The Inositol 5′-Phosphatase SHIP Binds to Immunoreceptor Signaling Motifs and Responds to High Affinity IgE Receptor Aggregation

Abstract

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-3,4,5-trisphosphate 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.The aggregation of immunoreceptors by antigen initiates a complex response leading to cellular activation (1). Receptors on T-, B-, and mast cells each contain subunits with similar primary amino acid sequence within their cytosolic domains, comprising the immunoreceptor-based tyrosine activation motif (ITAM), 1 whose consensus is (D/E)X 2 YXX(L/I)X 6 -8 YXX(L/I) (2, 3). Both tyrosine residues within the ITAM are rapidly phosphorylated by protein kinases after receptor aggregation. The bisphosphorylated ITAM then binds directly to cytosolic tyrosine kinases such as Syk in B-cells and mast cells and ZAP70 in T-cells, thereby activating their tyrosine kinase activity (4, 5). In mast cells, the Fc⑀RI subunits ␤ and ␥ each possess a single ITAM, which, when bisphosphorylated on tyrosine, binds to Syk (6 -8).We have used a novel genetic approach, the yeast tribrid system (8), to isolate cDNAs that encode proteins that interact with the tyrosine-phosphorylated Fc⑀RI ␥ ITAM. The yeast two-hybrid system facilitates the study of protein-protein interactions but is limited to the investigation of proteins that are properly expressed and modified in the host, Saccharomyces cerevisiae. S. cerevisiae does not employ tyrosine phosphorylation as a major regulatory modification of proteins (9, 10). This limits the utility of the two-hybrid system, especially in the area of signal transduction, where tyrosine phosphorylation is a critical component of the process. In order to study protein-protein interactions that are dependent on tyrosine phosphorylation or on other post-translational or allosteric modifications, the yeast tribrid system was developed (8, 11).In the yeast tribrid system, a third plasmid is introduced, which directs th...

Restoration of SHIP-1 activity in human leukemic cells modifies NF-κB activation pathway and cellular survival upon oxidative stress

“…Another hypothesis to explain the positive role of SHIP-1 in IKK redox SHIP-1 modifies leukemic cell response to oxidative stress G Gloire et al regulation in T cells might be the association of SHIP-1 with the TCR. Such an interaction was already reported in vitro by Osborne et al (1996), who showed that SHIP-1 binds phosphorylated ITAM of the CD3 complex via its SH2 domain. It is possible that SHIP-1 acts as a scaffold protein that, when present, mediates a sustained IKK activation through a TCR-mediated signaling pathway.…”

Cloning and characterization of APS, an adaptor molecule containing PH and SH2 domains that is tyrosine phosphorylated upon B-cell receptor stimulation

“…The APS-associated phosphoproteins were quite similar to those associated with Grb2 and c-Cbl. The major APS binding phosphoprotein, p145 was precipitated with all other antibodies, and it is probably SHIP which is reported to be associated with Shc and Grb2 (Damen et al, 1996;Osborne et al, 1996). A marginal amount of p120 c-Cbl was precipitated with APS.…”

“…Grb-IR was cloned by using the tyrosine kinase domain of the insulin receptor as bait (Liu and Roth, 1995). A yeast tribrid system has been developed to isolate cDNAs encoding proteins that interact with the tyrosine-phosphorylated FceRIg ITAM, and novel SH2 containing proteins, SH2-B and SHIP (SH2-domain containing inositol polyphosphate 5-phosphatase) were cloned by this method (Osborne et al, 1995(Osborne et al, , 1996. We used oncogenic c-kit kinase domain as bait, expecting high kinase activity of the bait.…”

Negative Signaling in Fc Receptor Complexes

“…SHIP1 activity was reported to associate with the phosphorylated ζ subunit and to negatively regulate FcγRIIIA-dependent ADCC in human NK cells (Galandrini et al, 2002). SHIP1 was found to bind in vitro to phopshopetides corresponding to the FcRβ ITAM (Kimura et al, 1997) and to interact with FcRβ when examined by yeast triple hybrid assay (Osborne et al, 1996). The possible in vivo recruitment of this phopshatase in FcεRI signaling complexes remains elusive as, so far, it was not reported to coprecipitate with FcεRI, including the FcRβ subunit, following receptor engagement in mast cells.…”