Chemotaxis in the absence of PIP3 gradients

Chemotaxing neutrophils and Dictyostelium amoebae produce in their plasma membranes the signaling lipid PI(3,4,5)P3 (PIP3) in gradients, which are orientated with the external chemotactic gradient and have been proposed to act as an internal compass, guiding movement of the cell. Evidence for and against this idea exists, but in all cases it depends on the use of inhibitors or gene knockouts, which may only incompletely abolish the PIP3 gradient. We have created a multiple gene-knockout strain in Dictyostelium lacking all five type-1 phosphoinositide 3-kinases encoded in the genome and the PTEN phosphatase and have thus removed all known ways for chemoattractant to produce PIP3 gradients in the plasma membrane. The resulting sextuple mutant is able to chemotax to cyclic-AMP with near wild-type efficiency and to trigger actin polymerization without apparent defect. There is, however, a consistent defect in movement speed in chemotaxis and especially in random movement. This work shows that polarization of membrane PIP3 is not necessary for accurate chemotaxis, but it can affect cell speed. A signaling pathway from receptor to cytoskeleton able to guide cells independently of polarized PIP3 and type-1 phosphoinositide 3-kinases must exist.     

Non-opsonic phagocytosis of Legionella pneumophila by macrophages is mediated by phosphatidylinositol 3-kinase

Background

Legionella pneumophila, is an intracellular pathogen that causes Legionnaires'' disease in humans, a potentially lethal pneumonia. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis.

Methodology/Principal Findings

Phagocytosis was measured by cell invasion assays. Construction of PI3K mutant by PCR cloning and expression of dominant negative mutant was detected by Western blot. PI3K activity was measured by ³²P labeling and detection of phospholipids products by thin layer chromatography. Infection of macrophages with virulent L. pneumophila stimulated the formation of phosphatidylinositol 3-phosphate (PIP3), a phosphorylated lipid product of PI3K whereas two structurally distinct phosphatidylinositol 3 kinase (PI3K) inhibitors, wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, reduced L. pneumophila entry into macrophages in a dose-dependent fashion. Furthermore, PI3K activation led to Akt stimulation, a serine/threonine kinase, which was also inhibited by wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. In contrast, PI3K and protein kinase B (PKB/Akt) activities were lower in macrophages infected with an avirulent bacterial strain. Only virulent L. pneumophila increased lipid kinase activity present in immunoprecipitates of the p85α subunit of class I PI3K and tyrosine phosphorylated proteins. In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells.

Conclusion/Significance

Entry of L. pneumophila is mediated by PI3K/Akt signaling pathway. These results suggest an important role for PI3K and Akt in the L. pneumophila infection process. They point to possible novel strategies for undermining L. pneumophila host uptake and reducing pathogenesis of Legionnaires'' disease.     

Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase

The c-ret gene encodes a receptor tyrosine kinase (RET) essential for the development of the kidney and enteric nervous system. Activation of RET requires the secreted neurotrophin GDNF (glial cell line-derived neurotrophic factor) and its high affinity receptor, a glycosyl phosphatidylinositol-linked cell surface protein GFRalpha1. In the developing kidney, RET, GDNF, and GFRalpha1 are all required for directed outgrowth and branching morphogenesis of the ureteric bud epithelium. Using MDCK renal epithelial cells as a model system, activation of RET induces cell migration, scattering, and formation of filopodia and lamellipodia. RET-expressing MDCK cells are able to migrate toward a localized source of GDNF. In this report, the intracellular signaling mechanisms regulating RET-dependent migration and chemotaxis are examined. Activation of RET resulted in increased levels of phosphatidylinositol 3-kinase (PI3K) activity and Akt/PKB phosphorylation. This increase in PI3K activity is essential for regulating the GDNF response, since the specific inhibitor, LY294002, blocks migration and chemotaxis of MDCK cells. Using an in vitro organ culture assay, inhibition of PI3K completely blocks the GDNF-dependent outgrowth of ectopic ureter buds. PI3K is also essential for branching morphogenesis once the ureteric bud has invaded the kidney mesenchyme. The data suggest that activation of RET in the ureteric bud epithelium signals through PI3K to control outgrowth and branching morphogenesis.     

Down-regulation of apolipoprotein M expression is mediated by phosphatidylinositol 3-kinase in HepG2 cells

Apolipoprotein M (apoM) is a novel apolipoprotein present mostly in high-density lipoprotein (HDL) in human plasma. In the present study, we demonstrate that insulin, insulin-like growth factor I (IGF-I), and IGF-I potential peptide (IGF-IPP) significantly inhibits apoM expression, in a dose- and a time-dependent manner, in the human hepatoma cell line, HepG2 cells. Insulin-induced down-regulation of apoM was blocked by AG1024 (a specific insulin receptor inhibitor) and LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), which indicates that it is mediated via the activation of PI3K pathway. In contrast, PD98059 (a MAP kinase inhibitor) did not influence insulin-induced down-regulation of apoM expression, and activation of neither PPAR-alpha agonist (GW7647) nor PPAR-gamma agonist (GW1929) influences apoM expression in HepG2 cells, which indicates that regulation of apoM expression is not related to the activation of PPAR-alpha and PPAR-gamma in hepatic cells, whereas, both PPAR-alpha and PPAR-gamma agonists could inhibit apoB expression. Moreover, in the present study, we demonstrated that PPAR beta/delta agonist (GW501516) could inhibit both apoM and apoB expression in the HepG2 cells. In conclusion, this study shows that apoM expression is regulated by PI3-kinase in HepG2-cells.     

Novel PtdIns(3)P-binding protein Etf1 functions as an effector of the Vps34 PtdIns 3-kinase in autophagy

Autophagy is the process whereby cytoplasmic cargo (e.g., protein and organelles) are sequestered within a double membrane-enclosed transport vesicle and degraded after vesicle fusion with the vacuole/lysosome. Current evidence suggests that the Vps34 phosphatidylinositol 3-kinase is essential for macroautophagy, a starvation-induced autophagy pathway (Kihara et al., 2001). Here, we characterize a requirement for Vps34 in constitutive autophagy by the cytoplasm-to-vacuole targeting (Cvt) pathway. First, we show that transient disruption of phosphatidylinositol (PtdIns) 3-phosphate (PtdIns[3]P) synthesis through inactivation of temperature-sensitive Vps34 or its upstream activator, Vps15, blocks the Cvt and macroautophagy pathways. Yet, PtdIns(3)P-binding FYVE domain-containing proteins, which mediate carboxypeptidase Y (CPY) transport to the vacuole by the CPY pathway, do not account for the requirement of Vps34 in autophagy. Using a genetic selection designed to isolate PtdIns(3)P-binding effectors of Vps34, we identify Etf1, an uncharacterized type II transmembrane protein. Although Etf1 does not contain a known 3-phosphoinositide-binding domain (i.e., FYVE or Phox), we find that Etf1 interacts with PtdIns(3)P and that this interaction requires a basic amino acid motif (KKPAKK) within the cytosolic region of the protein. Moreover, deletion of ETF1 or mutation of the KKPAKK motif results in strong sorting defects in the Cvt pathway but not in macroautophagy or in CPY sorting. We propose that Vps34 regulates the CPY, Cvt, and macroautophagy pathways through distinct sets of PtdIns(3)P-binding effectors and that Vps34 promotes protein trafficking in the Cvt pathway through activation/localization of the effector protein Etf1.     

Preconditioning enhanced glucose uptake is mediated by p38 MAP kinase not by phosphatidylinositol 3-kinase

Ischemia is reported to stimulate glucose uptake, but the signaling pathways involved are poorly understood. Modulation of glucose transport could be important for the cardioprotective effects of brief intermittent periods of ischemia and reperfusion, termed ischemic preconditioning. Previous work indicates that preconditioning reduces production of acid and lactate during subsequent sustained ischemia, consistent with decreased glucose utilization. However, there are also data that preconditioning enhances glucose uptake. The present study examines whether preconditioning alters glucose transport and whether this is mediated by either phosphatidylinositol 3-kinase (PI3K) or p38 MAP kinase. Langendorff-perfused rat hearts were preconditioned with 4 cycles of 5 min of ischemia and 5 min of reperfusion, with glucose as substrate. During the last reflow, glucose was replaced with 5 mM acetate and 5 mM 2-deoxyglucose (2DG), and hexose transport was measured from the rate of production of 2-deoxyglucose 6-phosphate (2DG6P), using (31)P nuclear magnetic resonance. Preconditioning stimulated 2DG uptake; after 15 min of perfusion with 2DG, 2DG6P levels were 165% of initial ATP in preconditioned hearts compared with 96% in control hearts (p < 0.05). Wortmannin, an inhibitor of PI3K, did not block the preconditioning induced stimulation of 2DG6P production, but perfusion with SB202190, an inhibitor of p38 MAP kinase, did attenuate 2DG6P accumulation (111% of initial ATP, p < 0. 05 compared with preconditioned hearts). SB202190 had no effect on 2DG6P accumulation in nonpreconditioned hearts. Preconditioning stimulation of translocation of GLUT4 to the plasma membrane was not inhibited by wortmannin. The data demonstrate that ischemic preconditioning increases hexose transport and that this is mediated by p38 MAP kinase and is PI3K-independent.     

Neuroprotection by brain-derived neurotrophic factor is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase.

Apoptosis is a form of programmed cell death that plays a pivotal role during development and in the homeostasis of the adult nervous systems. However, mechanisms that regulate neuronal apoptosis are not well defined. Here, we report that brain-derived neurotrophic factor (BDNF) protects cortical neurons against apoptosis induced by camptothecin or serum deprivation and activates the extracellular-signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Using pharmacological agents and transient transfection with dominant interfering or constitutive active components of the ERK or the PI 3-kinase pathway, we demonstrate that the ERK pathway plays a major role in BDNF neuroprotection against camptothecin. Furthermore, ERK is activated in cortical neurons during camptothecin-induced apoptosis, and inhibition of ERK increases apoptosis. In contrast, the PI 3-kinase pathway is the dominant survival mechanism for serum-dependent survival under normal culture conditions and for BDNF protection against serum withdrawal. These results suggest that the ERK pathway is one of several neuroprotective mechanisms that are activated by stress to counteract death signals in central nervous system neurons. Furthermore, the relative contribution of the ERK and PI 3-kinase pathways to neuronal survival may depend on the type of cellular injury.     

Endosomal cargo recycling mediated by Gpa1 and phosphatidylinositol 3-kinase is inhibited by glucose starvation

Cell surface protein trafficking is regulated in response to nutrient availability, with multiple pathways directing surface membrane proteins to the lysosome for degradation in response to suboptimal extracellular nutrients. Internalized protein and lipid cargoes recycle back to the surface efficiently in glucose-replete conditions, but this trafficking is attenuated following glucose starvation. We find that cells with either reduced or hyperactive phosphatidylinositol 3-kinase (PI3K) activity are defective for endosome to surface recycling. Furthermore, we find that the yeast Gα subunit Gpa1, an endosomal PI3K effector, is required for surface recycling of cargoes. Following glucose starvation, mRNA and protein levels of a distinct Gα subunit Gpa2 are elevated following nuclear translocation of Mig1, which inhibits recycling of various cargoes. As Gpa1 and Gpa2 interact at the surface where Gpa2 concentrates during glucose starvation, we propose that this disrupts PI3K activity required for recycling, potentially diverting Gpa1 to the surface and interfering with its endosomal role in recycling. In support of this model, glucose starvation and overexpression of Gpa2 alter PI3K endosomal phosphoinositide production. Glucose deprivation therefore triggers a survival mechanism to increase retention of surface cargoes in endosomes and promote their lysosomal degradation.     

Feeding induced by cannabinoids is mediated independently of the melanocortin system

Background

Cannabinoids, the active components of marijuana, stimulate appetite, and cannabinoid receptor-1 (CB1-R) antagonists suppress appetite and promote weight loss. Little is known about how CB1-R antagonists affect the central neurocircuitry, specifically the melanocortin system that regulates energy balance.

Methodology/Principal Findings

Here, we show that peripherally administered CB1-R antagonist (AM251) or agonist equally suppressed or stimulated feeding respectively in A^y , which lack a functional melanocortin system, and wildtype mice, demonstrating that cannabinoid effects on feeding do not require melanocortin circuitry. CB1-R antagonist or agonist administered into the ventral tegmental area (VTA) equally suppressed or stimulated feeding respectively, in both genotypes. In addition, peripheral and central cannabinoid administration similarly induced c-Fos activation in brain sites suggesting mediation via motivational dopaminergic circuitry. Amperometry-detected increases in evoked dopamine (DA) release by the CB1-R antagonist in nucleus accumbens slices indicates that AM251 modulates DA release from VTA terminals.

Conclusions/Significance

Our results demonstrate that the effects of cannabinoids on energy balance are independent of hypothalamic melanocortin circuitry and is primarily driven by the reward system.     

Membrane translocation of P-Rex1 is mediated by G protein betagamma subunits and phosphoinositide 3-kinase

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac that is directly activated by the betagamma subunits of heterotrimeric G proteins and by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), which is generated by phosphoinositide 3-kinase (PI3K). Gbetagamma subunits and PIP(3) are membrane-bound, whereas the intracellular localization of P-Rex1 in basal cells is cytosolic. Activation of PI3K alone is not sufficient to promote significant membrane translocation of P-Rex1. Here we investigated the subcellular localization of P-Rex1 by fractionation of Sf9 cells co-expressing P-Rex1 with Gbetagamma and/or PI3K. In basal, serum-starved cells, P-Rex1 was mainly cytosolic, but 7% of the total was present in the 117,000 x g membrane fraction. Co-expression of P-Rex1 with either Gbetagamma or PI3K caused only an insignificant increase in P-Rex1 membrane localization, whereas Gbetagamma and PI3K together synergistically caused a robust increase in membrane-localized P-Rex1 to 23% of the total. PI3K-driven P-Rex1 membrane recruitment was wortmannin-sensitive. The use of P-Rex1 mutants showed that the isolated Dbl homology/pleckstrin homology domain tandem of P-Rex1 is sufficient for synergistic Gbetagamma- and PI3K-driven membrane localization; that the enzymatic GEF activity of P-Rex1 is not required for membrane translocation; and that the other domains of P-Rex1 (DEP, PDZ, and IP4P) contribute to keeping the enzyme localized in the cytosol of basal cells. In vitro Rac2-GEF activity assays showed that membrane-derived purified P-Rex1 has a higher basal activity than cytosol-derived P-Rex1, but both can be further activated by PIP(3) and Gbetagamma subunits.     

Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways

Neurotrophins protect neurons against glutamate excitotoxicity, but the signaling mechanisms have not been fully elucidated. We studied the role of the phosphatidylinositol 3-kinase (PI3-K) and Ras/mitogen-activated protein kinase (MAPK) pathways in the protection of cultured hippocampal neurons from glutamate induced apoptotic cell death, characterized by nuclear condensation and activation of caspase-3-like enzymes. Pre-incubation with the neurotrophin brain-derived neurotrophic factor (BDNF), for 24 h, reduced glutamate-evoked apoptotic morphology and caspase-3-like activity, and transiently increased the activity of the PI3-K and of the Ras/MAPK pathways. Inhibition of the PI3-K and of the Ras/MAPK signaling pathways abrogated the protective effect of BDNF against glutamate-induced neuronal death and similar effects were observed upon inhibition of protein synthesis. Moreover, incubation of hippocampal neurons with BDNF, for 24 h, increased Bcl-2 protein levels. The results indicate that the protective effect of BDNF in hippocampal neurons against glutamate toxicity is mediated by the PI3-K and the Ras/MAPK signaling pathways, and involves a long-term change in protein synthesis.     

Localization of Tec29 to ring canals is mediated by Src64 and PtdIns(3,4,5)P3-dependent mechanisms

Two tyrosine kinases, Src64 and Tec29, regulate the growth of actin rich-ring canals in the Drosophila ovary. We have shown previously that Src64 directs the localization of Tec29 to ring canals, but the mechanism underlying this process was unknown. Here, we show that Tec29 localizes to ring canals via its Src homology 3 (SH3) and Src homology 2 (SH2) domains. Tec29 activity is required for its own ring canal localization, suggesting that a phosphotyrosine ligand for the SH2 domain is generated by Tec29 itself. Src64 regulates this process by phosphorylating Y677 within the kinase domain of Tec29, an event required for Tec29 activation. We also show that the pleckstrin homology (PH) domain of Tec29 has dual functions in mediating Src64 regulation. In the absence of Src64, the PH domain prevents Tec29 ring canal localization. In the presence of Src64, it enhances membrane targeting of Tec29 by a PI(3,4,5)P(3)-mediated mechanism. In the absence of its PH domain, Tec29 constitutively localizes to ring canals, but still requires Src64 for full activation.     

Activation of phosphatidylinositol-3-kinase by insulin is mediated by both A and B human insulin receptor types.

Activation of a phosphatidylinositol-3-kinase (PI-3-kinase) is one of the earliest consequences of insulin binding to the receptor. The human insulin receptor exists in two isoforms which differ in the length of the alpha-subunit (HIR-A = 719 aa, HIR-B = 731 aa). To test whether both isoforms transduce an insulin signal on PI-3-kinase we used rat-1-fibroblasts expressing HIR-A or HIR-B. We found that insulin stimulates 32P incorporation into PIP through both HIR-A and HIR-B to a similar extent (approx. 8-10 fold).     

Restructuring of focal adhesion plaques by PI 3-kinase. Regulation by PtdIns (3,4,5)-p(3) binding to alpha-actinin

Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of alpha-actinin and vinculin from the focal adhesion complex to the Triton X-100-soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns (3,4,5)-P(3)), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P(3) binds to alpha-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of alpha-actinin with the integrin beta subunit, and that PtdIns (3,4,5)-P(3) could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P(3), has important implications for the regulation of cellular adhesive strength and motility.     

Oxysterol-induced osteogenic differentiation of marrow stromal cells is regulated by Dkk-1 inhibitable and PI3-kinase mediated signaling

Osteoporosis and its complications cause morbidity and mortality in the aging population, and result from increased bone resorption by osteoclasts in parallel with decreased bone formation by osteoblasts. A widely accepted strategy for improving bone health is targeting osteoprogenitor cells in order to stimulate their osteogenic differentiation and bone forming properties through the use of osteoinductive/anabolic factors. We previously reported that specific naturally occurring oxysterols have potent osteoinductive properties, mediated in part through activation of hedgehog signaling in osteoprogenitor cells. In the present report, we further demonstrate the molecular mechanism(s) by which oxysterols induce osteogenesis. In addition to activating the hedgehog signaling pathway, oxysterol-induced osteogenic differentiation is mediated through a Wnt signaling-related, Dkk-1-inhibitable mechanism. Bone marrow stromal cells (MSC) treated with oxysterols demonstrated increased expression of osteogenic differentiation markers, along with selective induced expression of Wnt target genes. These oxysterol effects, which occurred in the absence of beta-catenin accumulation or TCF/Lef activation, were inhibited by the hedgehog pathway inhibitor, cyclopamine, and/or by the Wnt pathway inhibitor, Dkk-1. Furthermore, the inhibitors of PI3-Kinase signaling, LY 294002 and wortmanin, inhibited oxysterol-induced osteogenic differentiation and induction of Wnt signaling target genes. Finally, activators of canonical Wnt signaling, Wnt3a and Wnt1, inhibited spontaneous, oxysterol-, and Shh-induced osteogenic differentiation of bone marrow stromal cells, suggesting the involvement of a non-canonical Wnt pathway in pro-osteogenic differentiation events. Osteogenic oxysterols are, therefore, important small molecule modulators of critical signaling pathways in pluripotent mesenchymal cells that regulate numerous developmental and post-developmental processes.     

Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin

Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.     

Atg8 lipidation is coordinated in a PtdIns3P-dependent manner by the PROPPIN Atg21

In Saccharomyces cerevisiae Atg8 coupled to phosphatidylethanolamine is a key component of autophagosome biogenesis. Atg21 binds via 2 sites at the circumference of its β-propeller to PtdIns3P at the phagophore assembly site (PAS). It recruits and arranges both Atg8 and Atg16, which is part of the E3-like ligase complex Atg12–Atg5-Atg16. Binding of Atg8 to Atg21 requires the FK-motif within the N-terminal-helical domain of Atg8 and D146 at the top of the Atg21 β-propeller. Atg16 binds via D101 and E102 within its coiled-coil domain to Atg21.