Article Title: PTEN counteracts FBXL2 to promote IP3R3– and Ca2+–mediated apoptosis limiting tumour growth
Figure Lengend Snippet: IP3R3 is targeted for proteasomal degradation by FBXL2 a , HEK293T cells were transfected with the indicated Flag-tagged F-box proteins (FBPs). 24 h post-transfection, cells were treated with MG132 for 3 h before harvesting for immunoprecipitation and immunoblotting as indicated. WCL, whole-cell lysate from untransfected cells. b , Flag-tagged FBXL2 was stably expressed in HeLa cells. After harvesting, cells were lysed, and cell membranes (CM) were isolated and immunoprecipitated with either a normal mouse IgG or an anti-Flag antibody. Subsequently, immunoprecipitates were analysed by immunoblotting as indicated. The results of these experiments ( n = 2) indicate that FBXL2 is incorporated into a complete SCF ligase that binds its substrate(s) at cellular membranes. c , Flag-tagged FBXL2 or Flag-tagged FBXL2(CaaX/SaaX) were transiently expressed in HeLa cells. After harvesting, cells were lysed and cytoplasmic (CYTO) and cell membrane (CM) fractions were isolated and analysed by immunoblotting as indicated. Actin and calnexin are used as markers for cytoplasmic and cell membrane fractions, respectively. This experiment was performed twice. d , HEK293T cells were transfected with either an empty vector (EV) or the indicated Flag-tagged proteins. 24 h post-transfection, where indicated, cells were treated with MG132 for 3 h before harvesting for immunoprecipitation and immunoblotting. Ned. CUL1, neddylated CUL1. This experiment shows that FBXL2, but not FBXL2(CaaX/SaaX), interacts with endogenous, neddylated CUL1. Since the covalent linkage of NEDD8 to CUL1 stimulates the ubiquitin ligase activity of SCFs and is promoted by the binding of the substrate to the F-box protein subunit, this result suggests that FBXL2 localization to cell membranes is required for substrate binding, which in turn stimulates CUL1 neddylation and SCF activation. Moreover, FBXL2(ΔF-box) bound more IP3R3 than wild-type FBXL2, and this difference could be abolished by treatment with MG132, supporting the hypothesis that FBXL2(ΔF-box) cannot mediate the degradation of IP3R3 since it does not form an active SCF complex. e , HEK293T cells were transfected with either an empty vector or the indicated Flag-tagged proteins. The experiment was performed in the presence or absence of MG132 as indicated. Whole-cell lysates were immunoblotted as indicated. The asterisk indicates a non-specific band. This experiment was performed twice. The fact that proteasome inhibitors prevented the decrease of IP3R3 levels upon re-addition of serum suggests that IP3R3 is degraded by the proteasome in response to mitogens, in agreement with previous studies reporting IP3Rs as substrates of the proteasome 26 , 27 . f , Normal, non-transformed, non-immortalized, human diploid fibroblasts (NHFs) (passage 2) were serum-starved for 72 h and then re-stimulated with serum (SR) for 30 min in the absence or presence of either MG132 (a proteasome inhibitor) or lactacystin (another proteasome inhibitor) as indicated. The graph on the right shows the quantification of IP3R3 levels from three independent experiments. P values were calculated by one-way ANOVA. Error bars indicate s.e.m. g , NHFs (passage 2) were transfected with either three different siRNAs targeting FBXL2 (each independently) or a non-silencing siRNA (NS). The graph shows FBXL2 mRNA levels analysed using realtime PCR in triplicate measurements. Error bars indicate s.e.m. The values represent the ratios between FBXL2 and GAPDH mRNAs. h , During a 72 h serum starvation, NHFs (passage 2 or 3) were transfected with either an siRNA targeting FBXL2 (#1) or a non-silencing siRNA (NS). Cells were subsequently stimulated with medium containing serum and harvested at the indicated times for immunoblotting. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. i , During a 72 h serum starvation, NHFs (passage 3 or 4) were transfected with either siRNAs targeting FBXL2 (oligo #2 or #3) or a non-silencing siRNA (NS). Cells were subsequently stimulated with medium containing serum and harvested at the indicated time points for immunoblotting. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. j , Schematic representation of the FBXL2 genomic locus and gRNAs target location. Exon 2 and exon 3 refer to the human FBXL2 gene in NC_000003.12 (GRCh38.p7 (Gene Bank ID: 3129728)). k , Schematic representation of FBXL2 CRISPR–Cas9 mutagenesis outcomes. A first round of CRISPR– Cas9 gene editing yielded no homozygous FBXL2 -knockout clones. A secondary round of CRISPR–Cas9 gene editing was carried out in three FBXL2 +/− hTERT RPE-1 clones, which resulted in cell death, suggesting that FBXL2 is required for cell fitness and it is not possible to generate FBXL2 -knockout cells. Similar results were obtained in A549 cells (data not shown). l , FBXL2 +/+ and FBXL2 +/− RPE-1-hTERT cells (clones 2 and 3) were serum-starved for 72 h and subsequently stimulated with medium containing serum for 90 min, after which cell extracts were immunoblotted for the indicated proteins. The graph shows the quantification of IP3R3 levels from three independent experiments. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1 .
Article Snippet: Where indicated, cells were treated with 10 μM MG132 (Peptides International), 100 μM cycloheximide (Sigma), 10-15 μM GGTi-2418 (Moffit Cancer Center), 10 μM Eeyarestatin 1 (Eer1, TOCRIS bioscience), 100 μM histamine (Sigma), 10 μM MRS2578 (Sigma), 100 μM ATP (Sigma), and 1 μM cyclosporin-A (Sigma).
Techniques: Transfection, Immunoprecipitation, Stable Transfection, Isolation, Plasmid Preparation, Activity Assay, Binding Assay, Activation Assay, Transformation Assay, Polymerase Chain Reaction, CRISPR, Mutagenesis, Knock-Out, Clone Assay