Mutational analysis reveals multiple distinct sites within Fc gamma receptor IIB that function in inhibitory signaling

The low-affinity receptor for IgG, FcgammaRIIB, functions broadly in the immune system, blocking mast cell degranulation, dampening the humoral immune response, and reducing the risk of autoimmunity. Previous studies concluded that inhibitory signal transduction by FcgammaRIIB is mediated solely by its immunoreceptor tyrosine-based inhibition motif (ITIM) that, when phosphorylated, recruits the SH2-containing inositol 5'- phosphatase SHIP and the SH2-containing tyrosine phosphatases SHP-1 and SHP-2. The mutational analysis reported here reveals that the receptor's C-terminal 16 residues are also required for detectable FcgammaRIIB association with SHIP in vivo and for FcgammaRIIB-mediated phosphatidylinositol 3-kinase hydrolysis by SHIP. Although the ITIM appears to contain all the structural information required for receptor-mediated tyrosine phosphorylation of SHIP, phosphorylation is enhanced when the C-terminal sequence is present. Additionally, FcgammaRIIB-mediated dephosphorylation of CD19 is independent of the cytoplasmic tail distal from residue 237, including the ITIM. Finally, the findings indicate that tyrosines 290, 309, and 326 are all sites of significant FcgammaRIIB1 phosphorylation following coaggregation with B cell Ag receptor. Thus, we conclude that multiple sites in FcgammaRIIB contribute uniquely to transduction of FcgammaRIIB-mediated inhibitory signals.     

Two distinct tyrosine-based motifs enable the inhibitory receptor FcgammaRIIB to cooperatively recruit the inositol phosphatases SHIP1/2 and the adapters Grb2/Grap

FcgammaRIIB are low-affinity receptors for IgG that contain an immunoreceptor tyrosine-based inhibition motif (ITIM) and inhibit immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. When coaggregated with ITAM-bearing receptors, FcgammaRIIB become tyrosyl-phosphorylated and recruit the Src homology 2 (SH2) domain-containing inositol 5'-phosphatases SHIP1 and SHIP2, which mediate inhibition. The FcgammaRIIB ITIM was proposed to be necessary and sufficient for recruiting SHIP1/2. We show here that a second tyrosine-containing motif in the intracytoplasmic domain of FcgammaRIIB is required for SHIP1/2 to be coprecipitated with the receptor. This motif functions as a docking site for the SH2 domain-containing adapters Grb2 and Grap. These adapters interact via their C-terminal SH3 domain with SHIP1/2 to form a stable receptor-phosphatase-adapter trimolecular complex. Both Grb2 and Grap are required for an optimal coprecipitation of SHIP with FcgammaRIIB, but one adapter is sufficient for the phosphatase to coprecipitate in a detectable manner with the receptors. In addition to facilitating the recruitment of SHIPs, the second tyrosine-based motif may confer upon FcgammaRIIB the properties of scaffold proteins capable of altering the composition and stability of the signaling complexes generated following receptor engagement.     

Src homology 2 domain-containing inositol 5-phosphatase 1 mediates cell cycle arrest by FcgammaRIIB

We previously found that low affinity receptors for the Fc portion of IgG, FcgammaRIIB, which are widely expressed by hematopoietic cells, can negatively regulate receptor tyrosine kinase-dependent cell proliferation. We investigated here the mechanisms of this inhibition. We used as experimental models wild-type mast cells, which constitutively express the stem cell factor receptor Kit and FcgammaRIIB, FcgammaRIIB-deficient mast cells reconstituted with wild-type or mutated FcgammaRIIB, and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1)-deficient mast cells. We found that, upon coaggregation with Kit, FcgammaRIIB are tyrosyl-phosphorylated, recruit SHIP1, but not SHIP2, SH2 domain-containing protein tyrosine phosphatase-1 or -2, abrogate Akt phosphorylation, shorten the duration of the activation of mitogen-activated protein kinases of the Ras and Rac pathways, abrogate cyclin induction, prevent cells from entering the cell cycle, and block thymidine incorporation. FcgammaRIIB-mediated inhibition of Kit-dependent cell proliferation was reduced in SHIP1-deficient mast cells, whereas inhibition of IgE-induced responses was abrogated. Cell proliferation was, however, inhibited by coaggregating Kit with FcgammaRIIB whose intracytoplasmic domain was replaced with the catalytic domain of SHIP1. These results demonstrate that FcgammaRIIB use SHIP1 to inhibit pathways shared by receptor tyrosine kinases and immunoreceptors to trigger cell proliferation and cell activation, respectively, but that, in the absence of SHIP1, FcgammaRIIB can use other effectors that specifically inhibit cell proliferation.     

Partially distinct molecular mechanisms mediate inhibitory FcgammaRIIB signaling in resting and activated B cells.

FcgammaRIIB functions as an inhibitory receptor to dampen B cell Ag receptor signals and immune responses. Accumulating evidence indicates that ex vivo B cells require the inositol 5-phosphatase, Src homology domain 2-containing inositol 5-phosphatase (SHIP), for FcgammaRIIB-mediated inhibitory signaling. However, we report here that LPS-activated primary B cells do not require SHIP and thus differ from resting B cells. SHIP-deficient B cell blasts display efficient FcgammaRIIB-dependent inhibition of calcium mobilization as well as Akt and extracellular signal-related protein kinase phosphorylation. Surprisingly, FcgammaRIIB-dependent degradation of phosphatidylinositol 3,4,5-trisphosphate and conversion into phosphatidylinositol 3,4-bisphosphate occur in SHIP-deficient B cell blasts, demonstrating the function of an additional inositol 5-phosphatase. Further analysis reveals that while resting cells express only SHIP, B cell blasts also express the recently described inositol 5-phosphatase, SHIP-2. Finally, data suggest that both SHIP-2 and SHIP can mediate downstream biologic consequences of FcgammaRIIB signaling, including inhibition of the proliferative response.     

Phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3)/Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals.

Tec family non-receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B-cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N-terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase-dependent phospholipase Cgamma (PLCgamma) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2-containing inositol phosphatase (SHIP)-dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby PtdIns-3,4,5-P3 regulates receptor-dependent calcium signals through the function of Tec kinases.     

Molecular basis of the recruitment of the SH2 domain-containing inositol 5-phosphatases SHIP1 and SHIP2 by fcgamma RIIB

FcgammaRIIB are single-chain low affinity receptors for IgG that negatively regulate immunoreceptor tyrosine-based activation motif-dependent cell activation. They bear one immunoreceptor tyrosine-based inhibition motif (ITIM) that becomes tyrosyl-phosphorylated upon coaggregation of FcgammaRIIB with immunoreceptor tyrosine-based activation motif-bearing receptors and that recruits SH2 domain-containing inositol 5-phosphatases (SHIPs) in vivo. Synthetic FcgammaRIIB ITIM phosphopeptides, however, also bind SH2 domain-containing protein-tyrosine phosphatases (SHPs) in vitro. To identify SHIP-binding sites, we exchanged residues between the FcgammaRIIB ITIM and the N-terminal ITIM of a killer cell Ig-like receptor that does not bind SHIPs. Loss of function and gain of function substitutions identified the Y+2 leucine, in the FcgammaRIIB ITIM, as determining the binding of both SHIP1 and SHIP2, but not the binding of SHP-1 or SHP-2. Conversely, the Y-2 isoleucine that determines the in vitro binding of SHP-1 and SHP-2 affected neither the binding nor the recruitment of SHIP1 or SHIP2. One hydrophobic residue, in the ITIM of FcgammaRIIB therefore determines the affinity for SHIPs. This residue is symmetrical to the hydrophobic residue that determines the affinity of all ITIMs for SHPs. It defines a SHIP-binding site, distinct from a SHP-binding site, that enables FcgammaRIIB to recruit SHIP1 and SHIP2 and that is preferentially used in vivo.     

p135 src homology 2 domain-containing inositol 5'-phosphatase (SHIPbeta ) isoform can substitute for p145 SHIP in fcgamma RIIB1-mediated inhibitory signaling in B cells

The inositol 5'-phosphatase, SHIP (also referred to as SHIP-1 or SHIPalpha), is expressed in all cells of the hematopoietic lineage. Depending on the cell type being investigated and the state of differentiation, SHIP isoforms of several different molecular masses (170, 160, 145, 135, 125, and 110 kDa) have been seen in immunoblots. However, the function of the individual isoforms and the effect of expressing multiple isoforms simultaneously are not understood. Some of these SHIP isoforms have recently been characterized at the level of primary sequence. In this report, we investigated the function of the recently characterized 135-kDa SHIP isoform (SHIPbeta), which appears to possess the catalytic domain but lacks some of the protein-protein interaction motifs at the C terminus. By reconstituting SHIP-deficient DT40 B cells with either SHIPbeta or the better-characterized p145 SHIPalpha, we addressed the function of SHIPbeta in the complete absence of SHIPalpha. We observed that SHIPbeta had enzymatic activity comparable with SHIPalpha and that SHIPbeta was able to reconstitute F(c)gammaRIIB1-mediated inhibition of B cell receptor-induced signaling events such as calcium flux and Akt and mitogen-activated protein kinase activation. SHIPbeta was readily phosphorylated in response to B cell receptor cross-linking with the inhibitory receptor F(c)gammaRIIB1 and SHIPbeta also interacted with the adapter protein Shc. During these studies we also observed that the SHIPalpha or SHIPbeta interaction with Grb2 is not required for F(c)gammaRIIB1-mediated inhibition of calcium flux. These data suggest that SHIPbeta, which is normally expressed in B cells along with SHIPalpha, functions comparably with SHIPalpha and that these two isoforms are not likely to be antagonistic in their function in vivo.     

Dynamic interactions of Fc gamma receptor IIB with filamin-bound SHIP1 amplify filamentous actin-dependent negative regulation of Fc epsilon receptor I signaling

The engagement of high affinity receptors for IgE (FcepsilonRI) generates both positive and negative signals whose integration determines the intensity of mast cell responses. FcepsilonRI-positive signals are also negatively regulated by low affinity receptors for IgG (FcgammaRIIB). Although the constitutive negative regulation of FcepsilonRI signaling was shown to depend on the submembranous F-actin skeleton, the role of this compartment in FcgammaRIIB-dependent inhibition is unknown. We show in this study that the F-actin skeleton is essential for FcgammaRIIB-dependent negative regulation. It contains SHIP1, the phosphatase responsible for inhibition, which is constitutively associated with the actin-binding protein, filamin-1. After coaggregation, FcgammaRIIB and FcepsilonRI rapidly interact with the F-actin skeleton and engage SHIP1 and filamin-1. Later, filamin-1 and F-actin dissociate from FcR complexes, whereas SHIP1 remains associated with FcgammaRIIB. Based on these results, we propose a dynamic model in which the submembranous F-actin skeleton forms an inhibitory compartment where filamin-1 functions as a donor of SHIP1 for FcgammaRIIB, which concentrate this phosphatase in the vicinity of FcepsilonRI and thereby extinguish activation signals.     

CDw150 associates with src-homology 2-containing inositol phosphatase and modulates CD95-mediated apoptosis

CDw150, a receptor up-regulated on activated T or B lymphocytes, has a key role in regulating B cell proliferation. Patients with X-linked lymphoproliferative disease have mutations in a gene encoding a protein, DSHP/SAP, which interacts with CDw150 and is expressed in B cells. Here we show that CDw150 on B cells associates with two tyrosine-phosphorylated proteins, 59 kDa and 145 kDa in size. The 59-kDa protein was identified as the Src-family kinase Fgr. The 145-kDa protein is the inositol polyphosphate 5'-phosphatase, SH2-containing inositol phosphatase (SHIP). Both Fgr and SHIP interact with phosphorylated tyrosines in CDw150's cytoplasmic tail. Ligation of CDw150 induces the rapid dephosphorylation of both SHIP and CDw150 as well as the association of Lyn and Fgr with SHIP. CD95/Fas-mediated apoptosis is enhanced by signaling via CDw150, and CDw150 ligation can override CD40-induced rescue of CD95-mediated cell death. The ability of CDw150 to regulate cell death does not correlate with serine phosphorylation of the Akt kinase, but does correlate with SHIP tyrosine dephosphorylation. Thus, the CDw150 receptor may function to regulate the fate of activated B cells via SHIP as well as via the DSHP/SAP protein defective in X-linked lymphoproliferative disease patients.     

Heterologous regulation of chemokine receptor signaling by the lipid phosphatase SHIP in lymphocytes

The SH2 domain-containing inositol polyphosphate 5-phosphatase (SHIP) is known to play an important role in the negative regulation by FcgammaRIIB of PI3K-dependent signaling cascades activated by the B cell antigen receptor (BCR) as well as several tyrosine-kinase coupled cytokine receptors. However, to date the role of SHIP in the regulation of PI3K-dependent signals elicited by G-protein-coupled receptors (GPCR) such as chemokine receptors has not been investigated. In this study, we report that ligation of the G-protein-coupled chemokine receptor CXCR4 by SDF-1/CXCL12 has no effect on the tyrosine phosphorylation of SHIP in the murine B cell lymphoma A20. However, co-ligation of the B cell antigen receptor and FcgammaRIIB inhibits the PI3K-dependent phosphorylation of PKB and ERK1/2 in response to CXCL12. We have also utilised a constitutively active membrane-localised SHIP mutant expressed in the Jurkat leukaemic T cell line (which do not normally express SHIP), in order to investigate the effect of this mutant on CXCL12 stimulated PI3K-dependent signaling events. Experiments have revealed that CXCL12-mediated PKB phosphorylation, chemotaxis and lipid accumulation are inhibited in the presence of this SHIP mutant. Thus, it appears that heterologous activation of SHIP by non-G-protein-coupled receptor-mediated routes can impinge on PI3K-dependent signaling pathways activated by independently ligated G-protein-coupled chemokine receptors.     

The pseudo-immunoreceptor tyrosine-based activation motif of CD5 mediates its inhibitory action on B-cell receptor signaling

Genetic studies revealed that CD5 could be a negative regulator of the B-cell antigen receptor (BCR). We explore here the effect of human CD5 on BCR-triggered responses. B cells were obtained expressing a chimera composed of extracellular and transmembrane domains of Fcgamma type IIB receptor fused to CD5 cytoplasmic domain (CD5cyt). Coligation of the chimera with the BCR induces CD5cyt tyrosine phosphorylation. A rapid inhibition of BCR-induced calcium response is observed, as well as a partial but delayed inhibition of phospholipase Cgamma-1 phosphorylation. Activation of extracellular regulated kinase-2 is also severely impaired. Moreover, at the functional level, interleukin-2 production is abolished. Src homology 2 domain-bearing tyrosine phosphatase SHP-1 and Src homology 2 domain-bearing inositol 5'-phosphatase SHIP usually participate in negative regulation of the BCR. We show that they do not associate with the phosphorylated CD5 chimera. We finally demonstrate that the pseudo-immunoreceptor tyrosine based activation motif present in CD5cyt is involved because its deletion eliminates the inhibitory effect of the chimera, both at biochemical and functional levels. These results demonstrate the inhibitory role of CD5 pseudo-immunoreceptor tyrosine based activation motif tyrosine phosphorylation on BCR signaling. They further support the idea that CD5 uses mechanisms different from those already described to negatively regulate the BCR pathway.     

CD22 forms a quaternary complex with SHIP, Grb2, and Shc. A pathway for regulation of B lymphocyte antigen receptor-induced calcium flux

CD22 is a cell surface molecule that regulates signal transduction in B lymphocytes. Tyrosine-phosphorylated CD22 recruits numerous cytoplasmic effector molecules including SHP-1, a potent phosphotyrosine phosphatase that down-regulates B cell antigen receptor (BCR)- and CD19-generated signals. Paradoxically, B cells from CD22-deficient mice generate augmented intracellular calcium responses following BCR ligation, yet proliferation is decreased. To understand further the mechanisms through which CD22 regulates BCR-dependent calcium flux and proliferation, interactions between CD22 and effector molecules involved in these processes were assessed. The adapter proteins Grb2 and Shc were found to interact with distinct and specific regions of the CD22 cytoplasmic domain. Src homology-2 domain-containing inositol polyphosphate-5'-phosphatase (SHIP) also bound phosphorylated CD22, but binding required an intact CD22 cytoplasmic domain. All three molecules were bound to CD22 when isolated from BCR-stimulated splenic B cells, indicating the formation of a CD22.Grb2.Shc.SHIP quaternary complex. Therefore, SHIP associating with CD22 may be important for SHIP recruitment to the cell surface where it negatively regulates calcium influx. Although augmented calcium responses in CD22-deficient mice should facilitate enhanced c-Jun N-terminal kinase (JNK) activation, BCR ligation did not induce JNK activation in CD22-deficient B cells. These data demonstrate that CD22 functions as a molecular "scaffold" that specifically coordinates the docking of multiple effector molecules, in addition to SHP-1, in a context necessary for BCR-dependent SHIP activity and JNK stimulation.     

Recruitment and phosphorylation of SH2-containing inositol phosphatase and Shc to the B-cell Fc gamma immunoreceptor tyrosine-based inhibition motif peptide motif.

Recently, we and others have demonstrated that negative signaling in B cells selectively induces the tyrosine phosphorylation of a novel inositol polyphosphate phosphatase, p145SHIP. In this study, we present data indicating that p145SHIP binds directly a phosphorylated motif, immunoreceptor tyrosine-based inhibition motif (ITIM), present in the cytoplasmic domain of Fc gammaRIIB1. Using recombinant SH2 domains, we show that binding is mediated via the Src homology region 2 (SH2)-containing inositol phosphatase (SHIP) SH2 domain. SHIP also bound to a phosphopeptide derived from CD22, raising the possibility that SHIP contributes to negative signaling by this receptor as well as Fc gammaRIIB1. The association of SHIP with the ITIM phosphopeptide was activation independent, while coassociation with Shc was activation dependent. Furthermore, experiments with Fc gammaRIIB1-deficient B cells demonstrated a genetic requirement for expression of Fc gammaRIIB1 in the induction of SHIP phosphorylation and its interaction with Shc. Based on these results, we propose a model of negative signaling in which co-cross-linking of surface immunoglobulin and Fc gammaRIIB1 results in sequential tyrosine phosphorylation of the ITIM, recruitment and phosphorylation of p145SHIP, and subsequent binding of Shc.     

SHIP2 multiple functions: a balance between a negative control of PtdIns(3,4,5)P₃ level, a positive control of PtdIns(3,4)P₂ production, and intrinsic docking properties

The SH2 domain containing inositol 5-phosphatase 2 (SHIP2) belongs to the family of the mammalian inositol polyphosphate 5-phosphatases. The two closely related isoenzymes SHIP1 (or SHIP) and SHIP2 contain a N-terminal SH2 domain, a catalytic domain, potential PTB domain-binding sites (NPXY), and C-terminal proline-rich regions with consensus sites for SH3 domain interactions. In addition, SHIP2 contains a unique sterile alpha motif (SAM) domain that could be involved in SAM-SAM domain interactions with other proteins or receptors. SHIP2 also shows the presence of an ubiquitin interacting motif at the C-terminal end. SHIP2 is essentially a PI(3,4,5)P(3) 5-phosphatase that negatively controls PI(3,4,5)P(3) levels in intact cells and produce PI(3,4)P(2) . Depending on the cells and stimuli, PI(3,4)P(2) could accumulate at important levels and be a "second messenger" by its own. It could interact with a very large number of target proteins such as PKB or TAPP1 and 2 that control insulin sensitivity. In addition to its catalytic activity, SHIP2 is also a docking protein for a large number of proteins: Cytoskeletal, focal adhesion proteins, scaffold proteins, adaptors, protein phosphatases, and tyrosine kinase associated receptors. These interactions could play a role in the control of cell adhesion, migration, or endocytosis of some receptors. SHIP2 could be acting independently of its phosphatase activity being part of a protein network of some receptors, e.g., the EGF receptor or BCR/ABL. These non-catalytic properties associated to a PI phosphatase have also been reported for other enzymes of the metabolism of myo-inositol such as Ins(1,4,5)P(3) 3-kinases, inositol phosphate multikinase (IPMK), or PTEN.     

The pre-B cell receptor signaling for apoptosis is negatively regulated by Fc gamma RIIB.

Many studies have shown that FcgammaRIIB is a negative regulator of B cell receptor signaling, and even though FcgammaRIIB is expressed through all developmental stages of the B cell lineage, its involvement in pre-B cell receptor (pre-BCR) signaling has not been examined. To investigate FcgammaRIIB function at the pre-B cell stage, we have established pre-BCR positive pre-B cell lines from normal mice and FcgammaRIIB-deficient mice, named PreBR and Fcgamma(-/-)PreBR, respectively. These cell lines are able to differentiate into immature B cells in vitro by removal of IL-7. In PreBR, apoptosis was moderately induced by F(ab')(2) anti-mu Ab, but not by intact anti-mu Ab. Phosphorylation of SH2-containing inositol 5-phosphatase (SHIP) and Dok, which are involved in FcgammaRIIB signaling, was induced by anti-mu cross-linking in PreBR. In contrast, apoptosis was strongly induced by both the F(ab')(2) and intact anti-mu Abs in Fcgamma(-/-)PreBR, and the level of phosphorylation of SHIP or Dok was much lower in Fcgamma(-/-)PreBR than those observed in PreBR. Restoration of FcgammaRIIB to Fcgamma(-/-)PreBR followed by anti-mu cross-linking blocked severe apoptosis, and up-regulated SHIP and Dok phosphorylation. The results demonstrate that FcgammaRIIB negatively regulates pre-BCR-mediated signaling for apoptosis.     

The Src homology 2 domain containing inositol 5-phosphatase SHIP2 is recruited to the epidermal growth factor (EGF) receptor and dephosphorylates phosphatidylinositol 3,4,5-trisphosphate in EGF-stimulated COS-7 cells

The lipid phosphatase SHIP2 (Src homology 2 domain containing inositol 5-phosphatase 2) has been shown to be expressed in nonhemopoietic and hemopoietic cells. It has been implicated in signaling events initiated by several extracellular signals, such as epidermal growth factor (EGF) and insulin. In COS-7 cells, SHIP2 was tyrosine-phosphorylated at least at two separated tyrosine phosphorylation sites in response to EGF. SHIP2 was coimmunoprecipitated with the EGF receptor (EGFR) and also with the adaptor protein Shc. A C-terminal truncated form of SHIP2 that lacks the 366 last amino acids, referred to as tSHIP2, was also precipitated with the EGFR when transfected in COS-7 cells. The Src homology 2 domain of SHIP2 was unable to precipitate the EGFR in EGF-stimulated cells. Moreover, when transfected in COS-7 cells, it could not be detected in immunoprecipitates of the EGFR. When the His-tagged full-length enzyme was expressed in COS-7 cells and stained with anti-His6 monoclonal antibody, a signal was observed at plasma membranes in EGF-stimulated cells that colocalize with the EGFR by double staining. Upon stimulation by EGF, phosphatidylinositol 3,4,5-trisphosphate and protein kinase B activity were decreased in SHIP2-transfected COS-7 cells as compared with the vector alone. SHIP2 appears therefore in a tyrosine-phosphorylated complex with at least two other proteins, the EGFR and Shc.     

Enzymatic activity of the Src homology 2 domain-containing inositol phosphatase is regulated by a plasma membrane location

The negative regulatory role of the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) has been invoked in a variety of receptor-mediated signaling pathways. In B lymphocytes, co-clustering of antigen receptor surface immunoglobulin with FcgammaRIIb promotes the negative effects of SHIP, but how SHIP activity is regulated is unknown. To explore this issue, we investigated the effect of SHIP phosphorylation, receptor tyrosine engagement by its Src homology 2 domain, and membrane recruitment of SHIP on its enzymatic activity. We examined two SHIP phosphorylation kinase candidates, Lyn and Syk, and observed that the Src protein-tyrosine kinase, Lyn is far superior to Syk in its ability to phosphorylate SHIP both in vitro and in vivo. However, we found a minimal effect of phosphorylation or receptor tyrosine engagement of SHIP on its enzymatic activity, whereas membrane localization of SHIP significantly reduced cellular phosphatidylinositol 3,4, 5-triphosphate levels. Based on our results, we propose that a membrane localization of SHIP is the crucial event in the induction of its phosphatase effects.     

The SH2 domain-containing inositol 5'-phosphatase (SHIP) recruits the p85 subunit of phosphoinositide 3-kinase during FcgammaRIIb1-mediated inhibition of B cell receptor signaling

Coligation of FcgammaRIIb1 with the B cell receptor (BCR) or FcepsilonRI 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 FcgammaRIIb1 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 FcgammaRIIb1 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 FcgammaRIIb1 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 FcgammaRIIb1 coligation. The association of PI3K with SHIP may serve to activate PI3K and to regulate downstream events such as B cell activation-induced apoptosis.     

Effects of Src homology domain 2 (SH2)-containing inositol phosphatase (SHIP), SH2-containing phosphotyrosine phosphatase (SHP)-1, and SHP-2 SH2 decoy proteins on Fc gamma RIIB1-effector interactions and inhibitory functions

Coaggregation of Fc gamma RIIB1 with B cell Ag receptors (BCR) leads to inhibition of BCR-mediated signaling via recruitment of Src homology domain 2 (SH2)-containing phosphatases. In vitro peptide binding experiments using phosphotyrosine-containing sequences derived from the immunoreceptor tyrosine-based inhibitory motif (ITIM) known to mediate Fc gamma RIIB1 effects suggest that the receptor uses SH2-containing inositol phosphatase (SHIP) and SH2-containing phosphotyrosine phosphatase (SHP)-1, as well as SHP-2 as effectors. In contrast, coimmunoprecipitation studies of receptor-effector associations suggest that the predominant Fc gamma RIIB1 effector protein is SHIP. However, biologically significant interactions may be lost in such studies if reactants' dissociation rates (Kd) are high. Thus, it is unclear to what extent these assays reflect the relative recruitment of SHIP, SHP-1, and SHP-2 to the receptor in vivo. As an alternative approach to this question, we have studied the effects of ectopically expressed SHIP, SHP-1, or SHP-2 SH2-containing decoy proteins on Fc gamma RIIB1 signaling. Results demonstrate the SHIP is the predominant intracellular ligand for the phosphorylated Fc gamma RIIB1 ITIM, although the SHP-2 decoy exhibits some ability to bind Fc gamma RIIB1 and block Fc receptor function. The SHIP SH2, while not affecting Fc gamma RIIB1 tyrosyl phosphorylation, blocks receptor-mediated recruitment of SHIP, SHIP phosphorylation, recruitment of p52 Shc, phosphatidylinositol 3,4,5-trisphosphate hydrolysis, inhibition of mitogen-activated protein kinase activation, and, albeit more modestly, Fc gamma RIIB1 inhibition of Ca2+ mobilization. Taken together, results implicate ITIM interactions with SHIP as a major mechanism of Fc gamma RIIB1-mediated inhibitory signaling.     

Cloning of the genomic locus of mouse SH2 containing inositol 5-phosphatase (SHIP) and a novel 110-kDa splice isoform, SHIPdelta

The SH2 domain containing inositol 5'-phosphatase (SHIP) was initially described as a 145-kDa protein phosphorylated on tyrosines upon growth factor and cytokine stimulation. It was shown to be phosphorylated after Fc and B cell receptor activation and plays a role in negative signaling. Different isoforms of the SHIP protein result from alternative mRNA splicing, proteolysis, or a combination of both. The expression of discrete SHIP isoforms changes with the potential developmental-dependent maturation state of myeloid cells, suggesting mechanisms for the regulation of SHIP interactions with other signaling molecules. A p135 (SHIPbeta) spliced isoform is known to be expressed in developing myeloid cells. Now we have identified a new SHIP isoform, SHIPdelta, which is the product of an out-of-frame splice with a deletion of 167 nucleotides in the C-terminal region, resulting in an approximately 110-kDa protein. Biochemically, SHIPdelta differs from SHIPalpha by exhibiting little or no tyrosine phosphorylation or association with the signaling protein Shc after M-CSF activation of FD-Fms cells. In addition, we have characterized the structure of the entire SHIP genomic locus, which provides a basis for understanding the alternative splicing events. SHIP is expressed in hematopoiesis and spermatogenesis, and we also describe the promoter for the SHIP gene, which has potential for explaining the tissue-specific expression pattern.