-IN-8, or 10 μM CC-90001 with culturing in the medium with or without glutamine for 12 h, expression of ASCT2 and ZDHHC14 was measured by western blot. e MS analysis of representative ZDHHC14 peptides carrying Thr440 phosphorylation. f Schematic diagram showing that phosphorylation site of ZDHHC14 with conserved residue in Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus, and Felis catus. g For CHX chase assay, ZDHHC14-WT or phosphorylation mutant ZDHHC14T440A was expressed in NCI-H1299 cells, and then cells were treated with 50 μg/mL CHX for the indicated time, the expression level of ZDHHC14 was analyzed by western blot. The band density of ZDHHC14 was quantified and normalized to Actin (mean ± SD, n = 3). The p value was determined by two-way ANOVA. h NCI-H1299 (left) and NCI-H1975 (right) cells were transfected with ZDHHC14-WT or ZDHHC14 phosphorylation mutant ZDHHC14T440A, then cultured in the medium with or without glutamine for 12 h, expression of ASCT2, ZDHHC14, JNK1/2/3, Phospho-JNK1/2/3 was measured by western blot. i NCI-H1299 (left) and NCI-H1975 (right) cells expressing ZDHHC14-WT or ZDHHC14 phosphorylation mutant ZDHHC14T440A were treated with Anisomycin at the indicated concentrations for 12 h, and the expression of ZDHHC14 was analyzed by western blot. j NCI-H1299 cells were co-expressed with ASCT2-Flag and ZDHHC14-WT/ZDHHC14T440A mutant, then cultured in the medium with or without glutamine for 12 h, immunoprecipitation and Alkyne-azide click chemistry experiments examined the palmitoylation level of ASCT2.

Fig 3: ZDHHC14-ASCT2 axis regulates glutamine metabolism and tumorigenesis in NSCLC.a Glutamine uptake and glutamate production in NCI-H1975 (left) and NCI-H1299 (right) cells with stable empty vector, ZDHHC14-WT or catalytically inactive mutant ZDHHC14C195S expression were measured by assays for glutamine uptake (up) and glutamate production (bottom) detection (mean ± SD in three separate experiments). b Cell viability of the ZDHHC14-WT or catalytically inactive mutant ZDHHC14C195S expressing cells was analyzed for the indicated times. Data were presented as a percentage of control cell lines (mean ± SD in three separate experiments). The p value was determined by two-way ANOVA. c, d Representative pictures showed colony formation of NCI-H1299 (left) and NCI-H1975 (right) cells expressing stable empty vector, ZDHHC14-WT, or catalytically inactive mutant ZDHHC14C195S (c). Data were presented as a percentage of empty vector cell lines (mean ± SD in three separate experiments) (d). The p value was determined by one-way ANOVA. e–g NCI-H1299 cells expressing stable empty vector, ZDHHC14-WT or catalytically inactive mutant ZDHHC14C195S were injected subcutaneously into the flanks of Balb/c nude mice. The growth of tumors was examined every 4 days. Images of subcutaneous xenografts from the indicated NCI-H1299 cells; n = 6 mice per group (e). Tumor size and weight were recorded (f, g). Data were shown as mean ± SEM from six mice. The p value of tumor size was determined by two-way ANOVA, the p value of tumor weight was determined by one-way ANOVA. h–j Images (h), weight (i), and tumor size (j) of subcutaneous xenografts from the indicated NCI-H1299 cells; n = 6 mice per group. Data were shown as mean ± SEM from six mice. The p value of tumor size was determined by two-way ANOVA, the p value of tumor weight was determined by one-way ANOVA. k Analysis of the TCGA dataset for the expression of ASCT2 (left) and ZDHHC14 (right). The p value was determined by paired t-test. l Representative images of ASCT2 and ZDHHC14 IHC staining in consecutive NSCLC tissues. Scale bar = 100 μm.

Fig 4: Cys39 and Cys48 sites of ASCT2 palmitoylation are mainly involved in lysosomal-mediated protein degradation.a Schematic diagram showing the ASCT2 structure with multiple transmembrane domains and eight possible palmitoylation sites. b LC-MS/MS spectra of representative ASCT2 peptides carrying palmitoylated Cys39 residue. c Expression of empty vector, WT ASCT2, or each cysteine residue mutation in HEK293T cells lysates was tested by immunoprecipitation, and Alkyne-azide click chemistry experiments. Palmitoylation levels of ASCT2 with or without hydroxylamine (NH2OH) treatment were detected by in-gel fluorescence with silver staining the same gel. d WT-ASCT2 or ASCT2C39S/C48S mutants were expressed in HEK293T cells, and their palmitoylation levels were determined by Alkyne-azide click chemistry experiments. e ZDHHC14-mediated palmitoylation of WT or C39S/C48S mutant ASCT2 was assessed in HEK293T cells by immunoprecipitation and Alkyne-azide click chemistry. f Schematic diagram showing that palmitoylation sites of ASCT2 with conserved cysteine residues in Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus, and Felis catus. g, h NCI-H1975 cells stably expressing WT ASCT2 or various ASCT2 mutants were treated with 50 μg/mL CHX for the indicated time, and then the expression level of ASCT2 was analyzed by western blot (g). The band density of ASCT2 was quantified and normalized to Actin (mean ± SD, n = 3) (h). The p value was determined by two-way ANOVA. i Localization of ASCT2 and LAMP1 was measured by immunofluorescent staining in WT-ASCT2 or ASCT2C39S/C48S mutant expression NCI-H1975 cells treated with BafA1, with statistical results shown on the right. Values are mean ± SD from five images (n = 5). The p value was determined by two-tailed Student’s t-test. Nuclei were visualized with DAPI (blue). Scale bar = 10 μm.

Fig 5: ZDHHC14 promotes ASCT2 palmitoylation and lysosome degradation.a NCI-H1299 cells were cultured with 2-BP at the indicated concentrations for 24 h. The media were collected for analysis of glutamine uptake (left), and the cells were collected for analysis of glutamate production(right). b Table showing the palmitoylations of glutamine metabolism regulators identified by Palmitoylation LFQ Proteomics. c NCI-H1299 (top) and NCI-H1975 (bottom) cells were treated with 50 μM 2-BP at the indicated time and subjected to western blot with ASCT2 and Actin antibodies. d ASCT2-HA expression HEK293T cells were cultured in medium containing 50 μM 2-BP and Alk14 for 8 h, and the cell lysates were prepared for immunoprecipitation and Alkyne-azide click chemistry experiments. Palmitoylation levels of ASCT2 with or without hydroxylamine (NH2OH) treatment were detected using Alk14 labeling by in-gel fluorescence detection, with silver staining the same gel to ensure consistent loading. e NCI-H1299 cells were harvested for co-IP assays using anti-IgG or anti-ASCT2 antibodies and then analyzed by western blot. f Exogenous ASCT2 and ZDHHC14 were stained by immunofluorescence assay in NCI-H1299 cells. DAPI, nucleus. Scale bar = 10 μm. Intensity traces (yellow lines) were plotted below. g HEK293T cells were co-transfected with ASCT2-HA and increasing amounts of ZDHHC14-Myc plasmid, immunoprecipitation and Alkyne-azide click chemistry experiments tested the cysteine palmitoyltransferase activity of ZDHHC14 on ASCT2. h, i NCI-H1299 cells with ZDHHC14-Myc (h) or catalytically inactive mutant ZDHHC14C195S-Myc (i) overexpression were incubated with 50 μg/mL cycloheximide (CHX) in a time-course frame, respectively. The collected cell lysates were subjected to immunoblotting detection with ASCT2, Myc, and Actin antibodies. The band density of ASCT2 was quantified and normalized to Actin (mean ± SD, n = 3). The p value was determined by two-way ANOVA. j NCI-H1299 ZDHHC14-knockout cells were treated with 50 μg/mL CHX for the indicated time. The collected cell lysates were subjected to western blot with ASCT2, Myc, and Actin antibodies. The band density of ASCT2 was quantified and normalized to Actin (mean ± SD, n = 3). The p value was determined by two-way ANOVA. k NCI-H1299 (left) and NCI-H1975 (right) cells with stable empty vector or ZDHHC14 expression were incubated with DMSO, 0.1 μM bafilomycin A1 (BafA1) or 10 μM MG132 for 6 h. The collected cell lysates were measured using ASCT2 and Actin antibodies. l Representative immunofluorescence pictures showing the co-localization of endogenous ASCT2 and lysosome-associated membrane protein-1 (LAMP1) in NCI-H1299 cells treated with BafA1, with statistical results shown on the right. Values are mean ± SD from five images (n = 5). The p value was determined by two-tailed Student’s t-test. DAPI, nucleus. Scale bar = 10 μm.