Direct binding of p85 to sst2 somatostatin receptor reveals a novel mechanism for inhibiting PI3K pathway
2006; Springer Nature; Volume: 25; Issue: 17 Linguagem: Inglês
10.1038/sj.emboj.7601279
ISSN1460-2075
AutoresCorinne Bousquet, Julie Guillermet‐Guibert, Nathalie Saint‐Laurent, Élodie Archer-Lahlou, Frédéric Lopez, Marjorie Fanjul, Audrey Ferrand, Daniel Fourmy, Carole Pichereaux, Bernard Monsarrat, Lucien Pradayrol, Jean‐Pierre Estève, Christiane Susini,
Tópico(s)Lung Cancer Research Studies
ResumoArticle17 August 2006free access Direct binding of p85 to sst2 somatostatin receptor reveals a novel mechanism for inhibiting PI3K pathway Corinne Bousquet Corresponding Author Corinne Bousquet INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Julie Guillermet-Guibert Julie Guillermet-Guibert INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Nathalie Saint-Laurent Nathalie Saint-Laurent INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Elodie Archer-Lahlou Elodie Archer-Lahlou INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Frédéric Lopez Frédéric Lopez IFR 31, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Marjorie Fanjul Marjorie Fanjul INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Audrey Ferrand Audrey Ferrand INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Daniel Fourmy Daniel Fourmy INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Carole Pichereaux Carole Pichereaux Institut de Pharmacologie et de Biologie Structurale, CNRS, Toulouse, cedex, France Search for more papers by this author Bernard Monsarrat Bernard Monsarrat Institut de Pharmacologie et de Biologie Structurale, CNRS, Toulouse, cedex, France Search for more papers by this author Lucien Pradayrol Lucien Pradayrol INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Jean-Pierre Estève Jean-Pierre Estève INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Christiane Susini Christiane Susini INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Corinne Bousquet Corresponding Author Corinne Bousquet INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Julie Guillermet-Guibert Julie Guillermet-Guibert INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Nathalie Saint-Laurent Nathalie Saint-Laurent INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Elodie Archer-Lahlou Elodie Archer-Lahlou INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Frédéric Lopez Frédéric Lopez IFR 31, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Marjorie Fanjul Marjorie Fanjul INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Audrey Ferrand Audrey Ferrand INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Daniel Fourmy Daniel Fourmy INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Carole Pichereaux Carole Pichereaux Institut de Pharmacologie et de Biologie Structurale, CNRS, Toulouse, cedex, France Search for more papers by this author Bernard Monsarrat Bernard Monsarrat Institut de Pharmacologie et de Biologie Structurale, CNRS, Toulouse, cedex, France Search for more papers by this author Lucien Pradayrol Lucien Pradayrol INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Jean-Pierre Estève Jean-Pierre Estève INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Christiane Susini Christiane Susini INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France Search for more papers by this author Author Information Corinne Bousquet 1, Julie Guillermet-Guibert1, Nathalie Saint-Laurent1, Elodie Archer-Lahlou1, Frédéric Lopez2, Marjorie Fanjul1, Audrey Ferrand1, Daniel Fourmy1, Carole Pichereaux3, Bernard Monsarrat3, Lucien Pradayrol1, Jean-Pierre Estève1 and Christiane Susini1 1INSERM U531, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France 2IFR 31, Institut Louis Bugnard, CHU Rangueil, Toulouse, cedex 4, France 3Institut de Pharmacologie et de Biologie Structurale, CNRS, Toulouse, cedex, France ‡These authors contributed equally to this work *Corresponding author. INSERM U531, IFR31, Institut Louis Bugnard, BP 84225, CHU Rangueil, 31432 Toulouse cedex 4, France. Tel.: +33 5 61 32 24 07; Fax: +33 5 61 32 24 03; E-mail: [email protected]. The EMBO Journal (2006)25:3943-3954https://doi.org/10.1038/sj.emboj.7601279 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Phosphatidylinositol 3-kinase (PI3K) regulates many cellular functions including growth and survival, and its excessive activation is a hallmark of cancer. Somatostatin, acting through its G protein-coupled receptor (GPCR) sst2, has potent proapoptotic and anti-invasive activities on normal and cancer cells. Here, we report a novel mechanism for inhibiting PI3K activity. Somatostatin, acting through sst2, inhibits PI3K activity by disrupting a pre-existing complex comprising the sst2 receptor and the p85 PI3K regulatory subunit. Surface plasmon resonance and molecular modeling identified the phosphorylated-Y71 residue of a p85-binding pYXXM motif in the first sst2 intracellular loop, and p85 COOH-terminal SH2 as direct interacting domains. Somatostatin-mediated dissociation of this complex as well as p85 tyrosine dephosphorylation correlates with sst2 tyrosine dephosphorylation on the Y71 residue. Mutating sst2-Y71 disabled sst2 to interact with p85 and somatostatin to inhibit PI3K, consequently abrogating sst2's ability to suppress cell survival and tumor growth. These results provide the first demonstration of a physical interaction between a GPCR and p85, revealing a novel mechanism for negative regulation by ligand-activated GPCR of PI3K-dependent survival pathways, which may be an important molecular target for antineoplastic therapy. Introduction The balance between cell survival and cell death critically controls tissue homeostasis. Growth factors, cytokines and integrins regulate this balance by activating Class IA phosphatidylinositol 3-kinase (PI3K) and excessive activation of this enzyme is a hallmark of a wide range of tumors (Vivanco and Sawyers, 2002; Luo et al, 2003). Class IA PI3K is a heterodimer consisting of a regulatory subunit (p85) and a catalytic subunit (p110), which catalyzes phosphorylation of phosphatidylinositol (PtdIns)-4,5-P2 to PtdIns-3,4,5-P3. These lipids recruit the proteine kinase Akt to the plasma membrane by engaging its PH domain, an event that triggers phosphorylation and activation of Akt. PI3K regulates a diverse array of cellular functions including cell survival, growth, proliferation, intermediary metabolism and cytoskeletal rearrangements (Cantley, 2002; Vivanco and Sawyers, 2002). Physical interaction of p85 SH2 domain with tyrosine-phosphorylated molecules, in the context of a YXXM consensus motif present in either activated tyrosine kinase receptors or their adapter molecules, and phosphorylation of p85 Tyr688 are directly responsible for ligand-induced PI3K regulation (Cuevas et al, 2001; Chan et al, 2002). p85 also interacts directly with the ion channel NMDA receptor (Takagi et al, 2003), as well as with estrogen and glucocorticoid receptors (Simoncini et al, 2000; Leis et al, 2004), resulting in ligand-inducible PI3K regulation. In contrast, although several ligand-activated GPCR, including gastrin, or the angiotensin AT2 receptor, regulate PI3K activity (Daulhac et al, 1999; Cui et al, 2002), no physical interaction has been demonstrated to occur between these receptors and p85, but rather with receptor-activated signaling molecules. A range of mechanisms is involved in negative regulation of PI3K activity in response to growth factors. For example, dephosphorylation of p85 on Tyr688 by the tyrosine phosphatases SHP-1, or phosphorylation on Ser608 by the intrinsic protein kinase activity of the p110 catalytic subunit inhibits PI3K activity (Cuevas et al, 1999; Foukas et al, 2004). The tyrosine phosphatase SHP-2 negatively regulates some growth factor-induced PI3K activation by dephosphorylating p85-binding sites on Gab-1 (Zhang et al, 2002). Another negative feedback inhibition of insulin-induced PI3K activation might involve mTOR signaling, which affects IRS-1 phosphorylation and transcription (Harrington et al, 2005). The inhibitory GPCR sst2 receptor transduces somatostatin antiproliferative, apoptotic, antiangiogenic and anti-invasive effects (Weckbecker et al, 2003; Bousquet et al, 2004). Interestingly, sst2 behaves as a tumor suppressor gene for pancreatic cancer. A selective sst2 loss is observed in 90% of human pancreatic adenocarcinomas (Buscail et al, 1996). Re-expression of sst2 in human pancreatic cancer cells results both in increased apoptosis and cell-sensitization to death ligand-induced apoptosis (Guillermet et al, 2003), and in decreased pancreatic cancer cell proliferation and tumorigenicity (Vernejoul et al, 2002; Kumar et al, 2004). Here, we report a novel mechanism for limiting PI3K activity. We present evidence for the first time for a physical interaction between a GPCR, sst2 and the PI3K regulatory subunit p85, occurring through the tyrosine-phosphorylated Y71AKM sequence, present in the sst2 first intracellular loop (il1). Interestingly, the somatostatin analog RC-160 inhibits PI3K activity by disrupting sst2–p85 complexes, triggering inhibition of cell survival and tumor growth. This observation is of interest as excess activation of the PI3K pathway has been reported in pancreatic adenocarcinomas (Asano et al, 2004), and potently participates in the acquisition of both an invasive phenotype and resistance of pancreatic cancer cells to chemotherapy (Ng et al, 2000). Results and discussion The regulatory subunit p85 of PI3K directly interacts with the sst2 phosphorylated-Y71 residue To identify novel sst2 interacting partners, sst2 protein sequence was screened and a consensus YXXM motif, known to bind the PI3K regulatory p85 subunit when the Y residue is phosphorylated, was identified in sst2-il1 (sequence Y71AKM74). We therefore investigated by surface plasmon resonance (SPR) for a direct interaction between p85 and sst2. A recombinant GST-p85 protein was shown to directly and specifically interact with a peptide, encompassing the sst2-il1 sequence and immobilized on the chip, only when the residue Y71 is phosphorylated (pY71-peptide), yielding a differential response (see Materials and methods) increasing with the concentration of the injected GST-p85 (Figure 1A). The calculated KD was 16 nM. Endogenous p85 was then shown to specifically interact with the pY71-peptide, as demonstrated by the subsequent injection of a cell extract resulting in a significant differential response on the sst2-il1-pY71-peptide, which was further increased after injecting an anti-p85 antibody, but not unrelated antibodies (Figure 1B). Figure 1.Identification and characterization of a direct interaction between sst2 and p85. (A, B) SPR analysis using two biotinylated peptides immobilized onto the Biacore chip, covering sst2-il1 with the Y71 residue either phosphorylated or not, and injection of either (A) increasing concentrations of a recombinant GST-p85 protein, or (B) CHO cell extracts together with injection of anti-p85, -SHP-1 or Gab-1 antibody. Results are expressed as a differential response (RU quantified on the phosphorylated- versus nonphosphorylated-Y71-peptide-containing flow cell). (C) SPR analysis using the recombinant GST-p85 protein immobilized onto the Biacore chip, and injection of either mock- (left panel), wild-type (middle panel) or mutated Y71F flag-tagged sst2-transfected cell extracts (right panel). A competitive concentration of the sst2 phosphorylated-Y71-containing peptide was coinjected (dotted line) or not (plain line) with the cell extracts. When indicated, the anti-flag antibody was injected. Results are expressed as a normalized differential response (RU), 100% referring to the maximal response observed for each cell extract in the absence of competitor peptide or antibody. (D) GST-pull-down assay using GST or GST-p85, coupled to glutathione agarose-beads, incubated with either mock-, wild-type or mutated Y71F flag-tagged, sst2-transfected cell extracts. The presence of sst2 in the putative sst2–p85 complex was assessed by using the anti-flag antibody (left panel, upper). The total input of the wild-type sst2 protein present in the wild-type, flag-tagged, sst2-transfected cell extracts is visualized in lane 1 (Input). Equal levels of recombinant GST protein present in each lane were assessed by Coomassie blue staining of the gel (left panel, lower). Cell transfection efficiencies of both flag-tagged wild-type or mutated Y71F sst2 were checked in the cell extracts used for the pull-down assay by immunoprecipitation/Western blot using the anti-flag antibody (right panel). Results presented in Figure 1 are representative of three independent experiments. Download figure Download PowerPoint Strikingly, mass spectrometry (MS) analysis coupled with SPR successfully identified p85, recovered from binding to the sst2-il1 pY71-peptide of proteins derived from the injection of a p85-transfected cell extract, as interacting with sst2 with a 27% sequence covering (Supplementary Figure 1). This result validates this promising approach to uncover novel protein interacting partners. Mutation of the Y residue included in the consensus YXXM motif, whereby Y is phosphorylated, is known to block p85 interaction with this motif (Vivanco and Sawyers, 2002). To investigate whether wild-type but not mutated Y71F sst2 might interact with p85, a recombinant GST-p85 protein was immobilized onto the chip to serve as a bait, and either mock-, wild-type or mutated Y71F flag-tagged sst2-transfected cell extracts were injected (Figure 1C, left, middle or right panel, respectively). Among the proteins of the cell extracts that bind GST-p85, sst2 was identified specifically in the wild-type sst2-tranfected cell extract, as indicated by the further increase in the differential response observed in the wild-type (middle panel, plain line), but not mock or mutated Y71F sst2 conditions (left or right panels, respectively, plain lines), after injecting the anti-flag antibody. This anti-flag antibody-mediated response, observed in the presence of wild-type sst2, was however abrogated when a competitive concentration of the pY71-peptide was coinjected with the cell extract (middle panel, dotted line), whereas no effect was observed with mock or mutated Y71 sst2-expressing cell extracts. This result indicated that sst2 interaction with GST-p85 was displaced by using an excess of the sst2-il1-pY71-peptide. Finally, a pull-down experiment was performed to further confirm the sst2–p85 interaction (Figure 1D). Consistently, sst2 was recovered in the pull-down experiment using GST-p85 and wild-type sst2-expressing cell extract, but not in the presence of GST alone, or when mock or mutated Y71F sst2-transfected cell extracts were used. When compared to the total input of wild-type sst2-expressing cell extract, about 5% of sst2 was bound to GST-p85. These results establish that GST-p85 was able to recover sst2 as a binding partner in cell extracts, indicating that both full-length proteins interact effectively. These data also convincingly suggest that the phosphorylated status of the sst2-Y71 residue is a prerequisite for sst2 and p85 to interact. To validate our experimental results, we designed a molecular model for the sst2–p85 interaction by docking sst2-il1 and p85 C- or N-terminal SH2 domain (SH2c or SH2n). We hypothesized that a p85 SH2 domain is involved in the interaction, as suggested by the requirement for the sst2-pY71 residue. Both p85-SH2c and SH2n domains have similar specificity to the sequence context of pY-hydrophobic-X-hydrophobic, in accordance with pY+1 being an alanine and pY+3 a methionine in sst2-il1 (Vivanco and Sawyers, 2002). p85-SH2n domain was demonstrated not to be involved in the sst2–p85 interaction, as no interaction occurred between sst2-il1 and a recombinant GST-p85-SH2n by SPR (not shown). By contrast, docking p85-SH2c to sst2-il1 yielded a refined model of this complex whereby sst2-il1 is located in a groove of the p85-SH2c (Supplementary Figure 2A). Accordingly, pY71 is inserted deeper into a binding pocket through robust interactions between its phosphate group and p85-SH2c residues E635 and R649. Interestingly, sst2 R70 and K75 residues appeared also involved in the interaction with p85-SH2c, through hydrogen bonds with E635 and E666, and D708, respectively (Supplementary Figure 2B). According to the model, full-size recombinant GST-p85 proteins containing single or multiple mutations on these p85-SH2c residues (E635L, R649M, E666L and D708A) were tested using SPR and the Y71-containing sst2-il1 peptides, either phosphorylated or not (Supplementary Figure 2C). Single mutations of p85-SH2c R649, but not of E635, E666 or D708, abrogated the sst2–p85 interaction. Consistently, R649 is included in the p85-SH2c FLVR motif, which is critical for SH2 domains to interact with phosphotyrosine (Kuriyan and Cowburn, 1997). Mutations of the three E635, E666 and D708 residues together decreased the sst2–p85 interaction by 50%, suggesting that their interactions with sst2 R70 and K75 residues stabilize the complex. Preferential interaction of p85-SH2c, rather than p85-SH2n, with phosphotyrosine residues was also demonstrated for NMDA (Takagi et al, 2003) or PDGF receptor (Panayotou et al, 1993). Sst2 co-immunoprecipitates in cells with p85 and with a PI3K activity To assess whether sst2 and p85 interact within a cellular context, wild-type or mutated Y71F flag-tagged sst2 was transfected together with HA-tagged p85 in COS-7 cells (Figure 2A). In basal conditions (in the presence of serum without addition of the sst2 agonist RC-160), p85 co-immunoprecipitates with wild-type, but not specifically with mutated Y71F sst2 (Figure 2A), nor with sst3, another member of the somatostatin receptor family which does not comprise the consensus YXXM motif (Figure 2B). This result, therefore, confirmed the involvement of the sst2-Y71 residue in the sst2–p85 interaction. Such sst2–p85 interaction was also observed in basal conditions in two other cell models expressing p85, CHO cells stably transfected with sst2 (CHO/sst2) and rat pancreatic cancer AR4-2J cells which express endogenous sst2 (Ferjoux et al, 2003). In these two models, p85 was present in the anti-sst2, but not preimmune serum, immunoprecipitate (Figures 3A and 4A, lanes 1 and 2, respectively). Figure 2.COS-7 cells. p85 and sst2, but not sst3, co-immunoprecipitate. COS-7 cells were transfected with HA-tagged p85 together with wild-type or mutated Y71F flag-tagged sst2 (A, C), or with T7-tagged sst2 or sst3 (B). (A, B) Immunoprecipitations were performed with the anti-flag (A) or anti-T7 (B) antibody, or preimmune (Pre-I) serum as control, which was followed by a western-blot using the anti-HA antibody (upper panels), or the anti-flag (A) or anti-T7 (B) antibody (middle panels) to check for equal levels of both transfection and immunoprecipitation of sst2/sst3 in each lane. Equal level of HA-p85 transfection was assessed by direct Western blot with the anti-HA antibody on a separate gel loaded with the same cell extracts (lower panels). (C) Measure of sst2-associated PI3K activity in COS-7 cell extracts transfected with wild-type or mutated Y71F flag-tagged sst2 (derived from lanes 2 and 3 of Figure 2A, respectively) immunoprecipitated with the anti-flag antibody, or preimmune (Pre-I) serum. When indicated, cell extracts were pretreated with 5 μM of LY294002. Results presented in Figure 2 are representative of three independent experiments. Download figure Download PowerPoint Figure 3.CHO cells. Somatostatin analog regulates sst2–p85 interaction and inhibits PI3K pathway and cell survival in a pY71-dependent manner. CHO/sst2 cells (A–C) or CHO cells transfected with wild-type or mutated Y71F flag-tagged sst2 (D–F) were treated as indicated with RC-160. (A) Sst2–p85 interaction was assessed in sst2-, or in preimmune (Pre-I) serum as control, immunoprecipitates, by Western blot with the anti-p85 antibody. Equal level of immunoprecipitation in each lane was checked by immunoblotting the same membrane with the anti-sst2 antibody. Quantification of the sst2–p85 interaction was performed by densitometric analysis of the p85 immunoblot. Results are the means±s.e.m. (n=3) and expressed as 100% of the control RC-160-untreated cells. (B–D) Sst2-associated (B) or total (C, D) PI3K activities were assessed in sst2-, or p85 immunoprecipitates, respectively. Equal level of immunoprecipitation in each lane was checked by immunoblotting an additional gel loaded with the same immunoprecipitates either with the anti-sst2 antibody (B) or the anti-p85 antibody (C, D). Quantification of the PI3K activity was performed by densitometric analysis of the PIP3 autoradiography. Results are the means±s.e.m. (n=3) and expressed as 100% of the control RC-160-untreated cells. (E) Immunoblot of Akt S473 phosphorylation. Equal loading of cell extract in each lane was checked by immunoblotting the same membrane with the anti-Akt antibody. (F) Cell viability assay using the MTT test in starved cells (control), serum-treated cells (FCS) or serum and RC-160-treated cells (FCS+RC-160, 10−9 M) for 24 h, as indicated. Results are the means±s.e.m. of three independent experiments and expressed as 100% of control. Cell transfection efficiencies of both wild-type or mutated Y71F flag-tagged sst2 were checked in the same cell extracts by immunoprecipitation/Western blot using the anti-flag antibody (D, lower panel) (*P<0.05 and **P<0.01 for RC-160-treated CHO/sst2 cells versus serum-treated cells). Download figure Download PowerPoint Figure 4.AR4-2J cells. Somatostatin analog regulates sst2–p85 interaction and inhibits PI3K pathway and cell survival. AR4-2J cells were treated as indicated with RC-160. (A) Sst2–p85 interaction was assessed in sst2-, or in preimmune (Pre-I) serum as control, immunoprecipitates, by Western blot with the anti-p85 antibody. Equal level of immunoprecipitation in each lane was checked by immunoblotting the same membrane with the anti-sst2 antibody. Quantification of the sst2–p85 interaction was performed by densitometric analysis of the p85 immunoblot. Results are the means±s.e.m. (n=3) and expressed as 100% of the control RC-160-untreated cells. (B) p85 tyrosine phosphorylation was assessed in p85 immunoprecipitates using an antiphosphotyrosine antibody. Equal level of immunoprecipitation in each lane was checked by immunoblotting the same membrane with the anti-p85 antibody. Quantification of p85 tyrosine phosphorylation was performed by densitometric analysis of the p-Tyr immunoblot. Results are the means±s.e.m. (n=3) and expressed as 100% of the control RC-160-untreated cells. (C) Immunoblot of Akt S473 phosphorylation. Equal loading of cell extract in each lane was checked by immunoblotting the same membrane with the anti-Akt antibody. (D) Cell viability assay using the MTT test in starved (control), serum-treated (FCS) or serum and RC-160-treated cells (FCS+RC-160, 10−11–10−7 M) for 24 h, as indicated. Results are the means±s.e.m. of three independent experiments and expressed as 100% of control (*P<0.05 and **P<0.01 for RC-160-treated AR4-2J cells versus serum-treated cells). Download figure Download PowerPoint Several reports have demonstrated the presence of an autoinhibitory motif in p85, which negatively regulates p110 catalytic activity. This p85 autoinhibitory action is relieved by both the engagement of either p85 SH2 domain to a phosphorylated-Y residue (Chan et al, 2002), and the phosphorylation of p85-Y688 residue, included in p85-SH2c (Cuevas et al, 2001), resulting in PI3K activation. Several arguments indicate that p85 autoinhibitory activity was relieved in our COS-7 and CHO cell models under basal conditions. Firstly, when assessing whether an endogenous PI3K activity associates with sst2, a PI3K activity was observed both in the anti-flag immunoprecipitate derived from wild-type, flag-tagged, sst2-transfected COS-7 cell extract (Figure 2C), or in the anti-sst2 immunoprecipitate from CHO/sst2 cells (Figure 3B, lane 1). This activity was specific because it was abrogated by addition of the PI3K inhibitor LY294002 (Figure 2C) and contributed to about 5% of the total PI3K activity (as measured in an anti-p85 immunoprecipitate, not shown). Interestingly, a robust total PI3K activity was observed in p85 immunoprecipitate from CHO/sst2 cells under basal conditions (Figure 3C, lane 1). We therefore suggest that, in the absence of added somatostatin and in the presence of serum, by binding p85, sst2 is permissive, rather than inhibitory, for growth factor-induced PI3K activity. Somatostatin regulates sst2–p85 interaction and inhibits PI3K activity To assess whether the sst2–p85 interaction is regulated by the somatostatin analog RC-160, the heterologous and endogenously sst2-expressing CHO/sst2 and AR4-2J cell models, respectively, were treated with 1 nM RC-160 in the presence of serum (Figures 3A and 4A). Treatment with RC-160 resulted in a time-dependent dissociation of p85 from sst2, which was maximal at 3–5 min. Interestingly, we then demonstrated in CHO/sst2 cells that ligand-activated sst2 time-dependently inhibited both sst2-associated and total PI3K activities (Figure 3B and C), with a maximum at 3–5 min. Strikingly, RC-160-induced dissociation of p85 from sst2, rather than decreased p85 protein expression (which was stable during somatostatin treatment, not shown), was shown to be critical for somatostatin-mediated inhibition of total PI3K activity. Indeed, CHO cells expressing the mutated Y71F-sst2 receptor, which does not bind p85, did not show RC-160-dependent inhibition of PI3K at both 3 and 5 min of treatment, as opposed to wild-type sst2-expressing CHO cells (Figure 3D). Functionality of the mutated Y71F sst2, in terms of appropriate folding and coupling to protein G, was confirmed (measure of inositol phosphate production, not shown) (Chen et al, 1997). Because about 5% of the total PI3K activity was estimated to bind the sst2 receptor, we hypothesize that, in a dynamic process, a large amount of the cellular p85-PI3K pool can be catalytically served via the sst2 interaction to be inactivated. Accordingly, we have here unraveled an additional level of somatostatin-dependent PI3K inactivation through p85 tyrosine dephosphorylation. AR4-2J cell treatment with RC-160 reduced p85 tyrosine phosphorylation with a maximun at 3–5 min (Figure 4B). RC-160-mediated tyrosine dephosphorylation of p85 was observed in CHO/sst2 cells as well (not shown), this dephosphorylation occurring with a kinetic similar as that observed for RC-160-mediated inhibition of PI3K activity (Figure 3C). We hypothesize that tyrosine phosphatases including SHP-1 and/or SHP-2, which are activated by sst2 (Ferjoux et al, 2003), might be involved in p85 tyrosine dephosphorylation, as already described for SHP-1 (Cuevas et al, 1999). Somatostatin inhibits sst2 tyrosine phosphorylation on the Y71 residue We have previously demonstrated that sst2 is tyrosine-phosphorylated in the presence of serum, and that CHO/sst2 cell treatment with RC-160 inhibits this phosphorylation (Ferjoux et al, 2003). We therefore investigated whether the specific residue Y71 in sst2-il1 is tyrosine-phosphorylated and whether RC-160 regulates this phosphorylation. For this purpose, the specificity towards the sst2-il1-pY71-peptide of a phospho-(Tyr) p85 PI3K antibody (Li et al, 2003), which recognizes peptides and proteins containing phosphorylated tyrosine at the consensus YXXM motif, was tested using SPR (Figure 5A). This antibody was shown to specifically interact with the sst2-il1-pY71-peptide immobilized on the chip, but not with the corresponding unphosphorylated sst2-il1-Y71-peptide nor with an alternate tyrosine-phosphorylated pY312-peptide encompassing the COOH-terminal 304–321 amino acids of sst2 sequence. Accordingly, the YXXM motif is present only in the first intracellular loop of the sst2 sequence. We therefore performed Western blotting experiments using this antibody to explore the phosphorylated status of the sst2 Y71 residue. Using anti-flag immunoprecipitates of COS cells either transfected with the wild-type or mutated Y71F flag-tagged sst2 and cultured in the presence of serum, the phosphorylation of the Y71 residue was observed in the wild-type, but not specifically in the mutated Y71, sst2. Interestingly, RC-160 decreased this phosphorylation after 5 min of cell treatment (Figure 5B). This result was confirmed in the endogenously sst2-expressing AR4-2J cells, whereby sst2 immunoprecipitates showed Y71 phosphorylation which was time-dependently decreased by RC-160 cell treatment (Figure 5C) with a kinetic similar as that observed for RC-160-mediated dissociation o
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