The SH2-containing inositol-5'-phosphatase, SHIP, restrains bone marrow-derived mast cell (BMMC) degranulation, at least in part, by hydrolyzing phosphatidylinositol (PI)-3-kinase generated PI-3,4,5-P(3) (PIP3) to PI-3,4-P(2). To determine which domains within SHIP influence its ability to hydrolyze PIP3, bone marrow from SHIP(-/-) mice was retrovirally infected with various SHIP constructs. Introduction of wild-type SHIP into SHIP(-/-) BMMCs reverted the Steel factor (SF)-induced increases in PIP3, calcium entry, and degranulation to those observed in SHIP(+/+) BMMCs. A 5'-phosphatase dead SHIP, however, could not revert the SHIP(-/-) response, whereas a SHIP mutant in which the 2 NPXY motifs were converted to NPXFs (2NPXF) could partially revert the SHIP(-/-) response. SF stimulation of BMMCs expressing the 2NPXF, which could not bind Shc, led to the same level of mitogen-activated protein kinase (MAPK) phosphorylation as that seen in BMMCs expressing the other constructs. Surprisingly, C-terminally truncated forms of SHIP, lacking different amounts of the proline rich C-terminus, could not revert the SHIP(-/-) response at all. These results suggest that the C-terminus plays a critical role in enabling SHIP to hydrolyze PIP(3) and inhibit BMMC degranulation.

SHIP is tyrosine phosphorylated (Yphos) upon the addition of a wide variety of extracellular stimuli. However, little is known about the tyrosine kinase(s) involved. Using the Src-specific inhibitor, PP2, we found that SHIP's Yphos, following co-clustering of the B-cell receptor (BCR) with the FcγRIIB co-receptor in WEHI 231 cells, was dramatically reduced while the Syk inhibitor, piceatannol, had little or no effect. Comparable results were obtained following stimulation with interleukin-3 (IL-3) or Steel factor (SF) in Ba/F3 cells and bone marrow derived mast cells (BMMCs), respectively. To corroborate these results, BMMCs derived from Lyn −/− mice were examined. A time course of SF stimulation revealed that SHIP Yphos in Lyn −/− BMMCs was dramatically reduced though not completely abolished. SHIP Yphos was further reduced, however, by addition of PP2 but not piceatannol, suggesting that some or all of the residual SHIP Yphos was due to other Src family members. Binding studies using GST-beads containing the SH3 domain of different Src kinases and various truncated forms of SHIP's proline-rich C-terminus. Co-immunoprecipitation studies verified that SHIP and Lyn associate in WEHI 231 B-cells and that this interaction does not increase following stimulation. These results suggest that members of the Src family are key regulators of SHIP Yphos following activation of BCR and cytokine receptor systems.

We recently reported that the src homology 2 (SH2)-containing inositol 5-phosphatase, SHIP, acts as a gatekeeper of bone marrow derived mast cell (BMMC) degranulation by preventing inappropriate and excess release of inflammatory mediators (PNAS 95, 11330, 1998; EMBO J 17, 7311, 1998). It does so, at least in part, by hydrolysing PI-3-kinase generated PI-3,4,5-P3 to PI-3,4-P2. To determine which domains within SHIP influence its ability to hydrolyze PI-3,4,5-P3 and inhibit degranulation following Steel Factor (SF)-stimulation, we retrovirally infected bone marrow from SHIP−/− mice with various forms of SHIP tagged at the N- and C-termini with hemagglutinin (HA) and green fluorescent protein (GFP), respectively. The mutant forms included one lacking a functional 5-phosphatase domain, one in which the two NPXY motifs were converted to NPXF's and several truncated forms lacing different amounts of the proline rich C-terminus. As expected, introduction of wild type SHIP into SHIP−/− BMMCs reverted the SF-induced increases in PI-3,4,5-P3 and PI-3,4-P2 and degranulation to those observed in SHIP+/+ BMMCs. Also as expected, the phosphatase dead SHIP-mutant could not revert, while the NPXF mutant could partially revert, the SHIP−/− response. Surprisingly, however, the C-terminally truncated forms of SHIP could not revert the response at all, even though protein expression levels were similar for all the SHIP constructs. This suggests that the proline-rich C-terminus of SHIP plays a critical role in enabling SHIP to hydrolyse PI-3,4,5-P3 and inhibit BMMC degranulation.

The recently cloned, hemopoietic-specific, src homology 2 (SH2)-containing inositol phosphatase, SHIP, is rapidly gaining prominence as a potential regulator of all phosphatidylinositol (PI)-3 kinase mediated events since it has been shown both in vitro and in vivo to hydrolyze the 5' phosphate from phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P-3). Thus SHIP, and its more widely expressed counterpart, SHIP2, could play a central role in determining PI-3,4,5-P-3 and PI-3,4-P-2 levels in many cell types. To explore the in vivo function of SHIP further we recently generated a SHIP knock out mouse and in this review we discuss experiments carried out with bone marrow derived mast cells (BMMCs) from these animals. (C) 1999 Elsevier Science Ltd. All rights reserved.

The SH2-containing inositol phosphatase, SHIP, often appears as multiple bands in anti-SHIP immunoblots. To characterize these bands, antisera were generated against the N-terminal (anti-N), mid-region (anti-M), and C-terminal (anti-C) portions of SHIP. Immunoprecipitation and immunoblotting studies showed that 145-, 135-, 125-, and 110-kD bands were detected in lysates from the murine hematopoietic cell line, DA-ER, with either anti-N or anti-M antisera, whereas only the 145- and 135-kD bands were recognized by the anti-C antiserum. This finding suggested that the smaller proteins might be C-terminal truncations of the full-length SHIP. To confirm this and determine if these proteins arose through alternate splicing or posttranslational cleavage, a 5′-hemagglutin (HA)-tagged full-length SHIP cDNA was expressed in these cells. We observed, via Western analysis with anti-HA antibodies, the same 4 bands with either anti-N or anti-M and only the 145- and 135-kD bands with anti-C immunoprecipitation. After interleukin-3 stimulation of HA-SHIP–expressing DA-ER cells, only the 145-kD form coprecipitated with Shc, raising the possibility that different forms of SHIP may have distinct intracellular sites. This was confirmed by subcellular fractionation, which showed that only the 110-kD form is present in the cytoskeleton of DA-ER cells. This 110-kD form possesses the same PIP3 5-ptase activity as the 145-kD form and can be generated from the latter in vitro by digestion with calpain. It is therefore possible that the different forms of SHIP are generated in vivo by calpain-mediated C-terminal truncations and perform distinct functions within hematopoietic cells. © 1998 by The American Society of Hematology.

We recently purified and cloned a 145-kDa protein that becomes tyrosine phosphorylated and associated with Shc in response to multiple cytokines. Based on its predicated amino acid sequence and its enzymatic activity, we have called this protein SHIP, for rc omology 2-containing nositol hosphatase. To gain further insight into the intracellular pathways that this putative signal transduction intermediate might regulate we have investigated whether SHIP binds to intracellular proteins other than Shc. The results presented herein demonstrate that following interleukin-3 stimulation, SHIP binds to the tyrosine phosphatase, SHP2 (also called Syp, PTP1D, SHPTP2, and PTP2C) and that Shc is not present in these SHIP-SHP2 complexes. Time course studies reveal that SHIP's association with SHP2 is transient and is maximal at 10 min of stimulation with interleukin-3. We further show that the association of SHIP with SHP2 occurs through the direct interaction of the SH2 domain of SHIP with a pYXN(I/V) sequence within SHP2.

We recently cloned and sequenced a cDNA encoding a 145-kD protein from the murine hematopoietic cell line B6SUtA, that becomes tyrosine phosphorylated and associated with Shc after cytokine stimulation. Based on its domains and enzymatic activity, we named this protein SHIP for SH2-containing inositol phosphatase (Damen et al, Proc Natl Acad Sci USA 93:1689, 1996). We describe here the cloning of the human homologue of murine SHIP (mSHIP) from a human megakaryocytic cell line (MO7e) lambda gt11 cDNA library using two nonoverlapping mSHIP cDNA fragments as probes. Northern blot analysis suggests that human SHIP (hSHIP) is expressed as a 5.3-kb mRNA in human bone marrow and a wide variety of other tissues. Sequence analysis of this cDNA predicts a protein of 1188 amino acids exhibiting 87.2% overall sequence identity with mSHIP. Contained within the defined open reading frame is an N-terminal, group l src homology 2 (SH2) domain; three NXXY motifs that, if phosphorylated, could be bound by phosphotyrosine binding (PTB) domains; a C-terminal proline-rich region; and two centrally located inositol polyphosphate 5-phosphatase motifs. Fluorescence in situ hybridization, using the full-length hSHIP cDNA as a probe, mapped hSHIP to the long arm of chromosome 2 at the border between 2q36 and 2q37.