Fig 2: Co-recruitment network predicts the occurrence of physical association between pairs of recruited proteins ACBLB first-neighbors network within the CBL CN. Edge width is coded according to Pearson correlation coefficient (R < 0.8 or R > 0.8). The identified edges are depicted in red if they have been already reported as direct interaction partners in the BioGRID database. Proteins detected in the CBLB signalosome shown in Fig 3 are colored in red.BHistograms showing the number and corresponding percentage of CBL interactors also present in the CBLB signalosome according to their distance from CBLB in the CBL CN. The two depicted analyses correspond to the complete and to the high-confidence (R > 0.8) CNs.CZoom on the high-confidence (R > 0.8) CRKL and CSK first-neighbors subnetwork found within the CBL CN. The identified edges are depicted in red if they have been already reported as direct interaction in the BioGRID database. CSK and CRKL are highlighted in yellow.D–GExperimental validation of the PPIs predicted on the basis of the high-confidence CN shown in (C). CD4+ T cells were left unstimulated (-) or stimulated (+) with anti-CD3 and anti-CD4 antibodies and subsequently lysed. Equal amounts of cell lysates were incubated with isotype control (Iso Ctrl) or the specified antibodies and the resulting immunoprecipitates (IP) analyzed by immunoblot with the antibodies specified in the left margin. The validated PPIs predicted in (C) correspond to CSK-CBLB and CSK-CD5 (D), CRKL-PI3Kp85ß and CSK-PI3Kp85ß (E), PI3Kp85ß-PI3Kp110a and CSK-PI3Kp110a (F), and CRKL-PI3Kp110a (G). Molecular masses are shown (kDa). Data are representative of at least two experiments.HSummary of the PPIs found in the high-confidence CSK and CRKL first-neighbors subnetwork (see C) that were validated by coimmunoprecipitation (see D–G). Interactions validated and not validated by coimmunoprecipitation are shown using red and blue edges, respectively. Interactions untractable by coimmunoprecipitation analysis due to the lack of specific antibodies are shown as gray edges.Data information: In (C) and (H), PI3Kp85ß and PI3Kp110a are denoted as PIK3R2 and PIK3CA, respectively.

Fig 3: Asymmetric functional relationships between CBL and CBLB CD4+ T cells from wild-type (WT), CBLOST, and CBLBOST mice were left unstimulated (-) or stimulated for 1 min with anti-CD3 and anti-CD4 antibodies (+). Equal amounts of cell lysates were subjected to affinity purification on Strep-Tactin Sepharose beads, and the resulting protein eluates were analyzed by immunoblotting with anti-ubiquitin (UB), anti-CBLB, or anti-CBL antibodies. Also shown are a loading control corresponding to equal amounts of proteins from whole-cell lysates (WCLs) probed with anti-CD5, and molecular masses (kDa). TCR stimulation resulted in some CBLB degradation and in the detection of a higher molecular weight CBLB form, likely due to its ubiquitylation (marked with an asterisk). In contrast, no degradation of CBL was detectable upon TCR stimulation and its molecular weight remained unchanged. Data are representative of at least two experiments.Representation of the ubiquitin (UB) first-neighbors subnetwork within the CBL and CBLB CN. Edge width is coded according to Pearson correlation coefficient (R < 0.8 or R > 0.8). Red edges depict direct interactions reported in the BioGRID database. Ubiquitin (UB) is highlighted in yellow.Immunoprecipitation of CBL from cell lysates of CD4+ T cells isolated from WT or CBLB-deficient mouse (Cblb -/-) and kept unstimulated (-) or stimulated as in (A). Immunoprecipitates and whole-cell lysates (WCLs) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin. Molecular masses are shown (kDa). Data are representative of at least two experiments.Immunoprecipitation of CBLB from cell lysates of CD4+ T cells isolated from WT or CBL-deficient mouse (Cbl -/-) and kept unstimulated (-) or stimulated as in (A). Immunoprecipitates and whole-cell lysates (WCLs) were analyzed by immunoblot with antibodies specific for the proteins specified in the left margin. Molecular masses are shown (kDa). Data are representative of at least three experiments.

Fig 4: cIAP1 reduces EGFR stability, but promotes its gene transcription. a EGFR and cIAP1 interaction was tested in BT549 cells using PLA assay. b BT549 cells stably expressing Myc/Flag-tagged EGFR were serum-starved and stimulated with 20 ng/ml EGF for the indicated times. Cells were lysed and EGFR was immunoprecipitated with anti-Flag antibody. Western blot was performed to evaluate the interaction of ectopic EGFR with cIAP1 and c-Cbl. c BT549 and MCF10A cells stably expressing Myc/Flag-tagged EGFR were serum-starved, pre-treated with 100 (BT549) or 50 (MCF10A) µg/ml cycloheximide for 30 min and stimulated with 20 ng/ml EGF for the indicated times. Levels of ectopic EGFR were detected with anti-Myc antibody. d Ectopic EGFR was immunoprecipitated as described in Fig. 6b from BT549 cells transfected with control and cIAP1-specific siRNAs. Western blot shows total levels of c-Cbl and the amount of c-Cbl interacting with EGFR. e BT549 and f MCF10A cell stably expressing ectopic Myc/Flag-tagged EGFR were further transduced with lentiviral particles to overexpress c-Cbl or GFP as a control. Cells were silenced for cIAP1, serum-starved, and stimulated with 20 ng/ml EGF as described above, and analyzed by western blot to evaluate the levels of ectopic EGFR by using a Myc-tagged-specific antibody. g BT549 (left panel) and MCF10A (right panel) were silenced for cIAP1 and analyzed by real-time PCR to quantify the levels of EGFR expression fold relative to GAPDH. BT549: unstimulated siCtr vs. sicIAP1 *P = 0.0134, EGF 3 h siCtr vs. sicIAP1 *P = 0.0270; n = 3. MCF10A: unstimulated siCtr vs. sicIAP1 ***P = 0.0004, EGF 3 h siCtr vs. sicIAP1 **P = 0.0183; n = 4; two-tailed paired t-test

Fig 5: PIP4K2A negatively regulates PI3K signaling through degradation of p85 via Cbl-p85 ubiquitination. (A) Immunoblot analysis of PI3K complex (p85, p110a, and p110ß) in patient-derived GBM cells after transduced with either control or PIP4K2A. Actin was used as a loading control. (B) Densitometric analysis of the blots in A. Values are presented as mean ± SEM. (C) Representative confocal images of immunofluorescence staining of PIP4K2A and p85. Bar, 20 µm. (D) Co-IP of ubiquitin and p85 in GBM cells that were transduced with either PIP4K2A or control and treated with or without MG132. For IP immunoblotting data, antibodies used for IP and Western blotting (WB) were labeled as red and blue, respectively, and p85 was used as a loading control. For evaluation of PIP4K2A overexpression in input data, actin was used as a loading control. (E) Immunoblot analysis of p85 and PIP4K2A in GBM cells that were transduced with control, p85 shRNA, PIP4K2A, or p85 shRNA/PIP4K2A. Actin was used as a loading control. (F) Proliferation assay of GBM cells from F. Values are presented as mean ± SD. (G) Co-IP and immunoblot analysis of p85 and ubiquitin in GBM cells that were transduced with either control or Cbl shRNA and treated with or without MG132. p85 was used as a loading control. (H) Immunoblot analysis of p85, PIP4K2A, and Cbl in patient-derived GBM cells that were transduced with control, PIP4K2A, Cbl shRNA, or PIP4K2A/Cbl shRNA. Actin was used as a loading control. (I) Immunoblot analysis of PIP4K2A and anti-Flag (PI3KC-E545K mutant) in GBM cells that were transduced with control, PIP4K2A, PI3KC-E545K, or PIP4K2A/PI3KC-E545K. Actin was used as a loading control. (J) Proliferation assay of GBM cells from I. Values are presented as mean ± SD. (K) Kaplan–Meier survival curve of mice that were orthotopically implanted with GBM cells transduced with control (n = 6), PIP4K2A (n = 7), control/PI3KC-E545K (n = 6), or PIP4K2A/PI3KC-E545K (n = 7). (L) Representative H&E sections of the mouse brains from K. Bar, 2 mm. Data shown in A–J are representative of three independent and reproducible experiments. P values: B, F, and J, two-tailed Student’s t test; K, two-sided log-rank test. *, P = 0.05. Exp., exposure; WB, Western blot.