Noxa determines localization and stability of MCL-1 and consequently ABT-737 sensitivity in small cell lung cancer.

The sensitivity to ABT-737, a prototype BH3 mimetic drug, varies in a broad range in small cell lung cancer (SCLC) cells. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is the critical determinant of ABT-737 sensitivity. We show here that Noxa regulates the localization and stability of MCL-1, an anti-apoptotic member, which results in modulating ABT-737 sensitivity. Mutations in Noxa within the BH3 domain, the carboxyl terminus mitochondrial targeting domain, or of ubiquitinated lysines not only change the localization and stability of Noxa itself but also affect the mitochondrial localization and phosphorylation/ubiquitination status of MCL-1 and consequently modulate sensitivity to ABT-737. Results of studies utilizing these mutant proteins indicate that Noxa recruits MCL-1 from the cytosol to the mitochondria. Translocation of MCL-1 initiates its phosphorylation and subsequent ubiquitination, which triggers proteasome-mediated degradation. The precise regulatory mechanisms of Noxa/MCL-1 expression and stability could provide alternative targets to modulate apoptosis induced by BH3 mimetic drugs or other chemotherapeutic reagents.

Comprehensive microRNA expression profiling of the hematopoietic hierarchy.

The hematopoietic system produces a large number of highly specialized cell types that are derived through a hierarchical differentiation process from a common stem cell population. miRNAs are critical players in orchestrating this differentiation. Here, we report the development and application of a high-throughput microfluidic real-time quantitative PCR (RT-qPCR) approach for generating global miRNA profiles for 27 phenotypically distinct cell populations isolated from normal adult mouse hematopoietic tissues. A total of 80,000 RT-qPCR assays were used to map the landscape of miRNA expression across the hematopoietic hierarchy, including rare progenitor and stem cell populations. We show that miRNA profiles allow for the direct inference of cell lineage relations and functional similarity. Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage. The analysis of miRNA expression in single hematopoietic cells further demonstrates that miRNA expression is very tightly regulated within highly purified populations, underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity.

The role of SHIP1 in macrophage programming and activation.

The SHIP1 (SH2-containing inositol-5'-phosphatase 1) acts as a negative regulator of proliferation, survival and end cell activation in haemopoietic cells. It does so, at least in part, by translocating to membranes after extracellular stimulation and hydrolysing the phosphoinositide 3-kinase-generated second messenger, PtdIns(3,4,5)P(3) to PtdIns(3,4)P(2). SHIP1(-/-) mice have, as a result, an increased number of neutrophils and monocyte/macrophages because their progenitors display enhanced survival and proliferation. These mice also suffer from osteoporosis because of an increased number of hyperactive osteoclasts and a significant neutrophil infiltration of the lungs. Interestingly, SHIP1(-/-) mice do not display endotoxin tolerance and we have found that lipopolysaccharide-induced endotoxin tolerance is contingent on up-regulating SHIP1, through the production of autocrine-acting transforming growth factor-beta, in bone-marrow-derived macrophages and mast cells. Intriguingly, unlike bone-marrow-derived macrophages, SHIP1(-/-) peritoneal and alveolar macrophages produce 10-fold less NO than wild-type macrophages because these in vivo-generated macrophages have very high arginase I levels and this enzyme competes with inducible nitric oxide synthase for the substrate L-arginine. It is probable that, in the face of chronically increased PtdIns(3,4,5)P(3) levels in their myeloid progenitors, SHIP1(-/-) mice display a skewed development away from M1 (killer) macrophages (which have high inducible nitric oxide synthase levels and produce NO to kill microorganisms and tumour cells), towards M2 (healing) macrophages (which have high arginase levels and produce ornithine to promote host-cell growth and collagen formation). This skewing probably occurs to avoid septic shock and suggests that the phosphoinositide 3-kinase pathway plays a critical role in programming macrophages.

The role of SHIP in cytokine-induced signaling.

The phosphatidylinositol (PI)-3 kinase (PI3K) pathway plays a central role in regulating many biological processes via the generation of the key second messenger PI-3,4,5-trisphosphate (PI-3,4,5-P3). This membrane-associated phospholipid, which is rapidly, albeit transiently, synthesized from PI-4,5-P2 by PI3K in response to a diverse array of extracellular stimuli, attracts pleckstrin homology (PH) domain-containing proteins to membranes to mediate its many effects. To ensure that the activation of this pathway is appropriately suppressed/terminated, the ubiquitously expressed tumor suppressor PTEN hydrolyzes PI-3,4,5-P3 back to PI-4,5-P2 while the 145-kDa hemopoietic-restricted SH2-containing inositol 5'- phosphatase, SHIP (also known as SHIP1), the 104-kDa stem cell-restricted SHIP (sSHIP) and the more widely expressed 150-kDa SHIP2 hydrolyze PI-3,4,5-P3 to PI-3,4-P2. In this review we will concentrate on the properties of the three SHIPs, with special emphasis being placed on the role that SHIP plays in cytokine-induced signaling.

Role of Src homology 2-containing-inositol 5'-phosphatase (SHIP) in mast cells and macrophages.

The haemopoietic-restricted Src homology 2-containing inositol 5'-phosphatase (SHIP) acts as a negative regulator of myeloid cell proliferation, survival and end-cell activation. It does so, at least in part, by hydrolysing the phosphoinositide 3-kinase (PI3K)-generated second messenger, PtdIns(3,4,5) P (3) (PI-3,4,5-P(3)) to PtdIns(3,4) P (2). As a result, the myeloid progenitors in SHIP-knockout mice display enhanced survival and proliferation and the mice have increased numbers of neutrophils and monocytes/macrophages. Interestingly, although SHIP is not required for mast cell or macrophage development, it restrains their differentiation since progenitors from SHIP(-/-) mice differentiate into mature mast cells and macrophages significantly faster than their wild-type counterparts. This could suggest that elevated PI-3,4,5-P(3) levels accelerate myeloid differentiation. In bone-marrow-derived mast cells, SHIP prevents degranulation by IgE alone, restrains IgE-antigen-induced degranulation and limits the production of inflammatory cytokines. On the other hand, in peritoneal macrophages, SHIP is a positive regulator of NO production, since SHIP(-/-) peritoneal macrophages produce 5-10-fold less NO than their wild-type counterparts, even though they show greater lipopolysaccharide/interferon-gamma-induced nuclear factor kappa B activation and more rapid inducible NO synthase (iNOS) generation. This is a result of 10-fold higher levels of arginase I in the SHIP(-/-) macrophages, which redirects the iNOS substrate, L-arginine, from NO to ornithine production. This suggests that the chronically elevated PI-3,4,5-P(3) levels in SHIP(-/-) mice may convert M1 (killing) macrophages, which produce NO to kill micro-organisms and tumour cells, into M2 (healing) macrophages, which produce ornithine to promote host cell growth and fibrosis.

Mechanisms of resistance to imatinib (STI571) and prospects for combination with conventional chemotherapeutic agents.

Imatinib (STI571, Glivec) is a small molecule drug selected for its ability to inhibit the Bcr-Abl kinase, the pathogenic molecular abnormality in chronic myelogenous leukemia (CML). It also is an efficient inhibitor of the Kit and platelet-derived growth factor receptor tyrosine kinases. In vitro studies have demonstrated that this drug potently inhibits proliferation and induces apoptosis of cells that depend on activation of these kinases. Phase I clinical studies have demonstrated remarkable activity against CML. However, these studies, as well as a variety of experimental models, have suggested that clinical resistance to STI571 could develop. The mechanisms for the development of this resistance will be discussed along with the potential for circumventing STI571 resistance by combining it with traditional anti-neoplastic agents.

Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival.

Although IgE binding to mast cells is thought to be a passive presensitization step, we demonstrate herein that monomeric IgE (mIgE) in the absence of antigen (Ag) stimulates multiple phosphorylation events in normal murine bone marrow-derived mast cells (BMMCs). While mIgE does not induce degranulation or leukotriene synthesis, it leads to a more potent production of cytokines than IgE + Ag. Moreover, mIgE prevents the apoptosis of cytokine-deprived BMMCs, likely by maintaining Bcl-X(L) levels and producing autocrine-acting cytokines. The addition of Ag does not increase this IgE-induced survival. Since IgE concentrations as low as 0.1 microg/ml enhance BMMC survival, elevated plasma IgE levels in humans with atopic disorders may contribute to the elevated mast cell numbers seen in these individuals.

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.

Maturation of erythroid cells and erythroleukemia development are affected by the kinase activity of Lyn.

This study examined the impact of the tyrosine kinase Lyn on erythropoietin-induced intracellular signaling in erythroid cells. In J2E erythroleukemic cells, Lyn coimmunoprecipitated with numerous proteins, including SHP-1, SHP-2, ras-GTPase-activating protein, signal transducers and activators of transcription (STAT) 5a, STAT5b, and mitogen-activated protein kinase; however, introduction of a dominant-negative Lyn (Y397F Lyn) inhibited the interaction of Lyn with all of these molecules except SHP-1. Cells containing the dominant-negative Lyn displayed altered intracellular phosphorylation patterns, including mitogen-actiated protein kinase, but not erythropoietin receptor, Janus-activated kinase (JAK) 2, or STAT5. As a consequence, erythropoietin-initiated differentiation and basal proliferation were severely impaired. Y397F Lyn reduced the protein levels of erythroid transcription factors erythroid Kruppel-like factor and GATA-1 up to 90%, which accounts for the inability of J2E cells expressing Y397F Lyn to synthesize hemoglobin. Although Lyn was shown to bind several sites on the cytoplasmic domain of the erythropoietin receptor, it was not activated when a receptor mutated at the JAK2 binding site was ectopically expressed in J2E cells indicating that JAK2 is the primary kinase in erythropoietin signaling and that Lyn is a secondary kinase. In normal erythroid progenitors, erythropoietin enhanced phosphorylation of Lyn; moreover, exogenous Lyn increased colony forming unit-erythroid, but not burst forming uniterythroid, colonies from normal progenitors, demonstrating a stage-specific effect of the kinase. Significantly, altering Lyn activity in J2E cells had a profound effect on the development of erythroleukemias in vivo: the mortality rate was markedly reduced and latent period extended when either wild-type Lyn or Y397F Lyn was introduced into these cells. Taken together, these data show that Lyn plays an important role in intracellular signaling in nontransformed and leukemic erythroid cells.

Indolinone tyrosine kinase inhibitors block Kit activation and growth of small cell lung cancer cells.

Six indolinone tyrosine kinase inhibitors were characterized for their ability to inhibit Kit kinase and for their effects on the growth of small cell lung cancer (SCLC) cell lines. All of the six compounds were potent inhibitors of Kit kinase in a biochemical assay. A homology model of compound binding to the ATP binding site could account for the increased potency observed with the addition of a propionate moiety to the indolinone core but not the increase observed with addition of a chloride moiety. Although all of the compounds tested were potent in the biochemical assay, several exhibited significantly less potency in cellular kinase assays. Their effects on stem cell factor (SCF)-dependent Kit autophosphorylation and SCLC cell growth were also examined. Inhibition of SCF-stimulated Kit activation and cell growth in the H526 cell line was dose-dependent. At concentrations that inhibited SCF-stimulated H526 cell growth, there was little effect on insulin-like growth factor-1-stimulated growth, suggesting that these compounds exhibit reasonable selectivity for inhibition of Kit-mediated proliferation. Higher doses of the compounds were needed to inhibit serum-stimulated growth. Of the six compounds examined, SU5416 and SU6597 demonstrated the best cellular potency and, therefore, their effect on the growth of multiple SCLC cell lines in serum-containing media was examined. In addition to inhibiting proliferation, these compounds also induced significant cell death of several SCLC cell lines, but not of normal human diploid fibroblasts, in complete media. These observations suggest that Kit kinase inhibitors such as these may offer a new approach for inhibiting Kit-mediated proliferation of tumors such as SCLC, gastrointestinal stromal tumors, seminomas, and leukemias.

SHIP's C-terminus is essential for its hydrolysis of PIP3 and inhibition of mast cell degranulation.

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.

A novel assay for the measurement of Raf-1 kinase activity.

Raf-1 is a serine-threonine protein kinase that functions as a central component of the mitogen-activated protein kinase signal transduction pathway. Raf-1 activity is currently assayed in vitro by either measuring 32P incorporation into MEK, Raf-1's only characterized substrate, or by using the phosphorylated MEK to initiate a coupled assay culminating in the phosphorylation of myelin basic protein by MAP kinase. These assays are plagued by a potential lack of specificity in the case of the former, and the time consuming and error-prone nature of the later indirect assay. In this report, we demonstrate a novel single step assay for Raf-1 kinase activity based on phosphorylation of recombinant MEK-1, detected using an activation-specific MEK antibody that recognizes MEK only when specifically phosphorylated by Raf-1 on Ser 217 and Ser 221. The assay readily detected stem cell factor-mediated Raf-1 activation. MEK phosphorylation by immunoprecipitated Raf-1 plateaued at 10 min following initiation of the kinase reaction and was completely dependent on the inclusion of Raf-1. There was a linear correlation between the degree of MEK phosphorylation and the amount of Raf-1 protein immunoprecipitated. In addition to detecting growth factor-mediated activation, the assay was also able to detect paclitaxel-mediated Raf-1 activation. This assay is rapid, sensitive, and specific and therefore is a marked improvement over currently utilized techniques.

Lipid phosphatases in the immune system.

In the last 10 years we have come to appreciate the central role that phosphatidylinositol (PI)-3,4,5-P(3)plays in regulating a vast array of biological responses to extracellular signals. The level of this phospholipid is low in resting cells but increases rapidly in response to growth factor/cytokine-stimulated plasma membrane recruitment and activation of PI-3-kinase. Within the last 3 years three enzymes, SHIP, SHIP2 and PTEN, that play key roles in regulating the level of PI-3,4,5-P(3)have been cloned. In this review I have attempted to summarize our current knowledge regarding these three intriguing phosphatases.

The selective tyrosine kinase inhibitor STI571 inhibits small cell lung cancer growth.

At least 70% of small cell lung cancers express the Kit receptor tyrosine kinase and its ligand, stem cell factor (SCF). Numerous lines of evidence have demonstrated that this coexpression constitutes a functional autocrine loop, suggesting that inhibitors of Kit tyrosine kinase activity could have therapeutic efficacy in this disease. STI571, formerly known as CGP 57148B, is a p.o. bioavailable 2-phenylaminopyrimide derivative that was designed as an Abl tyrosine kinase inhibitor, but also has efficacy against the platelet-derived growth factor receptor and Kit in vitro. Pretreatment of the H526 small cell lung cancer (SCLC) cell line with STI571 inhibited SCF-mediated Kit activation with an IC50 of 0.1 microM as measured by inhibition of receptor tyrosine phosphorylation and 0.2 microM as measured by immune complex kinase assay. This paralleled the inhibition of SCF-mediated growth by STI571, which had an IC50 of approximately 0.3 microM. Growth inhibition in SCF-containing medium was accompanied by induction of apoptosis. STI571 efficiently blocked SCF-mediated activation of mitogen-activated protein kinase and Akt, but did not affect insulin-like growth factor-1 or serum-mediated mitogen-activated protein kinase or Akt activation. Growth of five of six SCLC cell lines in medium containing 10% FCS was inhibited by STI571 with an IC50 of approximately 5 microM. Growth inhibition in serum-containing medium appeared to be cytostatic in nature because no increase in apoptosis was observed. Despite this growth inhibition, STI571 failed to enhance the cytotoxicity of either carboplatinum or etoposide when coadministered. However, taken together with the minimal toxicity that this compound has shown in preclinical studies, these data suggest that STI571 could have a role in the treatment of SCLC, possibly to block or slow recurrence after chemotherapy-induced remissions.

Src family kinase activity is required for Kit-mediated mitogen-activated protein (MAP) kinase activation, however loss of functional retinoblastoma protein makes MAP kinase activation unnecessary for growth of small cell lung cancer cells.

Small cell lung cancer (SCLC) is characterized by multiple genetic alterations that include inactivation of the retinoblastoma protein (Rb), the establishment of several autocrine loops including that induced by coexpression of stem cell factor (SCF) and Kit, and the ectopic expression and activation of Src family kinases. Previous studies have shown that Lck associates with, and becomes activated by, Kit after SCF stimulation of SCLC cells. In the present study, we have demonstrated that PP1, a pharmacological inhibitor of Src kinases, blocked SCF-mediated activation of mitogen-activated protein (MAP) kinase, but it also inhibited Kit activation. However, MAP kinase activation was more sensitive than Kit activation to the effects of PP1. Overexpression of Lck reduced the sensitivity of MAP kinase activation to PP1 without altering the sensitivity of Kit activation, which suggested a role for Lck in SCF-mediated MAP kinase activation. Inducible expression of a dominant negative Lck inhibited MAP kinase activation in a dose-dependent manner, which confirmed that Src family kinase activity is required for SCF-induced MAP kinase activation. The growth of cells that expressed dominant negative Lck was unaffected, however, despite the inhibition of MAP kinase. Growth was also unaffected by the inhibition of the MAP kinase pathway using PD 98059, but sensitivity to the MAP/extracellular signal-regulated kinase kinase inhibitor could be partially restored by expression of wild-type Rb. Therefore, MAP kinase activation seems to be dispensable for the growth of SCLC only in the absence of Rb expression. These data suggest that the SCF/Kit autocrine loop, through activation of Lck and subsequently MAP kinase, and the mutational inactivation of Rb contribute to the loss of G1-S phase checkpoint regulation during the pathogenesis of SCLC. Furthermore, the data demonstrate that, in established SCLC cell lines, proliferative signal transduction initiated by Kit is mediated by pathways other than the classic MAP kinase pathway.

Thapsigargin-induced degranulation of mast cells is dependent on transient activation of phosphatidylinositol-3 kinase.

Thapsigargin, which elevates cytosolic calcium levels by inhibiting the sarcoplasmic/endoplasmic reticulum calcium-dependent ATPase, was tested for its ability to degranulate bone marrow-derived mast cells (BMMCs) from src homology 2-containing inositol phosphatase +/+ (SHIP+/+) and SHIP-/- mice. As was found previously with steel factor, thapsigargin stimulated far more degranulation in SHIP-/- than in SHIP+/+ BMMCs, and this was blocked with the phosphatidylinositol-3 (PI-3) kinase inhibitors, LY294002 and wortmannin. In contrast to steel factor, however, this heightened degranulation of SHIP-/- BMMCs was not due to a greater calcium influx into these cells, nor was the thapsigargin-induced calcium influx inhibited by LY294002, suggesting that the heightened thapsigargin-induced degranulation of SHIP-/- BMMCs was due to a PI-3 kinase-regulated step distinct from that regulating calcium entry. An investigation of thapsigargin-stimulated pathways in both cell types revealed that MAPK was heavily but equally phosphorylated. Interestingly, the protein kinase C inhibitor, bisindolylmaleimide (compound 3), totally blocked thapsigargin-induced degranulation in both SHIP+/+ and SHIP-/- BMMCs. As well, thapsigargin stimulated a PI-3 kinase-dependent, transient activation of protein kinase B, and this activation was far greater in SHIP-/- than in SHIP+/+ BMMCs. Consistent with this, thapsigargin was found to be a potent survival factor, following cytokine withdrawal, for both cell types and was more potent with SHIP-/- cells. These studies have both identified an additional PI-3 kinase-dependent step within the mast cell degranulation process, possibly involving 3-phosphoinositide-dependent protein kinase-1 and a diacylglycerol-independent protein kinase C isoform, and shown that the tumor-promoting activity of thapsigargin may be due to its activation of protein kinase B and subsequent promotion of cell survival.

Phosphatidylinositol 3,4,5-trisphosphate regulates Ca(2+) entry via btk in platelets and megakaryocytes without increasing phospholipase C activity.

The role of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P(3)) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P(3) was increased in platelets from mice deficient in the SH2 domain-containing inositol 5-phosphatase (SHIP), in response to collagen related peptide (CRP). Tyrosine phosphorylation and activation of phospholipase Cgamma2 (PLCgamma2) were unaltered in SHIP(-/-) platelets, whereas Btk was heavily tyrosine phosphorylated under basal conditions and maximally phosphorylated by low concentrations of CRP. There was an increase in basal Ca(2+), maximal expression of P-selectin, and potentiation of Ca(2+) and aminophospholipid exposure to CRP in SHIP(-/-) platelets in the presence of Ca(2+) (1 mM). Microinjection of PI3,4, 5P(3) into megakaryocytes caused a 3-fold increase in Ca(2+) in response to CRP, which was absent in X-linked immunodeficiency (Xid) mice, which have a mutation in the PH domain of Btk. There was a corresponding partial reduction in the sustained level of intracellular Ca(2+) in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca(2+) entry that involves PI3,4,5P(3) and Btk, and which is independent of increased PLC activity.

A dual role for Src homology 2 domain-containing inositol-5-phosphatase (SHIP) in immunity: aberrant development and enhanced function of b lymphocytes in ship -/- mice.

In this report, we demonstrate that the Src homology 2 domain-containing inositol-5-phosphatase (SHIP) plays a critical role in regulating both B cell development and responsiveness to antigen stimulation. SHIP(-/-) mice exhibit a transplantable alteration in B lymphoid development that results in reduced numbers of precursor B (fraction C) and immature B cells in the bone marrow. In vitro, purified SHIP(-/)- B cells exhibit enhanced proliferation in response to B cell receptor stimulation in both the presence and absence of Fcgamma receptor IIB coligation. This enhancement is associated with increased phosphorylation of both mitogen-activated protein kinase and Akt, as well as with increased survival and cell cycling. SHIP(-/)- mice manifest elevated serum immunoglobulin (Ig) levels and an exaggerated IgG response to the T cell-independent type 2 antigen trinitrophenyl Ficoll. However, only altered B cell development was apparent upon transplantation into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice. The in vitro hyperresponsiveness, together with the in vivo findings, suggests that SHIP regulates B lymphoid development and antigen responsiveness by both intrinsic and extrinsic mechanisms.

SHIPs ahoy.

In 1996 three groups independently cloned a hemopoietic specific, src homology 2-containing inositol 5'-phosphatase which, based on its structure, was called SHIP. More recently, a second more widely expressed SHIP-like protein has been cloned and called SHIP2. Both specifically hydrolyze phosphatidylinositol-3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in vitro. Moreover, SHIP has been shown in vivo to be the primary enzyme responsible for breaking down phosphatidylinositol-3,4,5-trisphosphate to phosphatidylinositol-3,4-bisphosphate in normal mast cells and, as a result, limits normal and prevents inappropriate mast cell degranulation. Because of their ability to break down phosphatidylinositol-3,4,5-trisphosphate, the SHIPs have the potential to regulate many, if not all, phosphatidylinositol-3-kinase induced events including, proliferation, differentiation, apoptosis, end cell activation, cell movement and adhesion and will thus likely be the subject of intensive research over the next few years.

Altered responsiveness to chemokines due to targeted disruption of SHIP.

SHIP has been implicated in negative signaling in a number of hematopoietic cell types and is postulated to downregulate phosphatidylinositol-3-kinase- (PI-3K-) initiated events in diverse receptor signaling pathways. Because PI-3K is implicated in chemokine signaling, we investigated whether SHIP plays any role in cellular responses to chemokines. We found that a number of immature and mature hematopoietic cells from SHIP-deficient mice manifested enhanced directional migration (chemotaxis) in response to the chemokines stromal cell-derived factor-1 (SDF-1) and B-lymphocyte chemoattractant (BLC). SHIP(-/-) cells were also more active in calcium influx and actin polymerization in response to SDF-1. However, colony formation by SHIP-deficient hematopoietic progenitor cell (HPCs) was not inhibited by 13 myelosuppressive chemokines that normally inhibit proliferation of HPCs. These altered biologic activities of chemokines on SHIP-deficient cells are not caused by simple modulation of chemokine receptor expression in SHIP-deficient mice, implicating SHIP in the modulation of chemokine-induced signaling and downstream effects.