SH2 Domain-containing Inositol 5-phosphatase Research Articles

Macrophage Pro-Inflammatory Response to Francisella novicida Infection Is Regulated by SHIP

Francisella tularensis, a Gram-negative facultative intracellular pathogen infecting principally macrophages and monocytes, is the etiological agent of tularemia. Macrophage responses to F. tularensis infection include the production of pro-inflammatory cytokines such as interleukin (IL)-12, which is critical for immunity against infection. Molecular mechanisms regulating production of these inflammatory mediators are poorly understood. Herein we report that the SH2 domain-containing inositol phosphatase (SHIP) is phosphorylated upon infection of primary murine macrophages with the genetically related F. novicida, and negatively regulates F. novicida–induced cytokine production. Analyses of the molecular details revealed that in addition to activating the MAP kinases, F. novicida infection also activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in these cells. Interestingly, SHIP-deficient macrophages displayed enhanced Akt activation upon F. novicida infection, suggesting elevated PI3K-dependent activation pathways in absence of SHIP. Inhibition of PI3K/Akt resulted in suppression of F. novicida–induced cytokine production through the inhibition of NFκB. Consistently, macrophages lacking SHIP displayed enhanced NFκB-driven gene transcription, whereas overexpression of SHIP led to decreased NFκB activation. Thus, we propose that SHIP negatively regulates F. novicida–induced inflammatory cytokine response by antagonizing the PI3K/Akt pathway and suppressing NFκB-mediated gene transcription. A detailed analysis of phosphoinositide signaling may provide valuable clues for better understanding the pathogenesis of tularemia.

The influence of anionic lipids on SHIP2 phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase activity

The SH2 domain containing inositol 5-phosphatase 2 (SHIP2) catalyzes the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3) to phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2) and participates in the insulin signalling pathway in vivo. In a comparative study of SHIP2 and the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), we found that their lipid phosphatase activity was influenced by the presence of vesicles of phosphatidylserine (PtdSer). SHIP2 PtdIns(3,4,5)P 3 5-phosphatase activity was greatly stimulated in the presence of vesicles of PtdSer. This effect appears to be specific for di-C8 and di-C16 fatty acids of PtdIns(3,4,5)P 3 as substrate. It was not observed with inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4) another in vitro substrate of SHIP2, nor with Type I Ins(1,4,5)P 3/Ins(1,3,4,5)P 4 5-phosphatase activity, an enzyme which acts on soluble inositol phosphates. Vesicles of phosphatidylcholine (PtdCho) stimulated only twofold PtdIns(3,4,5)P 3 5-phosphatase activity of SHIP2. Both a minimal catalytic construct and the full length SHIP2 were sensitive to the stimulation by PtdSer. In contrast, PtdIns(3,4,5)P 3 5-phosphatase activity of the Skeletal muscle and Kidney enriched Inositol Phosphatase (SKIP), another member of the mammaliam Type II phosphoinositide 5-phosphatases, was not sensitive to PtdSer. Our enzymatic data establish a specificity in the control of SHIP2 lipid phosphatase activity with PtdIns(3,4,5)P 3 as substrate which is depending on the fatty acid composition of the substrate.

A High-Throughput Microfluidic Assay for SH2 Domain-Containing Inositol 5-Phosphatase 2

SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) is a potential drug target for the treatment of type 2 diabetes. This enzyme serves as a negative regulator of insulin-mediated signal transduction by catalyzing the dephosphorylation of the second messenger lipid molecule phosphatidylinositol 3,4,5-triphosphate. Traditionally, assays for phosphoinositide phosphatases such as SHIP2 have relied on radiolabeled phosphatidylinositol-containing lipid membranes and chromatographic separation of labeled phospholipid substrate from product by thin-layer chromatography. We have expressed and purified catalytically active phosphatase domain constructs of SHIP2 from Escherichia coli and developed a sensitive and antibody- or binding protein-independent assay for SHIP2 amenable to high-throughput screening of phosphoinositide phosphatases or phosphoinositide kinases. This microfluidic assay, with Z' values approximately 0.8, is based upon the difference in mobility within an electric field between a fluorophore-labeled phosphatidylinositol 3,4,5-triphosphate substrate and the corresponding 3,4-bisphosphate product. High-throughput screening of a 91,060-member compound library in 384-well format resulted in the identification of SHIP2 inhibitors.

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

Murine FcγRIIB were demonstrated to recruit SH2 domain-containing inositol 5-phosphatases (SHIP1/2), when their ITIM is tyrosyl-phosphorylated upon co-aggregation with BCR, and SHIP1 to account for FcγRIIB-dependent negative regulation of murine B cell activation. Although human FcγRIIB share the same ITIM as murine FcγRIIB and similarly inhibit human B cell activation, which among the four known SH2 domain-containing (tyrosine or inositol) phosphatases is/are recruited by human FcγRIIB is unclear. Our recent finding that, besides the ITIM, a second tyrosine-based motif is mandatory for murine FcγRIIB to recruit SHIP1 challenged the possibility that human FcγRIIB recruit this phosphatase. Human FcγRIIB indeed lack this motif. Using an experimental model which enabled us to compare human FcγRIIB and murine FcγRIIB under strictly controlled conditions, we show that SHIP1 is recruited to the intracytoplasmic domain of human FcγRIIB and inhibits the same biological responses and intracellular signals as when recruited by murine FcγRIIB. Identical results were observed in murine and in human B cells. We demonstrate that SHIP is necessary for human FcγRIIB to inhibit BCR signaling, and cannot be replaced by SHP-1 or SHP-2. Although it contains no tyrosine, the C-terminal segment of human FcγRIIB was as mandatory as the tyrosine-containing C-terminal segment of murine FcγRIIB for SHIP1 to be recruited to the ITIM. This segment, however, did not recruit the adapters Grb2/Grap which were demonstrated to stabilize the recruitment of SHIP1 to the ITIM in murine FcγRIIB.

Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) negatively regulates TLR4-mediated LPS response primarily through a phosphatase activity- and PI-3K-independent mechanism

AbstractSrc homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1) plays important roles in negatively regulating the activation of immune cells primarily via the phosphoinositide 3-kinase (PI-3K) pathway by catalyzing the PI-3K product PtdIns-3,4,5P3 (phosphatidylinositol-3,4,5-triphosphate) into PtdIns-3,4P2. However, the role of SHIP1 in Toll-like receptor 4 (TLR4)-mediated lipopolysaccharide (LPS) response remains unclear. Here we demonstrate that SHIP1 negatively regulates LPS-induced inflammatory response via both phosphatase activity-dependent and -independent mechanisms in macrophages. SHIP1 becomes tyrosine phosphorylated and up-regulated upon LPS stimulation in RAW264.7 macrophages. SHIP1-specific RNA-interfering and SHIP1 overexpression experiments demonstrate that SHIP1 inhibits LPS-induced tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) production by negatively regulating the LPS-induced combination between TLR4 and myeloid differentiation factor 88 (MyD88); activation of Ras (p21ras protein), PI-3K, extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH2-terminal kinase (JNK); and degradation of IκB-α. SHIP1 also significantly inhibits LPS-induced mitogen-activated protein kinase (MAPK) activation in TLR4-reconstitited COS7 cells. Although SHIP1-mediated inhibition of PI-3K is dependent on its phosphatase activity, phosphatase activity-disrupted mutant SHIP1 remains inhibitory to LPS-induced TNF-α production. Neither disrupting phosphatase activity nor using the PI-3K pathway inhibitor LY294002 or wortmannin could significantly block SHIP1-mediated inhibition of LPS-induced ERK1/2, p38, and JNK activation and TNF-α production, demonstrating that SHIP1 inhibits LPS-induced activation of MAPKs and cytokine production primarily by a phosphatase activity- and PI-3K-independent mechanism. (Blood. 2005;105:4685-4692)

Vav Activation and Function as a Rac Guanine Nucleotide Exchange Factor in Macrophage Colony-Stimulating Factor-Induced Macrophage Chemotaxis

Signal transduction mediated by phosphatidylinositol 3-kinase (PI 3-kinase) is regulated by hydrolysis of its products, a function performed by the 145-kDa SH2 domain-containing inositol phosphatase (SHIP). Here, we show that bone marrow macrophages of SHIP(-/-) animals have elevated levels of phosphatidylinositol 3,4,5-trisphosphate [PI (3,4,5)P(3)] and displayed higher and more prolonged chemotactic responses to macrophage colony-stimulating factor (M-CSF) and elevated levels of F-actin relative to wild-type macrophages. We also found that the small GTPase Rac was constitutively active and its upstream activator Vav was constitutively phosphorylated in SHIP(-/-) macrophages. Furthermore, we show that Vav in wild-type macrophages is recruited to the membrane in a PI 3-kinase-dependent manner through the Vav pleckstrin homology domain upon M-CSF stimulation. Dominant inhibitory mutants of both Rac and Vav blocked chemotaxis. We conclude that Vav acts as a PI 3-kinase-dependent activator for Rac activation in macrophages stimulated with M-CSF and that SHIP regulates macrophage M-CSF-triggered chemotaxis by hydrolysis of PI (3,4,5)P(3).

FLT3/ITD Mutation Signaling Includes Suppression of SHP-1

Mutations in the FLT3 gene are the most common genetic alteration found in AML patients. FLT3 internal tandem duplication (ITD) mutations result in constitutive activation of FLT3 tyrosine kinase activity. The consequences of this activation are an increase in total phosphotyrosine content, persistent downstream signaling, and ultimately transformation of hematopoietic cells to factor-independent growth. The Src homology (SH)2 domain-containing protein-tyrosine phosphatase (SHP)-1 is involved in the down-regulation of a broad range of growth factor and cytokine-driven signaling cascades. Loss-of-function or deficiency of SHP-1 activity results in a hyperproliferative response of myelomonocytic cell populations to growth factor stimulation. In this study, we examined the possible role of SHP-1 in regulating FLT3 signaling. We found that transformation of TF-1 cells with FLT3/ITD mutations suppressed the activity of SHP-1 by approximately 3-fold. Suppression was caused by decreased SHP-1 protein expression, as analyzed at both the protein and RNA levels. In contrast, protein levels of SHP-2, a phosphatase that plays a stimulatory role in signaling through a variety of receptors, did not change significantly in FLT3 mutant cells. Suppressed SHP-1 protein levels in TF-1/ITD cells were partially overcome after cells were exposed to CEP-701, a selective FLT3 inhibitor. SHP-1 protein levels also increased in naturally occurring FLT3/ITD expressing AML cell lines and in primary FLT3/ITD AML samples after CEP-701 treatment. Furthermore, a small but reproducible growth/survival advantage was observed in both TF-1 and TF-1/ITD cells when SHP-1 expression was knocked down by RNAi. Taken together, these data provide the first evidence that suppression of SHP-1 by FLT3/ITD signaling may be another mechanism contributing to the transformation by FLT3/ITD mutations.

Absence of the lipid phosphatase SHIP2 confers resistance to dietary obesity

Genetic ablation of Inppl1, which encodes SHIP2 (SH2-domain containing inositol 5-phosphatase 2), was previously reported to induce severe insulin sensitivity, leading to early postnatal death. In the previous study, the targeting construct left the first eighteen exons encoding Inppl1 intact, generating a Inppl1(EX19-28-/-) mouse, and apparently also deleted a second gene, Phox2a. We report a new SHIP2 knockout (Inppl1(-/-)) targeted to the translation-initiating ATG, which is null for Inppl1 mRNA and protein. Inppl1(-/-) mice are viable, have normal glucose and insulin levels, and normal insulin and glucose tolerances. The Inppl1(-/-) mice are, however, highly resistant to weight gain when placed on a high-fat diet. These results suggest that inhibition of SHIP2 would be useful in the effort to ameliorate diet-induced obesity, but call into question a dominant role of SHIP2 in modulating glucose homeostasis.

Effect of Inositol-phosphatase on Fc Receptor-mediated Phagocytosis of Macrophages

Fc receptor-mediated phagocytosis is a complex process involving the activation of kinases and phosphatases. FcgammaRIIB has been known to transduces inhibitory signals through an immunoreceptor tyrosine-based inhibitory motif (ITIM) in cytoplasmic domains. In this study, we examined the involvement of inositol-phosphatase in the Fc receptor-mediated phagocytosis. Methods: J774 cells were infected using vaccinia viral vector containing SH2 domain-containing inositol-phosphatase (SHIP) cDNA and stimulated with the sensitized sheep red blood cells. Results: Stimulation of J774 cells induced the tyrosine phosphorylation of SHIP which was maximal at 5 minutes. Phosphatidylinositol-3 (PI-3) kinase inhibitor (wortmannin) inhibits J774 cell phagocytosis of sensitized sheep red blood cells in a dose-dependent manner. Heterologious expression of SHIP in J774 cells inhibits phagocytosis of sensitized sheep red blood cells in a dose-dependency manner, but catalytically dead mutants of SHIP has no effect on phagocytosis. Conclusion: These results strongly suggest that the active signals mediated by PI-3 kinase are opposed by inhibitory signals through SHIP in the regulation of Fc receptor-mediated phagocytosis.

Src Homology 2 Domain-Containing Inositol-5-Phosphatase 1 (SHIP1) Negatively Regulates TLR4 Mediated LPS Signaling through Phosphatase Activity and PI3K Independent Mechanism.

Lipopolysaccharide (LPS) is a cell wall component of Gram negative bacteria. LPS activates immune cells and triggers production of proinflammatory cytokines and other mediators through Toll-like receptor (TLR) 4. Src homology (SH) 2 domain-containing inositol-5-phosphatase 1 (SHIP1) plays important roles in negatively regulating the activation of immune cells primarily via phosphoinositide 3-kinase (PI3K) pathway by catalyzing PI3K product PtdIns-3,4,5P3 into PtdIns-3,4P2. However, little is known about the role of SHIP1 in TLR4-mediated LPS responses. We investigated the role of SHIP1 in LPS-induced MAPKs activation and IkappaB-alpha degradation by overexpression and RNA interfering experiments. SHIP1 becomes tyrosine phosphorylated and membrane translocated upon LPS stimulation in RAW264.7 macrophages. Stable SHIP1 transfection inhibits LPS-induced TNF-alpha and IL-6 production by 60% and 70% respectively. Transient SHIP1 transfection inhibits LPS-induced phospharylation of extracellular signal-regulated kinase (ERK1/2), p38, c-Jun NH2-terminal kinase (JNK) and degradation of IkappaB-alpha by about 20%, 50%, 40% and 40% respectively at the time point of 20 min after LPS stimulation. Disruption of SHIP1 phosphatase activity enhances LPS-induced activation of Akt, a downstream molecule of PI3K pathway, but fails to reverse SHIP1 mediated inhibition of LPS-induced activation of MAPKs and degradation of IkappaB-alpha. Consistently, LY 294002 and Wortmannin sufficiently inhibit LPS-induced Akt activation but can not diminish SHIP1 mediated inhibition of LPS-induced activation of MAPKs and degradation of IkappaB-alpha. On the contrary, SHIP1-specific RNA interfering inhibits SHIP1 expression and enhances LPS-induced phospharylation of ERK1/2, p38, JNK and degradation of IkappaB-alpha by about 2.6, 2.6, 3.0 and 8.0-fold respectively at the time point of 20 min after LPS stimulation. Even in the presence of LY 294002, SHIP1 RNA interfering could increase LPS-induced IL-6 production. Furthermore, SHIP1 overexpression nearly completely abolished TLR4 reconstitution conferred LPS responsiveness in COS7 cells. These results demonstrated that SHIP1 negatively regulates LPS responses in macrophage via phosphatase activity and PI3K-independent mechanism.

Phosphoproteomics Nabs New FGF Receptor Targets

Improvements in proteomic techniques and in mass spectrometry in particular are enabling large-scale analysis of signaling proteins that are covalently modified in stimulated cells. Hinsby et al. applied the latest techniques to identify proteins that participate in signaling from the fibroblast growth factor receptor (FGFR). They used a new linear ion trap Fourier transform mass spectrometer with attomole sensitivity to detect proteins phosphorylated in response to FGF in a human cell line transfected with the FGFR. Phosphoproteins were isolated by immunoprecipitation with antibody to phosphotyrosine. Then stable isotope labeling by amino acids in cell culture (SILAC) was used with mass spectrometry to allow direct comparison of peptides from stimulated and unstimulated cells (which could be distinguished by a 6-Da shift in mass of peptides from cells incubated with [13C]Arg). Of more than 800 proteins nonspecifically present in the immunoprecipitates, 28 were identified that showed response to FGF. A number of these were proteins not previously implicated in FGR signaling, including IRS-4, GIT1 and GIT2 (GTPase-activating proteins for the Arf small GRPases), SHIP2 (SH2 domain-containing inositol phosphatase), annexins VII and XI, liver-specific bHLH-Zip transcription factor (LISCH7), and WDR6, a protein with WD40 repeats that function in protein interactions. The authors propose that the described phosphoproteomics techniques will help provide unbiased mapping of signaling networks. A. M. Hinsby, J. V. Olsen, M. Mann, Tyrosine phosphoproteomics of fibroblast growth factor signaling. A role for insulin receptor substrate-4. J. Biol. Chem. 279 , 46438-46447 (2004). [Abstract] [Full Text]

5. IgE, mast cells, basophils, and eosinophils

IgE, mast cells, basophils, and eosinophils constitute essential elements in allergic inflammation. Allergen-specific IgE, synthesized in response to allergens in the environment, becomes fixed to FcepsilonRI on the membranes of mast cells and basophils. Aggregation of receptor-bound IgE molecules on re-exposure to specific allergen results in the production of mediators that produce the allergic response. Principal among the cells drawn to sites of mediator release is the eosinophil.

Co-aggregation of FcγRII with FcϵRI on Human Mast Cells Inhibits Antigen-induced Secretion and Involves SHIP-Grb2-Dok Complexes

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.

FcgammaRII expression on follicular dendritic cells and immunoreceptor tyrosine-based inhibition motif signaling in B cells.

Immune complexes (IC) initiate immunoreceptor tyrosine-based inhibition motif (ITIM) signaling and inhibit B cell activation by coligating B cell receptor for antigen (BCR) and FcgammaRII. Nevertheless, IC on follicular dendritic cells (FDC) stimulate rapid germinal center (GC) B cell proliferation suggesting that interactions between IC and FDC render IC capable of B cell activation. To understand this, we studied the kinetics of FDC FcgammaRII and complement receptors 1 and 2 (CR1&2) expressions during the GC reaction and determined whether FDC FcgammaRII could bind Fc in IC and block ITIM signaling. Mice were immunized with sheep red blood cells (SRBC), and CR1&2 and FcgammaRII levels in FDC reticula were monitored. The role of FDC FcgammaRII was studied using anti-BCR-stimulated A20 cells. Levels of FDC FcgammaRII in spleens of SRBC-injected mice increased within 24 h and were dramatically increased (approximately 50-fold) on days 3 and 5. In contrast, CR1&2 levels increased less than twofold. Addition of normal FDC, but not FDC lacking FcgammaRII, reduced and reversed anti-BCR-induced SH2 domain-containing inositol phosphatase (SHIP)-1 phosphorylation in A20 cells. FDC were able to induce normal recall responses even after overnight incubation of the lymphocytes with IC to stimulate ITIM signaling. Engagement of Ig Fc with numerous FcgammaRII on FDC appears to minimize IC-induced ITIM signaling. Thus, rapid up-regulation of FDC FcgammaRII may explain why poorly immunogenic IC are rendered highly immunogenic when presented by FDC in GC.

Brief calorie restriction increases Akt2 phosphorylation in insulin-stimulated rat skeletal muscle.

Skeletal muscle insulin sensitivity improves with short-term reduction in calorie intake. The goal of this study was to evaluate changes in the abundance and phosphorylation of Akt1 and Akt2 as potential mechanisms for enhanced insulin action after 20 days of moderate calorie restriction [CR; 60% of ad libitum (AL) intake] in rat skeletal muscle. We also assessed changes in the abundance of SH2 domain-containing inositol phosphatase (SHIP2), a negative regulator of insulin signaling. Fisher 344 x Brown Norway rats were assigned to an AL control group or a CR treatment group for 20 days. Epitrochlearis muscles were dissected and incubated with or without insulin (500 microU/ml). Total Akt serine and threonine phosphorylation was significantly increased by 32 (P < 0.01) and 30% (P < 0.005) in insulin-stimulated muscles from CR vs. AL. Despite an increase in total Akt phosphorylation, there was no difference in Akt1 serine or Akt1 threonine phosphorylation between CR and AL insulin-treated muscles. However, there was a 30% decrease (P < 0.05) in Akt1 abundance for CR vs. AL. In contrast, there was no change in Akt2 protein abundance, and there was a 94% increase (P < 0.05) in Akt2 serine phosphorylation and an increase of 75% (P < 0.05) in Akt2 threonine phosphorylation of insulin-stimulated CR muscles compared with AL. There was no diet effect on SHIP2 abundance in skeletal muscle. These results suggest that, with brief CR, enhanced Akt2 phosphorylation may play a role in increasing insulin sensitivity in rat skeletal muscles.

The effect of phosphatases SHP-1 and SHIP-1 on signaling by the ITIM- and ITAM-containing Fcgamma receptors FcgammaRIIB and FcgammaRIIA.

Inositol and tyrosine phosphatases have been implicated in inhibitory signaling by an Fc receptor for immunoglobulin G, FcgammaRIIB, in B cells, mast cells, and monocytes. Here, we propose a role for the Src homology 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1) in FcgammaRIIB-mediated inhibition of FcgammaR signaling. Coexpression of SHP-1 enhances FcgammaRIIB-mediated inhibition of FcgammaRIIA phagocytosis in COS-1 cells. SHP-1 also enhances the reduction in FcgammaRIIA tyrosine phosphorylation that accompanies this inhibition. Significantly, tyrosine phosphorylation of Syk kinase is substantially inhibited by SHP-1. Furthermore, the activation of SHP-1 tyrosine phosphorylation is observed following stimulation of FcgammaRII in COS-1 cells and in human monocytes. The SH2 domain containing inositol phosphatase (SHIP), SHIP-1 also enhances FcgammaRIIB-mediated inhibition of FcgammaRIIA, indicating that FcgammaRIIB can use more than one pathway for its inhibitory action. In addition, SHP-1 and SHIP-1 can inhibit FcgammaRIIA phagocytosis and signal transduction in the absence of FcgammaRIIB. The data support emerging evidence that SH2-containing phosphatases, such as SHP-1 and SHIP-1, can modulate signaling by "activating" receptors.

Fc gamma RIIB activation leads to inhibition of signalling by independently ligated receptors.

The inhibitory IgG receptor, Fc gamma RIIB, blocks signalling by co-aggregated antigen receptors on mature and activated B-cells. Fc gamma RIIB is also expressed by immature B-cells; however, its function on these cells has not been defined. In the present paper, we demonstrate that immature B-cells are highly sensitive to inhibitory signalling mediated by Fc gamma RIIB. Co-aggregation of Fc gamma RIIB with the B-cell antigen receptor (BCR) on immature B-cells leads to near ablation of late phase calcium mobilization. Concomitant with enhanced inhibitory signalling, we found that Src-homology-2-domain-containing inositol 5'-phosphatase (SHIP) is expressed at much higher levels in immature B-cells than in mature B-cells. Perhaps most importantly, we report that SHIP activated by BCR-Fc gamma RIIB co-aggregation inhibits independently ligated receptors whose signalling requires PtdIns(3,4,5) P (3). We found that stromal-derived factor 1 (SDF-1)-induced cell migration is impaired by prior activation of Fc gamma RIIB. This inhibition is reduced in SHIP-deficient B-cells. Therefore receptor-mediated signalling responses that are dependent on PtdIns(3,4,5) P (3) are subject to both direct and indirect inhibition by Fc gamma RIIB-activated SHIP.

Possible dominant-negative mutation of the SHIP gene in acute myeloid leukemia.

The SH2 domain-containing inositol 5'-phosphatase (SHIP) is crucial in hematopoietic development. To evaluate the possible tumor suppressor role of the SHIP gene in myeloid leukemogenesis, we examined primary leukemia cells from 30 acute myeloid leukemia (AML) patients, together with eight myeloid leukemia cell lines. A somatic mutation at codon 684, replacing Val with Glu, was detected in one patient, lying within the signature motif 2, which is the phosphatase active site. The results of an in vitro inositol 5'-phosphatase assay revealed that the mutation reduced catalytic activity of SHIP. Leukemia cells with the mutation showed enhanced Akt phosphorylation following IL-3 stimulation. K562 cells transfected with the mutated SHIP-V684E cDNA showed a growth advantage even at lower serum concentrations and resistance to apoptosis induced by serum deprivation and exposure to etoposide. These results suggest a possible role of the mutated SHIP gene in the development of acute leukemia and chemotherapy resistance through the deregulation of the phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3)/Akt signaling pathway. This is the first report of a mutation in the SHIP gene in any given human cancer, and indicates the need for more attention to be paid to this gene with respect to cancer pathogenesis.

Reversal of denervation-induced insulin resistance by SHIP2 protein synthesis blockade.

Short-term muscle denervation is a reproducible model of tissue-specific insulin resistance. To investigate the molecular basis of insulin resistance in denervated muscle, the downstream signaling molecules of the insulin-signaling pathway were examined in intact and denervated soleus muscle of rats. Short-term denervation induced a significant fall in glucose clearance rates (62% of control, P < 0.05) as detected by euglycemic hyperinsulinemic clamp and was associated with a significant decrease in insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR; 73% of control, P < 0.05), IR substrate 1 (IRS1; 69% of control, P < 0.05), and IRS2 (82% of control, P < 0.05) and serine phosphorylation of Akt (39% of control, P < 0.05). Moreover, denervation reduced insulin-induced association between IRS1/IRS2 and p85/phosphatidylinositol (PI) 3-kinase. Nevertheless, denervation caused an increase in PI 3-kinase activity associated with IRS1 (275%, P < 0.05) and IRS2 (180%, P < 0.05), but the contents of phosphorylated PI detected by HPLC were significantly reduced in lipid fractions. In the face of the apparent discrepancy, we evaluated the expression and activity of the 5-inositol, lipid phosphatase SH2 domain-containing inositol phosphatase (SHIP2), and the serine phosphorylation of p85/PI 3-kinase. No major differences in SHIP2 expression were detected between intact and denervated muscle. However, serine phosphorylation of p85/PI 3-kinase was reduced in denervated muscle, whereas the blockade of SHIP2 expression by antisense oligonucleotide treatment led to partial restoration of phosphorylated PI contents and to improved glucose uptake. Thus modulation of the functional status of SHIP2 may be a major mechanism of insulin resistance induced by denervation.

The Src homology 2 domain–containing inositol 5-phosphatase negatively regulates Fcγ receptor–mediated phagocytosis through immunoreceptor tyrosine-based activation motif–bearing phagocytic receptors

AbstractMolecular mechanisms by which the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) negatively regulates phagocytosis in macrophages are unclear. We addressed the issue using bone marrow–derived macrophages from FcγR- or SHIP-deficient mice. Phagocytic activities of macrophages from FcγRII(b)−/− and SHIP−/− mice were enhanced to a similar extent, relative to those from wild type. However, calcium influx was only marginally affected in FcγRII(b)−/−, but greatly enhanced in SHIP−/− macrophages. Furthermore, SHIP was phosphorylated on tyrosine residues upon FcγR aggregation even in macrophages from FcγRII(b)−/− mice or upon clustering of a chimeric receptor containing CD8 and the immunoreceptor tyrosine-based activation motif (ITAM)–bearing γ-chain or human-restricted FcγRIIa. These findings indicate that, unlike B cells, SHIP is efficiently phosphorylated in the absence of an immunoreceptor tyrosine-based inhibition motif (ITIM)–bearing receptor. We further demonstrate that SHIP directly bound to phosphorylated peptides derived from FcγRIIa with a high affinity, comparable to that of FcγRII(b). Lastly, FcγRIIa-mediated phagocytosis was significantly enhanced in THP-1 cells overexpressing dominant-negative form of SHIP in the absence of FcγRII(b). These results indicate that SHIP negatively regulates FcγR-mediated phagocytosis through all ITAM-containing IgG receptors using a molecular mechanism distinct from that in B cells.