The yeast Kes1p/Osh4p protein functions as a sterol/PI(4)P exchanger between lipid membranes, which suggests the possibility of creating a sterol gradient via phosphoinositide metabolism.

The mammalian target of rapamycin (mTOR) is a key regulator of growth and survival in many cell types. Its constitutive activation has been involved in the pathogenesis of various cancers. In this study, we show that mTOR inhibition by rapamycin strongly inhibits the growth of the most immature acute myeloid leukemia ( IntroductionAcute myeloid leukemia (AML) is a clonal disorder characterized by accumulation of malignant hematopoietic progenitor cells (HPCs) with impaired differentiation program. Despite important progress in the therapy of AML and high rates of complete remission after induction chemotherapy, most patients will relapse and die from the disease. Prevention of relapse is based on intensified programs, including high-dose chemotherapy and autologous or allogenic transplants that can benefit young patients. Thus, outcome of patients older than 60 years has not been improved for decades, underlying the need for potent and less toxic drugs for the treatment of this disease. 1 Recent studies have demonstrated that AML cells are characterized by recurrent mutations of genes involved in cell differentiation, survival, and proliferation. A pathogenesis model for AML suggests that mutations of both tyrosine kinase receptors and transcription factors, by conferring survival and/or proliferative advantage (class I mutation) and by impairing cell differentiation (class II mutation), are needed to cause leukemia. 2 Fms-like tyrosine kinase 3 (FLT3), c-KIT, and RAS mutations occur in 50% to 60% of AML cases, 3-7 leading to aberrant activation of major cell survival or proliferation pathways such as mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/ Akt, signal transducer and activator of transcription (STAT), or nuclear factor B (NF-kB). [8][9][10] These antiapoptotic signaling pathways also contribute to AML resistance to the cytotoxic agents currently used in this disease. 11,12 Thus, therapeutic interference with these pathways represents an attractive strategy in AML therapy. In this context, current clinical trials are evaluating new compounds directly targeting RAS or FLT3 (eg, farnesyl transferase inhibitors; CEP-701 and PKC 412). 13,14 Molecules integrating multiple signal transduction pathways may represent relevant therapeutic targets in AML. Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in the regulation of cell growth and proliferation by translational control of key proteins such as the cyclin-dependent kinase (CDK) inhibitor p27kip1, retinoblastoma protein, cyclin D1, c-myc, or STAT 3. mTOR is activated by different stimuli including nutrients or growth factors. 15,16 Once activated, mTOR can phosphorylate its downstream targets, the ribosomal p70S6 kinase (p70S6K) and the 4E-binding protein 1 (4E-BP1). There are 2 known isoforms of S6K, p70 and p85, generated from differential splicing from a common gene. The p85S6K isoform is identical to p70S6K, except for a 23-amino acid extension at the amino-terminus that specifically targets it to ...

Activation of the Wnt/b-catenin pathway has recently been shown to be crucial to the establishment of leukemic stem cells in chronic myeloid leukemia. We sought to determine whether b-catenin was correlated to clonogenic capacity also in the acute myeloid leukemia (AML) setting. Eighty-two patients were retrospectively evaluated for b-catenin expression by Western blot. b-Catenin was expressed (although at various protein levels) in 61% of patients, and was undetectable in the remaining cases. In our cohort, b-catenin expression was correlated with the clonogenic proliferation of AML-colony forming cells (AML-CFC or CFU-L) in methylcellulose in the presence of 5637-conditioned medium, and more strikingly with self-renewing of leukemic cells, as assessed in vitro by a replating assay. In survival analyses, b-catenin appeared as a new independent prognostic factor predicting poor event-free survival and shortened overall survival (both with Po0.05). Furthermore, variations in b-catenin protein levels were dependent on post-transcriptional mechanisms involving the Wnt/ b-catenin pathway only in leukemic cells. Indeed, b-catenin negative leukemic cells were found to increase b-catenin in response to Wnt3a agonist in contrast to normal counterparts. Altogether, our data pave the way to the evaluation of Wnt pathway inhibition as a new rationale for eradicating the clonogenic pool of AML cells.

Phosphoinositides are important regulators of diverse cellular functions, and phosphatidylinositol 3-monophosphate (PI3P) is a key element in vesicular trafficking processes. During its intraerythrocytic development, the malaria parasite Plasmodium falciparum establishes a sophisticated but poorly characterized protein and lipid trafficking system. Here we established the detailed phosphoinositide profile of P. falciparum-infected erythrocytes and found abundant amounts of PI3P, while phosphatidylinositol 3,5-bisphosphate was not detected. PI3P production was parasite dependent, sensitive to a phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, and predominant in late parasite stages. The Plasmodium genome encodes a class III PI3-kinase of unusual size, containing large insertions and several repetitive sequence motifs. The gene could not be deleted in Plasmodium berghei, and in vitro growth of P. falciparum was sensitive to a PI3-kinase inhibitor, indicating that PI3-kinase is essential in Plasmodium blood stages. For intraparasitic PI3P localization, transgenic P. falciparum that expressed a PI3P-specific fluorescent probe was generated. Fluorescence was associated mainly with the membrane of the food vacuole and with the apicoplast, a four-membrane bounded plastid-like organelle derived from an ancestral secondary endosymbiosis event. Electron microscopy analysis confirmed these findings and revealed, in addition, the presence of PI3P-positive single-membrane vesicles. We hypothesize that these vesicles might be involved in transport processes, likely of proteins and lipids, toward the essential and peculiar parasite compartment, which is the apicoplast. The fact that PI3P metabolism and function in Plasmodium appear to be substantially different from those in its human host could offer new possibilities for antimalarial chemotherapy.Phosphatidylinositol is a crucial phospholipid in eukaryotic cells. It is a structural membrane lipid, and phosphorylation of the hydroxyl groups of its inositol head group by specific lipid kinases leads to the production of seven different phosphoinositide species, which have been found to be enriched in distinct cellular compartments. They play key roles in a multitude of cellular processes, such as membrane traffic, cell motility, cytoskeletal reorganization, DNA synthesis, the cell cycle, adhesion, and signal transduction (9). Approximately 1% of total lipids in mammalian cells are phosphoinositides, mainly phosphatidylinositol 4-monophosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] (45). Derivatives phosphorylated at the 3 position are considerably less abundant in mammalian cells. Phosphatidylinositol 3-monophosphate (PI3P) is a ubiquitous lipid in eukaryotic cells and is present in small amounts in mammalian cells (classically Ͻ15% of PI4P), while PI3P is as abundant as PI4P in the yeast Saccharomyces cerevisiae (2). It has been suggested that one of the functions of these lipids is to establish membrane identity (46); PI4P predominates at the Gol...

Natural killer (NK) cells are cytotoxic innate lymphoid cells that are involved in immune defense. NK cell reactivity is controlled in part by MHC class I recognition by inhibitory receptors, but the underlying molecular mechanisms remain undefined. Using a mouse model of conditional deletion in NK cells, we show here that the protein tyrosine phosphatase SHP-1 is essential for the inhibitory function of NK cell MHC class I receptors. In the absence of SHP-1, NK cells are hyporesponsive to tumour cells in vitro and their early Ca 2 þ signals are compromised. Mice without SHP-1 in NK cells are unable to reject MHC class I-deficient transplants and to control tumours in vivo. Thus, the inhibitory activity of SHP-1 is needed for setting the threshold of NK cell reactivity.

During mitosis, cortical Moesin activity is restricted to promote cell elongation and cytokinesis, but localized Moesin recruitment is necessary for polar bleb retraction and cortical relaxation.

We previously reported that autosomal recessive demyelinating Charcot-Marie-Tooth (CMT) type 4B1 neuropathy with myelin outfoldings is caused by loss of MTMR2 (Myotubularin-related 2) in humans, and we created a faithful mouse model of the disease. MTMR2 dephosphorylates both PtdIns3P and PtdIns(3,5)P 2, thereby regulating membrane trafficking. However, the function of MTMR2 and the role of the MTMR2 phospholipid phosphatase activity in vivo in the nerve still remain to be assessed. Mutations in FIG4 are associated with CMT4J neuropathy characterized by both axonal and myelin damage in peripheral nerve. Loss of Fig4 function in the plt (pale tremor) mouse produces spongiform degeneration of the brain and peripheral neuropathy. Since FIG4 has a role in generation of PtdIns(3,5)P 2 and MTMR2 catalyzes its dephosphorylation, these two phosphatases might be expected to have opposite effects in the control of PtdIns(3,5)P 2 homeostasis and their mutations might have compensatory effects in vivo. To explore the role of the MTMR2 phospholipid phosphatase activity in vivo, we generated and characterized the Mtmr2/Fig4 double null mutant mice. Here we provide strong evidence that Mtmr2 and Fig4 functionally interact in both Schwann cells and neurons, and we reveal for the first time a role of Mtmr2 in neurons in vivo. Our results also suggest that imbalance of PtdIns(3,5)P 2 is at the basis of altered longitudinal myelin growth and of myelin outfolding formation. Reduction of Fig4 by null heterozygosity and downregulation of PIKfyve both rescue Mtmr2-null myelin outfoldings in vivo and in vitro.

During platelet activation, phosphoinositide 3-kinases (PI3Ks) produce lipid second messengers participating in the regulation of functional responses. Here, we generated a megakaryocyte-restricted p110␤ null mouse model and demonstrated a critical role of PI3K␤ in platelet activation via an immunoreceptor tyrosine-based activation motif, the glycoprotein VI-Fc receptor ␥-chain complex, and its contribution in response to Gprotein-coupled receptors. Interestingly, the production of phosphatidylinositol 3,4,5-trisphosphate and the activation of protein kinase B/Akt were strongly inhibited in p110␤ null platelets stimulated either via immunoreceptor tyrosine-based activation motif or G-protein-coupled receptors. Functional studies showed an important delay in fibrin clot retraction and an almost complete inability of these platelets to adhere onto fibrinogen under flow condition, suggesting that PI3K␤ is also acting downstream of ␣ IIb ␤ 3 . In vivo studies showed that these mice have a normal bleeding time and are not protected from acute pulmonary thromboembolism but are resistant to thrombosis after FeCl 3 injury of the carotid, suggesting that PI3K␤ is a potential target for antithrombotic drugs. IntroductionPlatelet activation is a highly regulated process involving various signaling pathways initiated by specific receptors coupled to heterotrimeric G proteins (GPCRs), integrins, or immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins. In all cases, key signaling enzymes, such as phospholipases C (PLC) and phosphoinositide 3-kinase (PI3K) isoforms, are activated. If the situation is clear for PLC (ie, the PLC␤ isoforms are activated by heterotrimeric Gq proteins, whereas the ␥ isoforms are stimulated via tyrosine phosphorylation and ITAM signaling), the implication of the different PI3K isoforms downstream of the major platelet receptors is still poorly known. Class Ia PI3Ks (␣,␤,␦), composed of a catalytic subunit (p110) and a regulatory subunit, are classically activated by their association with phosphotyrosine residues containing sequences via the SH2 domains of their regulatory subunit. 1 However, the p110␤ isoform may not follow this rule because its activation has been proposed to involve both G␤␥ and phosphotyrosyl peptides. 2-5 Using a selective inhibitor of p110␤, Jackson et al 6 have proposed a role of p110␤ in the regulation of ␣ IIb ␤ 3 integrin in a shear-dependent manner. Interestingly, this inhibitor prevented the formation of an occlusive thrombus generated in vivo. To firmly establish the role of the PI3K␤ in platelet activation and evaluate its impact on hemostasis in vivo, we created a mouse line in which this isoform has been inactivated by gene targeting selectively in the megakaryocyte lineage. Methods MaterialsCollagen was from Nycomed, U46619 from QBiogen Inc; integrilin from Glaxo Group Ltd; p110␣, ␤, ␥, and ␦ antibodies from Santa Cruz Biotechnology; p85 antibody from Upstate Biotechnology; TGX-221 from Cayman Chemical; and other reagents from Sigma-Aldrich. An...