Oncogenic Kit controls neoplastic mast cell growth through a Stat5/PI3-kinase signaling cascade

The D816V-mutated variant of Kit triggers multiple signaling pathways and is considered essential for malignant transformation in mast cell (MC) neoplasms. We here describe that constitutive activation of the Stat5-PI3K-Akt-cascade controls neoplastic MC development. Retrovirally transduced active Stat5 (cS5(F)) was found to trigger PI3K and Akt activation, and to transform murine bone marrow progenitors into tissue-infiltrating MCs. Primary neoplastic Kit D816V(+) MCs in patients with mastocytosis also displayed activated Stat5, which was found to localize to the cytoplasm and to form a signaling complex with PI3K, with consecutive Akt activation. Finally, the knock-down of either Stat5 or Akt activity resulted in growth inhibition of neoplastic Kit D816V(+) MCs. These data suggest that a downstream Stat5-PI3K-Akt signaling cascade is essential for Kit D816V-mediated growth and survival of neoplastic MCs.     

Lipid rafts are required for Kit survival and proliferation signals

In addition to its physiologic role as central regulator of the hematopoietic and reproductive systems, the Kit receptor tyrosine kinase (RTK) is pathologically overexpressed in some forms of leukemia and constitutively activated by oncogenic mutations in mast-cell proliferations and gastrointestinal stromal tumors. To gain insight into the general activation and signaling mechanisms of RTKs, we investigated the activation-dependent dynamic membrane distributions of wild-type and oncogenic forms of Kit in hematopoietic cells. Ligand-induced recruitment of wild-type Kit to lipid rafts after stimulation by Kit ligand (KL) and the constitutive localization of oncogenic Kit in lipid rafts are necessary for Kit-mediated proliferation and survival signals. KL-dependent and oncogenic Kit kinase activity resulted in recruitment of the regulatory phosphatidylinositol 3-kinase (PI3-K) subunit p85 to rafts where the catalytical PI3-K subunit p110 constitutively resides. Cholesterol depletion by methyl-beta-cyclodextrin prevented Kit-mediated activation of the PI3-K downstream target Akt and inhibited cellular proliferation by KL-activated or oncogenic Kit, including mutants resistant to the Kit inhibitor imatinib-mesylate. Our data are consistent with the notion that Kit recruitment to lipid rafts is required for efficient activation of the PI3-K/Akt pathway and Kit-mediated proliferation.     

G-CSF-induced tyrosine phosphorylation of Gab2 is Lyn kinase dependent and associated with enhanced Akt and differentiative, not proliferative, responses

The granulocyte colony-stimulating factor receptor (G-CSFR) transduces intracellular signals for myeloid cell proliferation, survival, and differentiation through the recruitment of nonreceptor protein tyrosine kinases Lyn and janus kinase 2 (Jak2). This results in the tyrosine phosphorylation of a small set of positive and negative adapters and effectors. Grb2-associated binder-2 (Gab2) is a newly described adapter molecule, preferentially expressed in hematopoietic cells and associated with phosphatidylinositol 3 (PI3) kinase. Studies suggest that Gab2 plays both positive and negative roles in cytokine receptor signaling. To investigate the role Gab2 plays in G-CSF receptor-mediated signaling, we have analyzed its activation state and correlated that with wild-type and mutant G-CSF receptors stably expressed in the murine factor-dependent Ba/F3 cell lines. G-CSF-induced tyrosine phosphorylation of Gab2 occurred in the wild-type and single Y-to-F mutants (Y704F, Y729F, and Y744F), but not in the ADA and W650R loss-of-function mutants. Cells expressing truncated proximal G-CSFR, the tyrosine-null (Y4F) G-CSFR, or Y764F mutant receptors had decreased phosphorylation of Gab2. Specific inhibitors of Src kinase (PD173 and PP1) but not Jak2 kinase (AG490) blocked Gab2 phosphorylation. Phosphorylation of Gab2 occurred in wild-type, but not Lyn-deficient, G-CSFR-transfected DT40 B cells. These data propose that Lyn, not Jak2, phosphorylates Gab2 and that maximal phosphorylation of Gab2 requires Y764, a Grb2-binding site. Serine phosphorylation of Akt, a marker of PI3-kinase activity, was detected in both wild-type and truncated proximal domain receptors, but not in the ADA and W650R mutants. Levels of phospho-Akt and phospho-extracellular signal-regulated kinase (phospho-ERK) were greater in proximal truncated than in wild-type G-CSFR cells, suggesting that Gab2 is dissociated from PI3 kinase or ERK activities. Overexpression of Gab2 enhanced the phosphorylation state of Akt, but not of ERK. This inhibited the proliferation of wild-type and truncated G-CSFR-transfected Ba/F3 cells and enhanced their myeloid differentiation. All together, these data indicate that G-CSF treatment leads to Lyn-mediated tyrosine phosphorylation of Gab2, which may serve as an important intermediate of enhanced Akt activity and myeloid differentiation, not growth/survival response.     

Interleukin 3-dependent survival by the Akt protein kinase

Interleukin 3 (IL-3)-dependent survival of hematopoietic cells is known to rely on the activity of multiple signaling pathways, including a pathway leading to activation of phosphoinositide 3-kinase (PI 3-kinase), and protein kinase Akt is a direct target of PI 3-kinase. We find that Akt kinase activity is rapidly induced by the cytokine IL-3, suggesting a role for Akt in PI 3-kinase-dependent signaling in hematopoetic cells. Dominant-negative mutants of Akt specifically block Akt activation by IL-3 and interfere with IL-3-dependent proliferation. Overexpression of Akt or oncogenic v-akt protects 32D cells from apoptosis induced by IL-3 withdrawal. Apoptosis after IL-3 withdrawal is accelerated by expression of dominant-negative mutants of Akt, indicating that a functional Akt signaling pathway is necessary for cell survival mediated by the cytokine IL-3. Thus Akt appears to be an important mediator of anti-apoptotic signaling in this system.     

Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic

Mutations in genes that encode components of the phosphatidyl-inositol 3-kinase (PI3-kinase) signaling pathway are common in human cancer. The recent discovery of nonrandom somatic mutations in the PIK3CA gene of many human tumors suggests an oncogenic role for the mutated enzyme. We have determined the growth-regulatory and signaling properties of the three most frequently observed PI3-kinase mutations: E542K, E545K, and H1047R. Expressed in chicken embryo fibroblasts, all three mutants induce oncogenic transformation with high efficiency. This transforming ability is correlated with elevated catalytic activity in in vitro kinase assays. The mutant-transformed cells show constitutive phosphorylation of Akt, of p70 S6 kinase, and of the 4E-binding protein 1. Phosphorylation of S6 kinase and of 4E-binding protein 1 is regulated by the target of rapamycin (TOR) kinase and affects rates of protein synthesis. The inhibitor of TOR, rapamycin, strongly interferes with cellular transformation induced by the PI3-kinase mutants, suggesting that the TOR and its downstream targets are essential components of the transformation process. The oncogenic transforming activity makes the mutated PI3-kinase proteins promising targets for small molecule inhibitors that could be developed into effective and highly specific anticancer drugs.     

Grb2-associated binder-1 plays a central role in the hepatocyte growth factor enhancement of hepatoma growth inhibition by K vitamin analog compound 5

Compound 5 (Cpd 5), a K vitamin analog, has been shown to inhibit Hep3B human hepatoma cell growth in cultures and rat hepatoma growth in vivo through prolonged epidermal growth factor receptor (EGFR)-extracellular response kinase (ERK) phosphorylation, and hepatocyte growth factor (HGF) synergizes with Cpd 5 to enhance the inhibition of Hep3B cell and rat hepatoma growth. To explore the mechanisms mediating the HGF/Cpd 5 synergy, we examined the possible involvement of the Grb2-associated binder-1 (Gab1) docking protein because it interacts with both EGFR and HGF receptor c-Met pathways. We found that HGF enhanced Cpd 5-induced c-Met phosphorylation at Tyr-1349, a binding site for Gab1, resulting in increased c-Met binding to Gab1, and induced strong and prolonged Gab1 tyrosine phosphorylation. Prolonged Gab1 phosphorylation by HGF/Cpd 5 in turn enhanced the ability of Gab1 to bind to protein tyrosine phosphatase SHP2 and enhanced the activation of its downstream mitogen-activated protein kinase pathway. In contrast, this same HGF/Cpd 5 treatment inhibited Gab1 binding to phosphatidylinositol 3-kinase (PI3K), leading to the inactivation of the PI3K-Akt pathway. The inhibition of Akt phosphorylation by HGF/Cpd 5 further activated the Raf-MEK-ERK signaling cascade via an Akt-Raf1 interaction, leading to strong and prolonged ERK phosphorylation. The transfection of Hep3B cells with mutated Gab1 (Gab1 Y627F), which had lost its ability to bind SHP2, antagonized HGF/Cpd 5-induced ERK phosphorylation, whereas the transfection of Hep3B cells with mutated Gab1 3YF, which lost its ability to bind PI3K, further enhanced HGF/Cpd 5-induced ERK phosphorylation and cell growth inhibition. CONCLUSION: Gab1 plays a central role in regulating HGF/Cpd 5 synergy in their actions on Hep3B cell growth inhibition.     

Autocrine IGF-1/IGF-1R signaling is responsible for constitutive PI3K/Akt activation in acute myeloid leukemia: therapeutic value of neutralizing anti-IGF-1R antibody

Background

Alterations in the PI3K/Akt pathway are found in a wide range of cancers and the development of PI3K inhibitors represents a promising approach to cancer therapy. Constitutive PI3K activation, reflecting an intrinsic oncogenic deregulation of primary blast cells, is detected in 50% of patients with acute myeloid leukemia. However, the mechanisms leading to this activation are currently unknown. As we previously reported IGF-1 autocriny in acute myeloid leukemia cells, we investigated whether IGF-1 signaling was involved in the constitutive activation of PI3K.

Design and Methods

We analyzed the IGF-1/IGF-1R signaling pathway and PI3K activity in 40 acute myeloid leukemia bone marrow samples. Specific inhibition of IGF-1/IGF-1R signaling was investigated using neutralizing anti-IGF-1R, anti-IGF-1 antibodies or IGF-1 short interfering RNA. The anti-leukemic activity of the neutralizing anti-IGF-1R was tested by analyzing its effects on leukemic progenitor clonogenicity, blast cell proliferation and survival.

Results

In all samples tested, we found that functional IGF-1R was constantly expressed in leukemic cells. In the acute myeloid leukemia samples with PI3K activation, we found that the IGF-1R was constitutively phosphorylated, although no IGF-1R activating mutation was detected. Specific inhibition of IGF-1R signaling with neutralizing anti-IGF-1R strongly inhibited the constitutive phosphorylation of both IGF-1R and Akt in 70% of the PI3K activated samples. Moreover, both incubation with anti-IGF-1 antibody and IGF-1 short interfering RNA inhibited Akt phosphorylation in leukemic cells. Finally, neutralizing anti-IGF-1R treatment decreased the clonogenicity of leukemic progenitors and the proliferation of PI3K activated acute myeloid leukemia cells.

Conclusions

Our current data indicate a critical role for IGF-1 autocriny in constitutive PI3K/Akt activation in primary acute myeloid leukemia cells and provide a strong rationale for targeting IGF-1R as a potential new therapy for this disease.     

Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor

Nerve growth factor (NGF) prevents apoptosis through stimulation of the TrkA receptor protein tyrosine kinase. The downstream activation of phosphatidylinositol 3-kinase (PI 3-kinase) is essential for the inhibition of apoptosis, although this enzyme does not bind to and is not directly activated by TrkA. We have found that the addition of NGF to PC-12 cells resulted in the phosphorylation of the Grb2-associated binder-1 (Gab1) docking protein and induced the association of several SH2 domain-containing proteins, including PI 3-kinase. A substantial fraction of the total cellular PI 3-kinase activity was associated with Gab1. PC-12 cells that overexpressed Gab1 show a decreased requirement for the amount of NGF necessary to inhibit apoptosis. The expression of a Gab1 mutant that lacked the binding sites for PI 3-kinase enhanced apoptosis and diminished the protective effect of NGF. Hence, Gab1 has a major role in connecting TrkA with PI 3-kinase activation and for the promotion of cell survival by NGF.     

Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins

The docking protein FRS2 is a major downstream effector that links fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein kinase signaling cascade. In this report, we demonstrate that FRS2 also plays a pivotal role in FGF-induced recruitment and activation of phosphatidylinositol 3-kinase (PI3-kinase). We demonstrate that tyrosine phosphorylation of FRS2alpha leads to Grb2-mediated complex formation with the docking protein Gab1 and its tyrosine phosphorylation, resulting in the recruitment and activation of PI3-kinase. Furthermore, Grb2 bound to tyrosine-phosphorylated FRS2 through its SH2 domain interacts primarily via its carboxyl-terminal SH3 domain with a proline-rich region in Gab1 and via its amino-terminal SH3 domain with the nucleotide exchange factor Sos1. Assembly of FRS2alpha:Grb2:Gab1 complex induced by FGF stimulation results in activation of PI3-kinase and downstream effector proteins such as the S/T kinase Akt, whose cellular localization and activity are regulated by products of PI3-kinase. These experiments reveal a unique mechanism for generation of signal diversity by growth factor-induced coordinated assembly of a multidocking protein complex that can activate the Ras/mitogen-activated protein kinase cascade to induce cell proliferation and differentiation, and PI3-kinase to activate a mediator of a cell survival pathway.     

Critical role for PI 3-kinase in the control of erythropoietin-induced erythroid progenitor proliferation

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors, namely insulin receptor substrate-2 (IRS2), Src homology 2 domain-containing inositol 5'-phosphatase (SHIP), Grb2-associated binder-1 (Gab1), and the Epo receptor (EpoR). Using different in vitro systems, we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors, Akt, FKHRL1, and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR), through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR, or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors, but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors, the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally, our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2), which, in turn, down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation.     

Regulation of Akt-dependent cell survival by Syk and Rac

Interleukin-2 (IL-2) prevents cell apoptosis and promotes survival, but the involved mechanisms have not been completely defined. Although phosphatidylinositide 3-kinase (PI 3-kinase) has been implicated in IL-2-mediated survival mechanisms, none of the 3 chains of the IL-2 receptor (IL-2R) expresses a binding site for PI 3-kinase. However, IL-2Rbeta does express a Syk-binding motif. By using an IL-2-dependent natural killer (NK) cell line, followed by validation of the results in fresh human NK cells, we identified Syk as a critical effector essential for IL-2-mediated prosurvival signaling in NK cells. Down-regulation of Syk by piceatannol treatment impaired NK cellular viability and induced prominent apoptosis as effectively as suppression of PI 3-kinase function by LY294002. Expression of kinase-deficient Syk or pretreatment with piceatannol markedly suppressed IL-2-stimulated activation of PI 3-kinase and Akt, demonstrating that Syk is upstream of PI 3-kinase and Akt. However, constitutively active PI 3-kinase reversed this loss of Akt function caused by kinase-deficient Syk or piceatannol. Thus, Syk appears to regulate PI 3-kinase, which controls Akt activity during IL-2 stimulation. More important, we observed Rac1 activation by IL-2 and found that it mediated PI 3-kinase activation of Akt. This conclusion came from experiments in which dominant-negative Rac1 significantly decreased IL-2-induced Akt activation, whereas constitutively active Rac1 reelevated Akt activity not only in Syk-impaired but also in PI 3-kinase-impaired NK cells. These results constitute the first report of a Syk --> PI3K --> Rac1 --> Akt signal cascade controlled by IL-2 that mediates NK cell survival.     

PI3-kinase/Akt/mTOR signaling: Impaired on/off switches in aging,cognitive decline and Alzheimer's disease

The normal on and off switching of the PI3-K (phosphoinositide 3-kinase)/Akt pathway, particularly by its major activators insulin and IGF-1 (insulin-like growth factor-1), is a powerful integrator of physiological responses rudimentary to successful aging. This is highlighted by extensive studies showing that reducing, but not obliterating, activation of the PI3-K/Akt/mTOR signal, at several levels, can extend healthy lifespan in organisms from yeast to mammals. Moreover, aberrant control of the PI3-K/Akt axis is emerging to be a primary causative node in all major diseases of aging: cancer, type 2 diabetes mellitus (T2DM), heart disease and neurodegeneration. Aging is the major risk factor for AD, the most common dementia disorder. The integrated coordination of neuronal responses through the PI3-K/Akt pathway has significant functional impact on key events that go awry in Alzheimer's disease (AD), including: synaptic plasticity, neuronal polarity, neurotransmission, proteostasis, use-dependent translation, metabolic control and stress responses including DNA repair. Investigation of the status of the PI3-K/Akt system in brains of individuals who have had AD shows aberrant and sustained activation of neuronal PI3-K/Akt/mTOR signaling to be an early feature of the disease. This is mechanistically linked to progressive desensitization of normal brain insulin and IGF-1 responses, aberrant proteostasis of Aβ and tau, synaptic loss and cognitive decline in the disease. Notably, concomitantly with feedback inhibition of insulin and IGF-1 responses, increased activation of the neuronal PI3-K/Akt/mTOR axis is a major candidate effector system for transmission of pathophysiological signals from Aβ to tau in the context of defects in synaptic transmission that lead to cognitive decline. Therapeutic approaches targeted at normalizing signaling through either the neuronal PI3-kinase/Akt/mTOR pathway or its activation by insulin and IGF-1 have been shown to be protective against the development of AD pathology and cognitive decline in animal models of AD and some of these therapies are entering clinical trials in patients with the disease.     

A biochemical mechanism for the oncogenic potential of the p110beta catalytic subunit of phosphoinositide 3-kinase

Class I PI3-kinases signal downstream of receptor tyrosine kinases and G protein-coupled receptors and have been implicated in tumorigenesis. Although the oncogenic potential of the PI3-kinase subunit p110α requires its mutational activation, other p110 isoforms can induce transformation when overexpressed in the wild-type state. In wild-type p110α, N345 in the C2 domain forms hydrogen bonds with D560 and N564 in the inter-SH2 (iSH2) domain of p85, and mutations of p110α or p85 that disrupt this interface lead to increased basal activity and transformation. Sequence analysis reveals that N345 in p110α aligns with K342 in p110β. This difference makes wild-type p110β analogous to a previously described oncogenic mutant, p110α-N345K. We now show that p110β is inhibited by p85 to a lesser extent than p110α and is not differentially inhibited by wild-type p85 versus p85 mutants that disrupt the C2-iSH2 domain interface. Similar results were seen in soft agar and focus-formation assays, where p110β was similar to p110α-N345K in transforming potential. Inhibition of p110β by p85 was enhanced by a K342N mutation in p110β, which led to decreased activity in vitro, decreased basal Akt and ribosomal protein S6 kinase (S6K1) activation, and decreased transformation in NIH 3T3 cells. Moreover, unlike wild-type p110β, p110β-K342N was differentially regulated by wild-type and mutant p85, suggesting that the inhibitory C2-iSH2 interface is functional in this mutant. This study shows that the enhanced transforming potential of p110β is the result of its decreased inhibition by p85, due to the disruption of an inhibitory C2-iSH2 domain interface.     

Role of Akt in growth and survival of PANC-1 pancreatic cancer cells.

INTRODUCTION: Akt is involved in different cellular processes such as cell growth, cell differentiation, and anti-apoptosis. AIMS: To investigate the role of Akt in cell growth and survival in PANC-1 pancreatic cancer cells. METHODOLOGY AND RESULTS: Insulin-like growth factor (IGF)-I induced Akt activation in a dose-dependent manner and stimulated anchorage-dependent and anchorage-independent cell growth of PANC-1 cells. In PANC-1 cells infected with adenovirus vectors carrying kinase-deficient Akt, anchorage-dependent and anchorage-independent cell growth was significantly reduced in the presence or absence of IGF-I compared with cells infected with adenovirus vectors carrying wild-type Akt, although IGF-I significantly stimulated cell growth in both transfected cell lines. Conversely, in PANC-1 cells infected with adenovirus vectors carrying kinase-deficient Akt, typical DNA laddering was undetectable in DNA fragmentation assay, and DNA 3;-OH reactivity was not detected in TUNEL assay. We then examined the role of phosphatidylinositol 3-kinase (PI3-K), an upstream mediator of Akt, on cell survival. In PANC-1 cells infected with adenovirus vector carrying a deletion mutant of the 85-kDa regulatory subunit of PI3-K and in cells treated with PI3-K inhibitor wortmannin, typical DNA laddering was evident in DNA fragmentation assay. In TUNEL assay, nuclear condensation and DNA 3;-OH reactivity was observed in approximately 30% of these cells. CONCLUSION: The present results indicate that Akt is implicated in cell growth, but not in survival in PANC-1 cells. These results suggest that there may be an alternative survival signal cascade from PI3-K in PANC-1 cells.     

Role of phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways in granulocyte macrophage-colony-stimulating factor failure to delay fas-induced neutrophil apoptosis in elderly humans

Fas-stimulated neutrophils from elderly individuals show impaired granulocyte macrophage-colony-stimulating factor (GM-CSF)-induced apoptosis cell rescue. Herein, this defect was found to be associated with a significant reduction in GM-CSF-mediated Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Using Akt and ERK1/2 inhibitors, we demonstrated that both kinases were critical for GM-CSF antiapoptotic effects. Whereas Akt inhibition also affected GM-CSF-dependent ERK1/2 phosphorylation, ERK1/2 inhibition did not affect GM-CSF-induced Akt phosphorylation, suggesting that phosphoinositide 3-kinase (PI3-K)/Akt and ERK1/2 are activated in series and that PI3-K is located upstream of ERK1/2 along the GM-CSF-dependent signaling pathway. No age-associated changes in GM-CSF receptor expression were observed. Interestingly, both suppressors of cytokine signaling (SOCS)1 and SOCS3 proteins were significantly higher in unstimulated neutrophils from elderly individuals and, unlike in young individuals, did not further increase following GM-CSF cell triggering. These results indicate that defective PI3-K/Akt/ERK1/2 activation, likely dependent on elevated SOCS1 and SOCS3 levels, may affect the GM-CSF capacity to delay neutrophil apoptosis in elderly persons.     

CD38-mediated growth suppression of B-cell progenitors requires activation of phosphatidylinositol 3-kinase and involves its association with the protein product of the c-cbl proto-oncogene

The signalling pathways that arrest the cell cycle and trigger cell death are only partially known. Dimerization of CD38, a 45-kD transmembrane type II glycoprotein highly expressed in immature B cells, inhibits cell growth and causes apoptosis in normal and leukemic B-cell progenitors, but the molecular mechanisms underlying these cellular responses are unknown. In the present study, we found that CD38 ligation in the immature B-cell lines 380, REH, and RS4;11 caused rapid tyrosine phosphorylation of the protein product of the proto- oncogene c-cbl. Dimerization of CD38 was accompanied by the association of cbl with the p85 subunit of phosphatidylinositol 3-kinase (Pl 3-K), resulting in markedly increased Pl 3-K activity in antiphosphotyrosine and anti-cbl immunoprecipitates. Wortmannin and LY294002, two potent inhibitors of Pl 3-K, rescued immature B cells from CD38-mediated growth suppression. This effect was observed not only in model B-cell lines, but also in cultures of leukemic lymphoblasts from patients, and in normal bone marrow B-cell progenitors as well. Concentrations of inhibitors that reversed cellular responses to CD38 significantly decreased Pl 3-K activity. By contrast, rapamycin, a p70 S6-kinase inhibitor, did not rescue immature B cells from CD38-mediated suppression. These results suggest that Pl 3-K activity is essential for CD38-mediated inhibition of lymphopoiesis and that cbl and Pl 3-K are regulatory molecules whose activation can result in suppression of cell proliferation and apoptosis in immature lymphoid cells.     

A role of Gab2 association in Flt3 ITD mediated Stat5 phosphorylation and cell survival

The haematopoietic growth factor receptor Flt3 has been implicated as major cause of transformation in acute myeloid leukaemia. Intracellular signals mediated by wild-type Flt3 are involved in cell differentiation and survival whereas signalling via the mutant Flt3 ITD (internal tandem duplication) promotes enhanced cell growth. In this study, we identified tyrosines 768, 955 and 969 of Flt3 as phosphorylation sites and mediators of growth factor receptor binding protein 2 (Grb2) interaction, leading to the association of Grb2 associated binder 2 (Gab2) and contributing to proliferation and survival. Ba/F3 cells were transfected with either the wild-type Flt3 or the ITD, with or without a triple mutation of the Grb2 binding sites, and characterised in terms of proliferation and viability. Interestingly, the Flt3 ITD promoted increased survival but after introducing the triple mutation, this phenotype was lost. When looking into different downstream pathways, this effect was mainly caused by decreased phosphoinositide 3-kinase and Stat5 signalling, and the Flt3 ITD carrying the Grb2 binding mutations showed less Akt and Stat5 activation compared to the regular Flt3 ITD receptor. These findings not only reveal novel phosphorylation sites in Flt3 but contribute to the understanding of the molecular mechanism by which Flt3 ITD functions in pathological conditions.     

Insulin activates ATP-sensitive potassium channels via phosphatidylinositol 3-kinase in cultured vascular smooth muscle cells

The effects of insulin on the vasculature are significant because insulin resistance is associated with hypertension. To increase the understanding of the effects of insulin on the vasculature, we analyzed changes in potassium ion transport in cultured vascular smooth muscle cells (VSMCs). Using the potential-sensitive fluorescence dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)], we found that insulin induced membrane hyperpolarization after 2 min in A10 cells. Insulin-induced hyperpolarization was suppressed by glibenclamide, an ATP-sensitive potassium (K(ATP)) channel blocker. Using a cell-attached patch clamp experiment, the K(ATP) channel was activated by insulin in both A10 cells and isolated VSMCs from rat aortas, indicating that insulin causes membrane hyperpolarization via K(ATP) channel activation. These effects were not dependent on intracellular ATP concentration, but wortmannin, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, significantly suppressed insulin-induced K(ATP) channel activation. In addition, insulin enhanced phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and protein kinase B (Akt) after 2 min. These data suggest that K(ATP) channel activation by insulin is mediated by PI3-K. Furthermore, using a nitric oxide synthase (NOS) inhibitor, we found that NOS might play an important role downstream of PI3-K in insulin-induced K(ATP) channel activation. This study may contribute to our understanding of mechanisms of insulin resistance-associated hypertension.