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• W2005131087 abstract "Coligation of FcγRIIb1 with the B cell receptor (BCR) or FcεRI on mast cells inhibits B cell or mast cell activation. Activity of the inositol phosphatase SHIP is required for this negative signal. In vitro, SHIP catalyzes the conversion of the phosphoinositide 3-kinase (PI3K) product phosphatidylinositol 3,4,5-trisphosphate (PIP3) into phosphatidylinositol 3,4-bisphosphate. Recent data demonstrate that coligation of FcγRIIb1 with BCR inhibits PIP3-dependent Btk (Bruton's tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. In this study, we provide evidence that coligation of FcγRIIb1 with BCR induces binding of PI3K to SHIP. This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Furthermore, the generation of phosphatidylinositol 3,4-bisphosphate was only partially reduced during coligation of BCR with FcγRIIb1 despite a drastic reduction in PIP3. In contrast to the complete inhibition of Tec kinase-dependent calcium signaling, activation of the serine/threonine kinase Akt was partially preserved during BCR and FcγRIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis. Coligation of FcγRIIb1 with the B cell receptor (BCR) or FcεRI on mast cells inhibits B cell or mast cell activation. Activity of the inositol phosphatase SHIP is required for this negative signal. In vitro, SHIP catalyzes the conversion of the phosphoinositide 3-kinase (PI3K) product phosphatidylinositol 3,4,5-trisphosphate (PIP3) into phosphatidylinositol 3,4-bisphosphate. Recent data demonstrate that coligation of FcγRIIb1 with BCR inhibits PIP3-dependent Btk (Bruton's tyrosine kinase) activation and the Btk-dependent generation of inositol trisphosphate that regulates sustained calcium influx. In this study, we provide evidence that coligation of FcγRIIb1 with BCR induces binding of PI3K to SHIP. This interaction is mediated by the binding of the SH2 domains of the p85 subunit of PI3K to a tyrosine-based motif in the C-terminal region of SHIP. Furthermore, the generation of phosphatidylinositol 3,4-bisphosphate was only partially reduced during coligation of BCR with FcγRIIb1 despite a drastic reduction in PIP3. In contrast to the complete inhibition of Tec kinase-dependent calcium signaling, activation of the serine/threonine kinase Akt was partially preserved during BCR and FcγRIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis. Coengagement of FcγRIIb1 with the B cell receptor (BCR) 1The abbreviations used are: BCR, B cell receptor; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PIP2, phosphatidylinositol 3,4-bisphosphate; GST, glutathioneS-transferase; DPBS, Dulbecco's phosphate-buffered saline; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography 1The abbreviations used are: BCR, B cell receptor; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PIP2, phosphatidylinositol 3,4-bisphosphate; GST, glutathioneS-transferase; DPBS, Dulbecco's phosphate-buffered saline; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography by an immune complex consisting of antigen and a specific antibody provides a feedback mechanism for the down-regulation of B cell activation (1Chan P.L. Sinclair N.R. Immunology. 1971; 21: 967-981PubMed Google Scholar, 2Phillips N.E. Parker D.C. J. Immunol. 1983; 130: 602-606PubMed Google Scholar). A distinct effect of BCR/FcγRIIb1 coligation is the loss of sustained calcium influx and a selective reduction in the tyrosine phosphorylation of certain proteins (3Bijsterbosch M.K. Klaus G.G. J. Exp. Med. 1985; 162: 1825-1836Crossref PubMed Scopus (159) Google Scholar, 4Wilson H.A. Greenblatt D. Taylor C.W. Putney J.W. Tsien R.Y. Finkelman F.D. Chused T.M. J. Immunol. 1987; 138: 1712-1718PubMed Google Scholar, 5Choquet D. Partiseti M. Amigorena S. Bonnerot C. Fridman W.H. Korn H. J. Cell Biol. 1993; 121: 355-363Crossref PubMed Scopus (103) Google Scholar, 6Kiener P.A. Lioubin M.N. Rohrschneider L.R. Ledbetter J.A. Nadler S.G. Diegel M.L. J. Biol. Chem. 1997; 272: 3838-3844Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 7Hippen K.L. Buhl A.M. D'Ambrosio D. Nakamura K. Persin C. Cambier J.C. Immunity. 1997; 7: 49-58Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The molecular events responsible for this phenotype are not clearly understood. Cocross-linking of BCR with FcγRIIb1 results in recruitment of SHIP (SH2 domain-containing inositol-polyphosphate 5′-phosphatase) to the immunoreceptor tyrosine-based inhibition motif present in the cytoplasmic tail of FcγRIIb1 (8Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (643) Google Scholar). Two approaches provided evidence for a functional requirement for SHIP during FcγRIIb1-mediated inhibitory signaling. Ectopic expression of a chimeric KIR/FcγRIIb1 protein, containing the extracellular and transmembrane regions of KIR and the cytoplasmic tail of FcγRIIb1, in natural killer cells inhibited the lysis of target cells bearing the HLA class I ligand for the extracellular KIR portion of the chimeric receptor (9Gupta N. Scharenberg A.M. Burshtyn D.N. Wagtmann N. Lioubin M.N. Rohrschneider L.R. Kinet J.-P. Long E.O. J. Exp. Med. 1997; 186: 473-478Crossref PubMed Scopus (74) Google Scholar). Coexpression of a dominant-negative mutant of SHIP, but not the tyrosine phosphatase Shp-1, reverted the inhibitory signal delivered by FcγRIIb1 in natural killer cells. Conversely, dominant-negative Shp-1, but not SHIP, reverted the negative signal mediated by KIR (9Gupta N. Scharenberg A.M. Burshtyn D.N. Wagtmann N. Lioubin M.N. Rohrschneider L.R. Kinet J.-P. Long E.O. J. Exp. Med. 1997; 186: 473-478Crossref PubMed Scopus (74) Google Scholar). The second approach made use of chicken DT40 B cells in which the SHIP or Shp-1 genes had been deleted by targeted homologous recombination (10Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). FcγRIIb1dependent inhibition was lost in the absence of SHIP, but remained intact in cells lacking Shp-1 (10Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). SHIP is a 145-kDa cytosolic protein that contains a single SH2 domain, a catalytic region that bears significant homology to inositol 5′-phosphatases, and several binding sites for other signaling proteins in its C-terminal region (11Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (377) Google Scholar, 12Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (560) Google Scholar, 13Kavanaugh W.M. Pot D.A. Chin S.M. Deuter-Reinhard M. Jefferson A.B. Norris F.A. Masiarz F.R. Cousens L.S. Majerus P.W. Williams L.T. Curr. Biol. 1996; 6: 438-445Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). SHIP interacts with Shc (14Chacko G.W. Tridandapani S. Damen J.E. Liu L. Krystal G. Coggeshall K.M. J. Immunol. 1996; 157: 2234-2238PubMed Google Scholar), which couples proximal signaling to the Grb2/Sos/Ras activation pathway. SHIP tyrosine phosphorylation and association with Shc increases upon BCR/FcγRIIb1 coligation (14Chacko G.W. Tridandapani S. Damen J.E. Liu L. Krystal G. Coggeshall K.M. J. Immunol. 1996; 157: 2234-2238PubMed Google Scholar). It was proposed that SHIP inhibits the BCR activation signal by competing with Grb2 for binding to Shc, thereby breaking the Ras signaling pathway (15Tridandapani S. Kelley T. Cooney D. Pradhan M. Coggeshall K.M. Immunol. Today. 1997; 18: 424-427Abstract Full Text PDF PubMed Scopus (69) Google Scholar). BCR ligation leads to phosphorylation of the tyrosines at positions 484 and 515 in CD19, which then recruit and activate phosphoinositide 3-kinase (PI3K) (16Tuveson D.A. Carter R.H. Soltoff S.P. Fearon D.T. Science. 1993; 260: 986-989Crossref PubMed Scopus (281) Google Scholar, 17Weng W.-K. Jarvis L. LeBien T.W. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar). Coligation of BCR with FcγRIIb1 leads to initial phosphorylation of CD19, followed by its rapid dephosphorylation (6Kiener P.A. Lioubin M.N. Rohrschneider L.R. Ledbetter J.A. Nadler S.G. Diegel M.L. J. Biol. Chem. 1997; 272: 3838-3844Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 7Hippen K.L. Buhl A.M. D'Ambrosio D. Nakamura K. Persin C. Cambier J.C. Immunity. 1997; 7: 49-58Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). One model proposes that a tyrosine phosphatase, such as Shp-1, dephosphorylates CD19, thereby blocking BCR-mediated activation by preventing PI3K activation (7Hippen K.L. Buhl A.M. D'Ambrosio D. Nakamura K. Persin C. Cambier J.C. Immunity. 1997; 7: 49-58Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). However, CD19 dephosphorylation cannot always account for FcγRIIb1-mediated negative signaling because such signaling operates in mast cells and natural killer cells that do not express CD19 (8Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (643) Google Scholar, 9Gupta N. Scharenberg A.M. Burshtyn D.N. Wagtmann N. Lioubin M.N. Rohrschneider L.R. Kinet J.-P. Long E.O. J. Exp. Med. 1997; 186: 473-478Crossref PubMed Scopus (74) Google Scholar). Furthermore, dephosphorylation by Shp-1 is not required for FcγRIIb1-mediated inhibition (8Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (643) Google Scholar, 9Gupta N. Scharenberg A.M. Burshtyn D.N. Wagtmann N. Lioubin M.N. Rohrschneider L.R. Kinet J.-P. Long E.O. J. Exp. Med. 1997; 186: 473-478Crossref PubMed Scopus (74) Google Scholar, 10Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 18Nadler M.J.S. Chen B. Anderson J.S. Wortis H.H. Neel B.G. J. Biol. Chem. 1997; 272: 20038-20043Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In vitro, SHIP cleaves the 5′-phosphate from phosphatidylinositol 3,4,5-trisphosphate (PIP3) and inositol 1,3,4,5-tetrakisphosphate to give rise to phosphatidylinositol 3,4-bisphosphate (PIP2) and inositol 1,3,4-trisphosphate, respectively (12Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (560) Google Scholar). Unlike other 5′-phosphatases, SHIP preferentially utilizes substrates that are phosphorylated on the D3 position of the inositol ring, thereby linking its activity to the PI3K pathway. Coengagement of FcγRIIb1 with BCR leads to a drastic reduction of cellular PIP3 at any time point of cross-linking as detected by thin-layer chromatography (19Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z.M. Lin S.Q. Gout I. Cantley L.C. Rawlings D.J. Kinet J.-P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar). PIP3 may either not be produced because of inactivation of PI3K, as proposed in the CD19 dephosphorylation model, or be rapidly turned over. Recruitment of SHIP by FcγRIIb1 may serve to achieve a rapid conversion of PIP3 to PIP2. Therefore, the possibility of a physical association between SHIP and PI3K was investigated. Coengagement of BCR with FcγRIIb1 resulted in a tyrosine phosphorylation-dependent recruitment of the p85 subunit of PI3K to SHIP. This interaction is mediated by direct binding of the SH2 domain of PI3K to a signature motif in the C-terminal region of SHIP. In addition, production of PIP2 and activation of Akt (also called protein kinase B) were observed during BCR/FcγRIIb1 coengagement. The B cell line A20 was maintained in RPMI 1640 medium with 10% fetal bovine serum, 2 mm glutamine, and 50 μm β-mercaptoethanol. NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium with 10% calf serum and 2 mm glutamine. F(ab′)2, intact rabbit anti-mouse IgG, and peroxidase-conjugated goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Anti-PI3K p85 and p110 subunit antibodies, unconjugated and biotin-conjugated anti-phosphotyrosine 4G10 antibodies, and a glutathione S-transferase (GST) fusion protein of the PI3K p85 C-terminal SH2 domain were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Recombinant GST protein and wortmannin were obtained from Sigma. Antibodies against Akt and phospho-Akt (specific for phosphoserine 473) were from New England Biolabs Inc. (Beverly, MA), and anti-Flag antibody (M2) was from Eastman Kodak Co. A rabbit antiserum against the peptide sequence VPACGVSSLNEMINP in the C-terminal region of SHIP was generated (Research Genetics, Huntsville, AL). Peroxidase conjugates of streptavidin and sheep anti-mouse IgG were from Amersham Pharmacia Biotech. Different deletion mutants of SHIP were obtained from M. Lioubin and L. Rohrschneider (Fred Hutchinson Cancer Research Center, Seattle, WA). The N-terminal SH2 domain is designated as n, the catalytic domain as cat, and the C-terminal region following the catalytic domain as c. Thus, the truncated mutants contain different combinations of n, cat, and c regions, i.e. ncat, nc, and catc. SHIPncat has amino acids 5–866; SHIPnc has a deletion in the catalytic domain corresponding to amino acids 500–809 and a replacement with amino acids EF arising from an EcoRI site located at the site of deletion; and SHIPcatc contains amino acids 174–1190. All constructs have a Flag tag followed by a NotI site at the amino terminus, which adds amino acids MGDYKDDDDKRPH onto the amino terminus of each. The cDNAs were cloned into plasmid pSCF4, a modified pSC65 plasmid (a gift of B. Moss), which contains a Kozak sequence and a Flag sequence followed by a multiple cloning site. Recombinant vaccinia viruses were generated and amplified as described (20Earl P.L. Moss B. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1988: 16.17.1-16.17.16Google Scholar). A20 cells (2 × 107) were washed twice with serum-free Iscove's medium, resuspended, and incubated with F(ab′)2fragment or intact rabbit anti-mouse IgG for the indicated times at 37 °C. Stimulation was stopped by addition of cold Dulbecco's phosphate-buffered saline (DPBS) and by rapid centrifugation of the cells in a Picofuge (Stratagene, La Jolla, CA). The cells were lysed in Tris-buffered saline, pH 8.0, containing 0.5% Triton X-100, 5 mm EDTA, 2 mm iodoacetamide, 5 μg/ml pepstatin A, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium metavanadate, and 10 mm sodium fluoride. Lysates were immunoprecipitated with the indicated antibodies and protein G-agarose beads. Recombinant vaccinia viruses encoding SHIPncat, SHIPnc, or SHIPcatc were used to infect NIH 3T3 cells as described (21Scharenberg A.M. Lin S. Cuenod B. Yamamura H. Kinet J.-P. EMBO J. 1995; 14: 3385-3394Crossref PubMed Scopus (142) Google Scholar). Briefly, NIH 3T3 cells (5 × 106) were infected in suspension at 5 plaque-forming units/cell with the indicated recombinant viruses in 2 ml of infection medium consisting of Dulbecco's modified Eagle's medium, 2 mm glutamine, 10 mm HEPES, and 0.5% bovine serum albumin for 3.5 h at 37 °C. The cells were washed once with DPBS and incubated in 1 ml of DPBS or pervanadate solution (10 mmH2O2 + 0.1 mm sodium metavanadate in DPBS) for 15 min at 37 °C. Subsequently, the cells were washed with cold DPBS and lysed, and the lysates were used for immunoprecipitation as described above. Synthetic peptides corresponding to amino acid sequences in the C-terminal region of SHIP and in the PI3K-binding motif in CD19 (SLGSQS(pY)EDMRG) were purchased from Quality Controlled Biochemicals (Hopkinton, MA). The SHIP peptides used were EMINPNYIGMGP, EMINPN(pY)IGMGP, and EMINPN(pY)IGRGP. All peptides were synthesized with an N-terminal biotin tag for coupling with streptavidin-agarose beads. The peptides were dissolved at 0.1 mg/ml in PBS, pH 7.4, and incubated with streptavidin-agarose beads (1-ml packed volume) overnight at 4 °C. The beads were washed four times with PBS, pH 7.4, and suspended in 1 ml of PBS. Lysates of unstimulated A20 cells were prepared as described above and incubated with 100 μl of the above peptide-streptavidin-agarose conjugate overnight at 4 °C. Beads were washed and boiled with SDS-PAGE sample buffer, and the bound material was separated by SDS-PAGE and subjected to silver staining or immunoblotting. Immunoprecipitates were separated on SDS-polyacrylamide gels and transferred to Immobilon P membranes. The blots were probed with the indicated antibodies and developed using the ECL detection reagents from Amersham Pharmacia Biotech. In the far Western blotting procedure, membranes were overlaid with 4 μg/ml recombinant GST protein or GST fused to the C-terminal SH2 domain of PI3K p85 in phosphate-buffered saline containing 5% bovine serum albumin, 0.1% Tween 20, and 1 mmdithiothreitol. The membranes were washed with buffer without dithiothreitol, reblocked, and incubated with rabbit polyclonal anti-GST antibodies. After washing, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG and developed with ECL reagents. A20 cells were labeled with32P and stimulated as described above. This was followed by extraction and deacylation of lipids and high performance liquid chromatography (HPLC) analysis of the glycerophosphoinositol head groups (22Auger K.R. Serunian L.A. Soltoff S.P. Libby P. Cantley L.C. Cell. 1989; 57: 167-175Abstract Full Text PDF PubMed Scopus (671) Google Scholar, 23Serunian L.A. Auger K.R. Cantley L.C. Methods Enzymol. 1991; 198: 78-87Crossref PubMed Scopus (130) Google Scholar). A20 cells were stimulated with F(ab′)2 or intact anti-IgG antibodies, and immunoprecipitates of PI3K were resolved by SDS-PAGE and probed for associated phosphotyrosine-containing proteins by Western blotting. A distinct phosphoprotein band migrating at ∼145 kDa coimmunoprecipitated with PI3K as early as 5 s after stimulation with intact antibody, but not with the F(ab′)2 antibody (Fig. 1A). Probing with anti-SHIP antiserum revealed the presence of SHIP at that position (Fig. 1B). To test whether SHIP was directly associated with PI3K or whether it was immunoprecipitated as part of the receptor complex by the stimulating intact anti-Ig antibody, the lysates were incubated with protein G-agarose beads alone prior to SDS-PAGE and Western blotting with anti-SHIP antibodies. Under those conditions, no 145-kDa band was seen in the protein G precipitates (data not shown). Cocross-linking of BCR with FcγRIIb1 through an intact IgG also enhanced the level of p85 in immunoprecipitates of SHIP (data not shown). Thus, coligation of BCR with FcγRIIb1 leads to the recruitment of PI3K to SHIP. Tyrosine phosphorylation of SHIP upon BCR/FcγRIIb1 coligation exceeds that obtained by cross-linking BCR alone (8Ono M. Bolland S. Tempst P. Ravetch J.V. Nature. 1996; 383: 263-266Crossref PubMed Scopus (643) Google Scholar, 14Chacko G.W. Tridandapani S. Damen J.E. Liu L. Krystal G. Coggeshall K.M. J. Immunol. 1996; 157: 2234-2238PubMed Google Scholar). Therefore, PI3K association with SHIP observed during coligation could be mediated by the binding of PI3K SH2 domains to phosphorylated tyrosine residues in SHIP. The p85 subunit of PI3K has two SH2 domains, one each at the N and C termini. The phosphotyrosine-containing motif recognized by these two domains includes a pYXXM sequence for the C-terminal SH2 domain and a more stringent pY(I/V/L)XM sequence for the N-terminal SH2 domain (24Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2370) Google Scholar). A GST fusion protein of the C-terminal SH2 domain was used to test for direct binding to SHIP. A20 cells were stimulated with F(ab′)2 or intact antibodies, and either SHIP or phosphotyrosine-containing proteins were immunoprecipitated from the lysates. In both cases, the GST-p85 SH2 fusion protein bound in a far Western blot to a protein of 145 kDa present in lysates of A20 cells stimulated with intact antibody (Fig. 2A, Expts. 1 and 2). Thus, p85 can bind directly to SHIP and to a tyrosine-phosphorylated protein that comigrated with SHIP on SDS-PAGE. GST alone did not bind SHIP under the same conditions (Fig. 2B), but it reacted with two nonspecific bands migrating at ∼135 and 140 kDa in anti-SHIP immunoprecipitates of both unstimulated and F(ab′)2- and intact anti-Ig-stimulated cell lysates. The presence of SHIP in the anti-phosphotyrosine and anti-SHIP immunoprecipitates is shown in Fig. 2C. Increased tyrosine phosphorylation of SHIP under conditions of BCR and FcγRIIb1 coligation is evident. Direct binding of the PI3K SH2 domain to SHIP by far Western blotting was also greater after receptor coligation than after cross-linking BCR alone. The SHIP cDNA was broadly divided into three regions encoding the SH2 domain designated as n, the central catalytic region containing the sequences conserved in several 5′-phosphatases designated as cat, and the C-terminal region designated as c (which contains sites for interaction with the PTB domains of Shc (12Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (560) Google Scholar, 25Liu L. Damen J.E. Hughes M.R. Babic I. Jirik F.R. Krystal G. J. Biol. Chem. 1997; 272: 8983-8988Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) and multiple prolines that interact with Grb2 (11Lioubin M.N. Algate P.A. Tsai S. Carlberg K. Aebersold R. Rohrschneider L.R. Genes Dev. 1996; 10: 1084-1095Crossref PubMed Scopus (377) Google Scholar)). Deletion mutants containing different combinations of these three domains (Fig. 3), namely ncat (∼105 kDa), nc (∼110 kDa), and catc (∼120 kDa), were inserted into recombinant vaccinia viruses and tested for their ability to bind PI3K. The deletion mutants were expressed in NIH 3T3 fibroblasts, immunoprecipitated following a stimulation with pervanadate, and subjected to far Western blotting with the GST-p85 SH2 fusion protein. All three mutants were tyrosine-phosphorylated upon pervanadate treatment (Fig. 4A), but only the nc and catc mutants bound the SH2 domain of PI3K (Fig. 4B). As these two molecules share only the C-terminal sequence of SHIP, the binding site must be in that region. The level of expression of all three deletion mutants was comparable (Fig. 4C). The deletion mutants ncat and nc also coimmunoprecipitated a protein at 52 kDa upon pervanadate stimulation (Fig. 4A), which could be the Shc adaptor protein associated with the N-terminal SH2 domain of SHIP. Amino acids 917–920 (YIGM) in the C-terminal region of SHIP correspond to a perfect motif for binding the N- and C-terminal SH2 domains of PI3K (24Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2370) Google Scholar). The tyrosine at position 917 is phosphorylated upon BCR/FcγRIIb1 coligation (25Liu L. Damen J.E. Hughes M.R. Babic I. Jirik F.R. Krystal G. J. Biol. Chem. 1997; 272: 8983-8988Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Twelve-amino acid-long peptides containing SHIP sequence 917–920 were synthesized with either unphosphorylated (YIGM) or phosphorylated (pYIGM) Tyr-917. Another phosphorylated peptide carrying the substitution M920R (pYIGR) and a phosphopeptide corresponding in sequence to the C-terminal PI3K-binding motif in CD19 (SLGSQSYEDMRG) were also synthesized for negative and positive controls, respectively. All biotinylated peptides were coupled to streptavidin-agarose beads and used to pull down proteins in A20 lysates. Two proteins of ∼85 and 110 kDa bound only to the CD19 peptide and the pYIGM peptide and not to the streptavidin-agarose beads alone or to pYIGR and unphosphorylated YIGM peptides (Fig. 5A). Immunoblotting with anti-PI3K antibodies revealed that these proteins comigrated with the p85 (Fig. 5B) and p110 (Fig. 5C) subunits of PI3K, respectively. Thus, the in vitro data shown in Figs. 4 and 5 suggest a possible mechanism by which SHIP binds PI3K upon B cell stimulation with intact anti-Ig antibodies or immune complexes. A potential outcome of the association of SHIP with PI3K in A20 cells stimulated with intact anti-Ig is the efficient production of PIP2, provided that SHIP and PI3K retain their catalytic activities. To test this possibility, A20 cells were stimulated with F(ab′)2 or intact antibodies for different times, and the total cellular levels of PIP2 and PIP3 were determined using a sensitive HPLC assay. Production of PIP2 upon FcγRIIb1 coligation was approximately two-thirds of that upon BCR stimulation alone (Fig. 6, upper panel). In contrast, there was a marked inhibition of the PI3K product PIP3 at early time points and complete loss at sustained time points (Fig. 6,lower panel). Activation of the serine/threonine kinase Akt requires binding of its pleckstrin homology domain to membrane-bound phosphatidylinositides (26Hemmings B.A. Science. 1997; 275: 628-630Crossref PubMed Scopus (434) Google Scholar, 27Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1043) Google Scholar). In particular, binding to PIP2 results in activation of Akt in vitro (28Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 628-630Crossref PubMed Scopus (1290) Google Scholar, 29Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (443) Google Scholar). Full Akt activation requires sequential phosphorylation by two kinases, the second of which phosphorylates serine 473 in Akt after binding to PIP3(30Downward J. Science. 1998; 279: 673-674Crossref PubMed Scopus (181) Google Scholar). We used phosphorylation of serine 473 as an indicator of Akt activation after signaling via BCR. A20 cells were stimulated with F(ab′)2 or intact antibodies for 2, 5, or 10 min, and active Akt was immunoprecipitated and immunoblotted using antibodies specific for phosphoserine 473. Fig. 7A reveals a large increase in the activity of Akt, which was clearly diminished during coligation of BCR with FcγRIIb1. To test whether PI3K activity is required for Akt activation upon B cell stimulation, two inhibitors, wortmannin and LY294002, were used. At low concentrations, these inhibitors block PI3K activity without affecting phosphoinositide 4-kinases (31Okada T. Sakuma L. Fukui Y. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3563-3567Abstract Full Text PDF PubMed Google Scholar). A20 cells were pretreated with wortmannin (Fig. 7B) or LY294002 (data not shown) and then stimulated with F(ab′)2 or intact antibodies for 2 min. Both BCR- and BCR/FcγRIIb1-induced Akt activities were completely lost upon inhibition of PI3K (Fig. 7B). Thus, PI3K activity persists during BCR/FcγRIIb1 coligation and is required for Akt activation. Coengagement of BCR with FcγRIIb1 results in a diminished transient calcium flux and a loss of sustained calcium flux (3Bijsterbosch M.K. Klaus G.G. J. Exp. Med. 1985; 162: 1825-1836Crossref PubMed Scopus (159) Google Scholar, 4Wilson H.A. Greenblatt D. Taylor C.W. Putney J.W. Tsien R.Y. Finkelman F.D. Chused T.M. J. Immunol. 1987; 138: 1712-1718PubMed Google Scholar, 5Choquet D. Partiseti M. Amigorena S. Bonnerot C. Fridman W.H. Korn H. J. Cell Biol. 1993; 121: 355-363Crossref PubMed Scopus (103) Google Scholar). The sustained calcium flux in BCR-triggered B cells requires activation of Btk, a member of the Tec kinase family that, in turn, activates phospholipase Cγ (19Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z.M. Lin S.Q. Gout I. Cantley L.C. Rawlings D.J. Kinet J.-P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar, 32Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (423) Google Scholar, 33Fluckiger A.-C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.-P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (355) Google Scholar, 34Bolland S. Pearse R.N. Kurosaki T. Ravetch J.V. Immunity. 1998; 8: 509-516Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Activation of Btk is dependent on the binding of its pleckstrin homology domain to PIP3 (19Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z.M. Lin S.Q. Gout I. Cantley L.C. Rawlings D.J. Kinet J.-P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar). A noticeable effect of FcγRIIb1 coligation is a drastic reduction of PIP3, otherwise produced very rapidly upon BCR triggering (19Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z.M. Lin S.Q. Gout I. Cantley L.C. Rawlings D.J. Kinet J.-P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar). The loss of PIP3 could be due to a reduced PI3K activity and conversion to PIP2 by a non-rate-limiting SHIP, to an increased SHIP activity, or to a complete loss of PI3K activity. However, the production of the SHIP metabolite PIP2 suggests that PI3K remains active during FcγRIIb1-mediated inhibition of the BCR activation signal. In addition, the direct association of PI3K with SHIP, demonstrated here, may serve to enhance the conversion of PIP3 to PIP2. Far Western blotting with the C-terminal SH2 domain of the p85 subunit of PI3K mapped the site of interaction to the C-terminal region of SHIP. Synthetic phosphopeptides that included sequences flanking tyrosine 917 of SHIP bound PI3K in cell lysates. The inducible association of PI3K with tyrosine-phosphorylated SHIP described here is different from the constitutive association of PI3K with an unidentified PIP3 5-phosphatase activity in human platelets (35Jackson S.P. Schoenwaelder S.M. Matzaris M. Brown S. Mitchell C.A. EMBO J. 1995; 14: 4490-4500Crossref PubMed Scopus (74) Google Scholar). The novel 5-phosphatase reported in that study is distinct from SHIP since its catalytic activity in vitro was limited to the substrate PIP3. Production of PIP2 during BCR/FcγRIIb1 coligation was consistently less than during BCR-mediated activation. This is probably due, in part, to a lower activity of PI3K and hence lower production of the SHIP substrate PIP3. As CD19 is dephosphorylated rapidly after BCR/FcγRIIb1 coligation (6Kiener P.A. Lioubin M.N. Rohrschneider L.R. Ledbetter J.A. Nadler S.G. Diegel M.L. J. Biol. Chem. 1997; 272: 3838-3844Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 7Hippen K.L. Buhl A.M. D'Ambrosio D. Nakamura K. Persin C. Cambier J.C. Immunity. 1997; 7: 49-58Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), a major source of PI3K activation is lost. Recruitment of PI3K by tyrosine-phosphorylated SHIP may serve to compensate for this loss. However, SHIP is not absolutely required for PI3K activation in avian DT40 B cells because a sustained calcium signal was observed after BCR/FcγRIIb1 coengagement in a SHIP-negative DT40 mutant cell (10Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). It is also possible that the rapid conversion of PIP3 by SHIP affects PI3K activation directly, or indirectly through a diminished PIP3-dependent activation of Ras (via Sos) (36Chen R.H. Corbalan-Garcia S. Bar-Sagi D. EMBO J. 1997; 16: 1351-1359Crossref PubMed Scopus (114) Google Scholar,37Rameh L.E. Arvidsson A. Carraway III, K.L. Couvillon A.D. Rathbun G. Crompton A. VanRenterghem B. Czech M.P. Ravichandran K.S. Burakoff S.J. Wang D.S. Chen C.S. Cantley L.C. J. Biol. Chem. 1997; 272: 22059-22066Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). To clearly address whether the catalytic activity of SHIP and/or PI3K is responsible for the observed pattern of PIP3 and PIP2 production, an inhibitor of SHIP phosphatase activity would be necessary. PIP2 and PIP3 control the activation of Akt by recruiting the pleckstrin homology domains of Akt and of another serine/threonine kinase that phosphorylates Akt (26Hemmings B.A. Science. 1997; 275: 628-630Crossref PubMed Scopus (434) Google Scholar, 27Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1043) Google Scholar, 28Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 628-630Crossref PubMed Scopus (1290) Google Scholar, 29Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (443) Google Scholar, 30Downward J. Science. 1998; 279: 673-674Crossref PubMed Scopus (181) Google Scholar). Akt delivers an anti-apoptotic signal by phosphorylating the pro-apoptotic molecule BAD, a member of the Bcl-2 protein family (38Yang E. Zha J. Jockel J. Boise L.H. Thompson C.B. Korsmeyer S.J. Cell. 1995; 80: 285-291Abstract Full Text PDF PubMed Scopus (1879) Google Scholar, 39White E. Genes Dev. 1996; 10: 1-15Crossref PubMed Scopus (1323) Google Scholar). Our data show residual activation of Akt during BCR/FcγRIIb1 coligation as measured by Akt phosphorylation on serine 473. This remaining Akt activation is in contrast to the complete loss of the sustained calcium flux mediated by the PIP3-dependent Tec kinase Btk during BCR/FcγRIIb1 coligation (19Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z.M. Lin S.Q. Gout I. Cantley L.C. Rawlings D.J. Kinet J.-P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar). The wortmannin sensitivity of Akt activation strongly suggests that PI3K activity is also retained. Although apoptosis of B cells after BCR/FcγRIIb1 coligation can occur and may even exceed that observed after BCR-mediated activation (40Ashman R.F. Peckham D. Stunz L.L. J. Immunol. 1996; 157: 5-11PubMed Google Scholar), the SHIP/PI3K/Akt pathway described here may lead to at least some anti-apoptotic signal. An anti-apoptotic effect of SHIP after BCR/FcγRIIb1 coligation has been suggested by the observation of increased apoptosis of DT40 cells deficient in SHIP and of DT40 cells expressing a mutant FcγRIIb1 that fails to bind SHIP (10Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). A pro-apoptotic mediator that binds to FcγRIIb1 was proposed to explain these observations (10Ono M. Okada H. Bolland S. Yanagi S. Kurosaki T. Ravetch J.V. Cell. 1997; 90: 293-301Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). On the other hand, the reduced survival of DT40 cells expressing the mutated FcγRIIb1 that fails to recruit SHIP may have been caused by the lack of SHIP-mediated PIP2production and, in turn, by a reduced Akt-mediated survival signal. In conclusion, this study demonstrates an association of the p85 subunit of PI3K with the inositol phosphatase SHIP in response to coligation of BCR with the inhibitory receptor FcγRIIb1. PI3K activity and PIP2 production were not abrogated by FcγRIIb1 ligation to BCR. We suggest that the physical association of SHIP and PI3K may provide a novel mode of PI3K activation and an enhanced conversion of PIP3 to PIP2. We thank M. Lioubin and L. Rohrschneider for deletion mutants of SHIP, L. Samelson for helpful suggestions, and M. Weston for technical assistance." @default.
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