Fig 2: Mapping of the FBXL2-binding domain in IP3R3, and evidence that the N-terminal suppressor domain inhibits the IP3R3-FBXL2 interactiona, Schematic representation of IP3R3 mutants. Binding of IP3R3 to FBXL2 is indicated with the symbol (+). b, HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated Flag-tagged IP3R3 truncated mutants. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin and proteins were immunoblotted as indicated. This experiment was performed twice. c, HEK293T cells were transfected with GFP-tagged FBXL2 and HA-tagged In IP3R3(1-602) constructs. Sixteen hours after transfection, cells were incubated with MG132 for 3 h before stimulation with ATP for 30 min. Whole-cell lysates were immunoprecipitated (IP) with an anti-HA resin and proteins were immunoblotted as indicated. This experiment was performed twice. d, HeLa cells stably transfected with Flag-tagged FBXL2 under the control of a doxycycline-inducible promoter were treated with doxycycline (0.4 μg ml−1) for 16 h and incubated with or without MG132 (during the last 3 h), in the presence or absence of MRS 2578, an antagonist of P2Y6 receptor (during the last 90 min), as indicated. Cells were subsequently treated with or without ATP for 30 min. Whole-cell lysates were immunoprecipitated (IP) with an anti-Flag resin and proteins were immunoblotted as indicated. Right panel shows quantifications of IP3R3 levels compared to untreated cells (UT), which were set as 100%. P values were calculated by one-way ANOVA and multiple-comparisons test. Error bars indicate s.e.m. The fact (shown in b) that fragments encoding IP3R3(436–587) and IP3R3(227–602) interact with FBXL2 better than the IP3R3(1–602) fragment suggests that the N terminus of IP3R3 inhibits the interaction between FBXL2 and IP3R3. It has been shown that removal of the N-terminal suppressor domain (amino acids 1–226) increases the binding of IP3 to the IP3 binding core domain (amino acids 227–579), and IP3 binding evokes conformational changes that open the suppressor domain29–32. These conformational changes may allow FBXL2 to access the degron of IP3R3 (that is, the amino acid region required for binding to FBXL2). Thus, in agreement with previous data indicating that IP3 causes the ubiquitination and downregulation of IP3Rs (ref. 27), our results suggest that IP3 promotes the binding of FBXL2 to IP3R3 and that FBXL2 preferentially binds IP3R3 in its open conformation (upon IP3 binding). Accordingly, treatment of cells with ATP, which induces IP3 production and repositioning of the N-terminal suppressor domain, increased the binding between FBXL2 and IP3R3, particularly if proteasomal degradation was inhibited by MG132. This suggests that once IP3 (produced after ATP stimulation) unmasks the IP3R3 degron, FBXL2 binds IP3R3 and this interaction is preserved when FBXL2-mediated degradation of IP3R3 is inhibited. So, ATP and MG132 appear to synergize with each other in promoting and preserving the FBXL2–IP3R3 interaction, respectively. e, Serum-starved (SS) and exponentially (EXP) growing NHFs (passage 2 and 3) were treated with or without ATP as indicated. Cells were subsequently harvested at the indicated time points for immunoblotting. The graph shows the quantification of IP3R3 levels. Error bars indicate s.e.m. f, HEK293T cells were transfected with GFP-tagged FBXL2 and the indicated Flag-tagged IP3R3 deletion mutants (in the context of the 436–587 domain of IP3R3). Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin, and proteins were immunoblotted as indicated. This experiment was performed twice. g, The experiment was performed twice as in f, except that different IP3R3 mutants were used. h, Alignment of the amino acid regions containing the FBXL2-binding motif in IP3R3 orthologues. i, Alignment of the amino acid regions containing the FBXL2-binding motif in human IP3R3 with the corresponding region in IP3R1 and IP3R2. j, HEK293T cells were transfected with the indicated constructs. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin and immunoblotted as indicated. The bracket on the right marks a ladder of bands corresponding to polyubiquitinated IP3R3. This experiment was performed twice. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1.

Fig 3: A non-degradable IP3R3 mutant sensitizes tumour cells to photodynamic therapya, Schematic representation of the ITPR3 genomic locus and gRNA target location. Exon 15 refers to human ITPR3 gene (GRCh38.p7 (Gene Bank ID: 31297280)). Silent mutations are shown as lower case letters and indicated by asterisks. b, Wild-type genomic DNA template and knock-in mutant sequences identified by TOPO-TA cloning of ITPR3 PCR from three independent A549 and PC3 clones are depicted. c, Schematic representation of the ITPR3 CRISPR–Cas9 mutagenesis outcomes for A549 and PC3 cells. d, Whole-cell lysates from A549 parental cells and three independent IP3R3(Q-FR/A-AA) knock-in clones were immunobloted as indicated. e, A549 parental cells and IP3R3(Q-FR/A-AA) knock-in cells (clones 1 and 3) were incubated with cycloheximide (CHX) for the indicated times. Cells were subsequently harvested for immunoblotting as indicated. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. f, Whole-cell lysates from PC3 parental cells and an IP3R3(Q-FR/A-AA) knock-in clone (#1) were immunoblotted as indicated. g, PC3 parental cells and the IP3R3(Q-FR/A-AA) knock-in PC3 clone 1 were treated with phthalocyanine, a photosensitizer used for PDT in patients with cancer. Left panel, cytosolic Ca2+ concentrations measured with Fura-2. Middle panel, quantifications of areas under the curve represented as percentage increase compared to empty-vector-transfected cells, which were set as 100%. Right panel, immunoblots of cell lysates from a representative experiment. The P value was calculated by unpaired t-test. Error bars indicate s.e.m. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1.

Fig 4: PTEN competes with FBXL2 for the binding to IP3R3, and the levels of IP3R3 and PTEN directly correlate in cancer cell lines and human prostate tumoursa, Left, schematic representation of IP3R3 mutants used for binding site mapping. Binding of IP3R3 to FBXL2 and PTEN is indicated with the symbol (+). Right, HEK293T cells were transfected with GFP-tagged PTEN and the indicated HA-tagged IP3R3 truncated mutants. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-HA resin and immunocomplexes were probed with antibodies to the indicated proteins. This experiment was performed twice. b, HEK293T cells were transfected with GFP-tagged IP3R3 or IP3R3(Q-FR/A-AA). Whole-cell lysates were immunoprecipitated (IP) with an anti-GFP resin and proteins were immunoblotted as indicated. c, HEK293T cells were co-transfected, as indicated, with GFP-tagged PTEN, HA-IP3R3(436–587), and increasing amounts of Flag-tagged FBXL2. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-HA resin and proteins were immunoblotted as indicated. This experiment was performed twice. d, HEK293T cells were co-transfected, as indicated, with GST-tagged PTEN, GFP-tagged IP3R3, and increasing amounts of Flag-tagged FBXL2. Whole-cell lysates were immunoprecipitated (IP) with anti-GFP resin and proteins were immunoblotted as indicated. This experiment was performed twice. e, HEK293T cells were co-transfected, as indicated, with Flag-tagged FBXL2, HA-tagged IP3R3(436–587), and increasing amounts of either GFP-tagged PTEN or the indicated GFP-tagged cancer-associated PTEN mutants. Whole-cell lysates were immunoprecipitated (IP) with anti-HA resin and proteins were immunoblotted as indicated. This experiment was performed twice. f, HEK293T cells were transfected with either an siRNA targeting PTEN or a non-silencing siRNA (NS), as indicated. After 48 h, cells were transfected with Flag-tagged FBXL2. Sixteen hours after the second transfection, whole-cell lysates were immunoprecipitated (IP) with anti-Flag resin. Immunocomplexes and WCLs were immunoblotted as indicated. g, Whole-cell lysates from the indicated cancer cell lines were immunoblotted as indicated. This experiment was performed twice. h, FBXL2 mRNA levels in the indicated cell lines analysed using real-time PCR in triplicate measurements (± s.e.m.). The values represent the ratios between FBXL2 and GAPDH mRNAs. This experiment was performed once. i, H460 and A549 cells were incubated with cycloheximide (CHX) for the indicated times. Cells were subsequently harvested and processed for immunoblotting as indicated. Long (l.e.) and short (s.e.) exposures are shown for IP3R3. The graph shows the quantification of IP3R3 levels from three independent experiments. Error bars indicate s.e.m. j, U87 and A549 cells were treated with GGTi-2418 for 16 h where indicated. Cells were subsequently harvested and whole-cell lysates were immunoblotted as indicated. This experiment was performed twice. k, Representative immunohistochemistry staining images of human prostate tumour specimens with no, low or high levels of PTEN protein. Levels of IP3R3 in consecutive tissue slides are shown. Scale bars correspond to 50 μm. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1.

Fig 5: IP3R3 degradation is prevented by PTEN, independently of its phosphatase activitya, HEK293T cells were transfected with the indicated Flag-tagged constructs encoding tumour suppressors and oncoproteins with established roles in Ca2+ homeostasis and that are known to localize to the endoplasmic reticulum-mitochondria interface (AKT1, BCL2, p53, PML4, PTEN and KRAS4B). Of those, AKT1, BCL2, PML4 and PTEN are known IP3R3 interactors33–39. Twenty-four hours post-transfection, cells were harvested for anti-Flag immunoprecipitations and immunoblotting as indicated. Asterisks denote Flag-tagged proteins. This experiment was performed twice. b, c, Whole-cell lysates (WCL) from HEK293T cells were immunoprecipitated with the indicated antibodies and immunoblotted as indicated. This experiment was performed twice. d, Pten+/+ and Pten−/− MEFs were incubated with cycloheximide (CHX) for the indicated times. Cells were subsequently harvested for immunoblotting as indicated. The graph on the right shows the quantification of IP3R3 levels from two independent experiments. e, FBXL2 mRNA levels in Pten+/+ and Pten−/− MEFs analysed using real-time PCR in triplicate measurements (± s.e.m.). The values represent the ratios between FBXL2 and GAPDH mRNAs. This experiment was performed twice. f, Pten+/+ and Pten−/− MEFs were transfected for 48 h with either an siRNA targeting FBXL2 (#1) or a non-silencing siRNA (NS). Subsequently, MEFs were transfected with Flag-TR-TUBE cDNA. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-Flag resin and immunoblotted as indicated. The bracket on the right marks a ladder of bands corresponding to polyubiquitinated IP3R3. This experiment was performed twice. g, U87 and A549 cells were transfected with either GFP-tagged PTEN, GFP-tagged PTEN(C124S), or an empty vector (EV) as indicated. Cells were subsequently harvested and whole-cell lysates were immunoblotted as indicated. Long (l.e.) and short (s.e.) exposures are shown for IP3R3. h, Pten−/− MEFs were processed as in (Fig. 2c). Concentrations of mitochondrial Ca2+ were measured with mitochondria-targeted aequorin in response to agonist stimulation (ATP). Top, representative traces. Bottom, quantification of three independent experiments. P values were calculated by one-way ANOVA and multiple comparisons test. Error bars indicate s.e.m. i, HEK293T cells were transfected with either GFP-tagged wild-type PTEN or the indicated GFP-tagged cancer-associated PTEN mutants. Whole-cell lysates (WCL) were immunoprecipitated (IP) with anti-GFP resin and proteins were immunoblotted as indicated. This experiment was performed twice. Unless otherwise noted, experiments were performed at least three times. For gel source data, see Supplementary Fig. 1.