The SH2 domain-containing inositol 5-phosphatase SHIP1 is recruited to the intracytoplasmic domain of human FcγRIIB and is mandatory for negative regulation of B cell activation (original) (raw)
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Immunology Letters, 2000
FcgRIIB are single-chain low-affinity receptors for IgG that bear an immunoreceptor tyrosine-based inhibition motif (ITIM) in their intracytoplasmic domain and that negatively regulate immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. In B cells, coaggregation of the B cell receptor (BCR) and FcgRIIB leads to an inhibition of B cell activation. Inhibitory properties of FcgRIIB have been related to the recruitment of SHIP, an SH2 domain-containing inositol 5-phosphatase (referred to as SHIP1), via ITIM phosphorylated FcgRIIB. Here, we demonstrate that the second SH2 domain-containing inositol 5-phosphatase SHIP2 could also bind to the FcgRIIB ITIM. As a model, a FcgRIIB deficient B cell line (IIA1.6), transfected with a cDNA encoding either w.t. FcgRIIB1% or FcgRIIB1% whose ITIM tyrosine was mutated has been used. SHIP2 tyrosine phosphorylation and association to the adaptator protein Shc were only found in transfectants expressing w.t. FcgRIIB1%. SHIP2 was also found to bind to a phosphopeptide corresponding to the ITIM sequence of FcgRIIB. There was no binding to the nonphosphorylated peptide. Finally, both SHIP2 and SHIP1 were coprecipitated with FcgRIIB1% upon coaggregation with BCR in IIA1.6 transfectants.
Immunology Letters, 1997
Co-clustering of FckRIIb and B-cell receptor (BCR) inhibits cell activation by interrupting BCR stimulated signal transduction. The immunoreceptor tyrosine-based inhibitory motif (ITIM) of FckRIIb becomes tyrosyl phosphorylated (P-ITIM) upon co-clustering with BCR then P-ITIM interacts with several signalling molecules, some of which negatively regulate the cell activation process. The molecules recruited by the P-ITIM of human FckRIIb have not been characterised yet. In order to affinity isolate the potential functional partner molecules of human FckRIIb, synthetic peptides were designed to cover almost the entire intracellular FckRIIb domain, including FckRIIb1 and FckRIIb2 specific sequences and stretches containing the phosphorylated and non-phosphorylated ITIM. We report here that several tyrosyl phosphorylated proteins bind to the P-ITIM peptide from both resting and activated B-cell lysates, the 53-56 kDa being the most prominent one. A fraction of the 53 -56 kDa bands were identified as the protein tyrosine kinase (PTK), Lyn which also bound to ITIM peptide, pointing to its role in initiating FckRIIb-mediated negative regulation. Among the P-ITIM associated tyr phosphorylated components, the 145 kDa one was identified as the inositol polyphosphate 5-phosphatase, SHIP and the 72 kDa protein as the protein tyrosine phosphatase (PTP) SHP2, whereas SHP1 was not detected. Phosphatase activity assays showed that P-ITIM bound about five times higher SHIP and four times higher PTP activity than the ITIM containing peptide. Furthermore, we detected PKC and MAPK in both ITIM and P-ITIM peptides precipitated samples. Since human B-cells express both FckRIIb1 and FckRIIb2, differing in a 19 amino acid insert in the cytoplasmic tail of the former, we investigated the components binding to FckRIIb1 and FckRIIb2 specific sequences. Synthetic peptide representing FckRIIb1 and FckRIIb2 specific sequences weakly bound unidentified tyr phosphorylated proteins at 50-56 kDa, while the insert itself did not bind a detectable amount of protein. Neither of the ITIM or P-ITIM bound molecules were observed in samples precipitated with peptides corresponding to FckRIIb1 or FckRIIb2 specific sequences. These observations suggest that protein kinases associate with both ITIM and P-ITIM of human FckRIIb, Lyn being responsible for the tyrosyl phosphorylation of ITIM. SHIP and SHP2 phosphatases selectively bind to the phosphorylated ITIM. Based on these data we assume that SHIP and SHP2 recruited in vivo to the FckRIIb co-clustered BCR are responsible for the FckRIIb mediated negative regulation of human B-cell activation. © 1997 Elsevier Science B.V.
Immunology Letters, 1997
Co-clustering of FckRIIb and B-cell receptor (BCR) inhibits cell activation by interrupting BCR stimulated signal transduction. The immunoreceptor tyrosine-based inhibitory motif (ITIM) of FckRIIb becomes tyrosyl phosphorylated (P-ITIM) upon co-clustering with BCR then P-ITIM interacts with several signalling molecules, some of which negatively regulate the cell activation process. The molecules recruited by the P-ITIM of human FckRIIb have not been characterised yet. In order to affinity isolate the potential functional partner molecules of human FckRIIb, synthetic peptides were designed to cover almost the entire intracellular FckRIIb domain, including FckRIIb1 and FckRIIb2 specific sequences and stretches containing the phosphorylated and non-phosphorylated ITIM. We report here that several tyrosyl phosphorylated proteins bind to the P-ITIM peptide from both resting and activated B-cell lysates, the 53-56 kDa being the most prominent one. A fraction of the 53 -56 kDa bands were identified as the protein tyrosine kinase (PTK), Lyn which also bound to ITIM peptide, pointing to its role in initiating FckRIIb-mediated negative regulation. Among the P-ITIM associated tyr phosphorylated components, the 145 kDa one was identified as the inositol polyphosphate 5-phosphatase, SHIP and the 72 kDa protein as the protein tyrosine phosphatase (PTP) SHP2, whereas SHP1 was not detected. Phosphatase activity assays showed that P-ITIM bound about five times higher SHIP and four times higher PTP activity than the ITIM containing peptide. Furthermore, we detected PKC and MAPK in both ITIM and P-ITIM peptides precipitated samples. Since human B-cells express both FckRIIb1 and FckRIIb2, differing in a 19 amino acid insert in the cytoplasmic tail of the former, we investigated the components binding to FckRIIb1 and FckRIIb2 specific sequences. Synthetic peptide representing FckRIIb1 and FckRIIb2 specific sequences weakly bound unidentified tyr phosphorylated proteins at 50-56 kDa, while the insert itself did not bind a detectable amount of protein. Neither of the ITIM or P-ITIM bound molecules were observed in samples precipitated with peptides corresponding to FckRIIb1 or FckRIIb2 specific sequences. These observations suggest that protein kinases associate with both ITIM and P-ITIM of human FckRIIb, Lyn being responsible for the tyrosyl phosphorylation of ITIM. SHIP and SHP2 phosphatases selectively bind to the phosphorylated ITIM. Based on these data we assume that SHIP and SHP2 recruited in vivo to the FckRIIb co-clustered BCR are responsible for the FckRIIb mediated negative regulation of human B-cell activation. © 1997 Elsevier Science B.V.
Molecular and cellular biology, 1997
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 phosp...
Journal of Biological Chemistry, 2000
Fc␥RIIB are single-chain low affinity receptors for IgG that negatively regulate immunoreceptor tyrosinebased activation motif-dependent cell activation. They bear one immunoreceptor tyrosine-based inhibition motif (ITIM) that becomes tyrosyl-phosphorylated upon coaggregation of Fc␥RIIB with immunoreceptor tyrosinebased activation motif-bearing receptors and that recruits SH2 domain-containing inositol 5-phosphatases (SHIPs) in vivo. Synthetic Fc␥RIIB ITIM phosphopeptides, however, also bind SH2 domain-containing protein-tyrosine phosphatases (SHPs) in vitro. To identify SHIP-binding sites, we exchanged residues between the Fc␥RIIB 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 Fc␥RIIB 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 Fc␥RIIB 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 Fc␥RIIB to recruit SHIP1 and SHIP2 and that is preferentially used in vivo.
Immunology letters, 1997
Co-clustering of Fc gamma RIIb and B-cell receptor (BCR) inhibits cell activation by interrupting BCR stimulated signal transduction. The immunoreceptor tyrosine-based inhibitory motif (ITIM) of Fc gamma RIIb becomes tyrosyl phosphorylated (P-ITIM) upon co-clustering with BCR then P-ITIM interacts with several signalling molecules, some of which negatively regulate the cell activation process. The molecules recruited by the P-ITIM of human Fc gamma RIIb have not been characterised yet. In order to affinity isolate the potential functional partner molecules of human Fc gamma RIIb, synthetic peptides were designed to cover almost the entire intracellular Fc gamma RIIb domain, including Fc gamma RIIb2 specific sequences and stretches containing the phosphorylated and non-phosphorylated ITIM. We report here that several tyrosyl phosphorylated proteins bind to the P-ITIM peptide from both resting and activated B-cell lysates, the 53-56 kDa being the most prominent one. A fraction of the ...
Journal of Biological …, 1996
Immunoreceptors such as the high affinity IgE receptor, Fc⑀RI, and T-cell receptor-associated proteins share a common motif, the immunoreceptor tyrosine-based activation motif (ITAM). We used the yeast tribrid system to identify downstream effectors of the phosphorylated Fc⑀RI ITAM-containing subunits  and ␥. One novel cDNA was isolated that encodes a protein that is phosphorylated on tyrosine, contains a Src-homology 2 (SH2) domain, inositolpolyphosphate 5-phosphatase activity, three NXXY motifs, several proline-rich regions, and is called SHIP. Mutation of the conserved tyrosine or leucine residues within the Fc⑀RI  or ␥ ITAMs eliminates SHIP binding and indicates that the SHIP-ITAM interaction is specific. SHIP also binds to ITAMs from the CD3 complex and T cell receptor chain in vitro. SHIP protein possesses both phosphatidylinositol-3,4,5trisphosphate 5-phosphatase and inositol-1,3,4,5-tetrakisphosphate 5-phosphatase activity. Phosphorylation of SHIP by a protein-tyrosine kinase, Lck, results in a reduction in enzyme activity. Fc⑀RI activation induces the association of several tyrosine phosphoproteins with SHIP. SHIP is constitutively tyrosine-phosphorylated and associated with Shc and Grb2. These data suggest that SHIP may serve as a multifunctional linker protein in receptor activation.
Journal of Biological Chemistry, 2004
FcγRIIB 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, FcγRIIB become tyrosyl-phosphorylated and recruit the Src homology 2 (SH2) domain-containing inositol 5′-phosphatases SHIP1 and SHIP2, which mediate inhibition. The FcγRIIB 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 FcγRIIB 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 FcγRIIB, but one adapter is suffi...
The Journal of Immunology, 2000
Coaggregation of FcγRIIB1 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 FcγRIIB1 effects suggest that the receptor uses SH2-containing inositol phophatase (SHIP) and SH2-containing phophotyrosine phosphatase (SHP)-1, as well as SHP-2 as effectors. In contrast, coimmunoprecipitation studies of receptor-effector associations suggest that the predominant FcγRIIB1 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 ectopi...