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PubMed2005

Role of phosphatidylinositol 3-kinase-gamma in mediating lung neutrophil sequestration and vascular injury induced by E. coli sepsis.

We addressed the in vivo role of phosphatidylinositol 3-kinase-gamma (PI3K-gamma) in signaling the sequestration of polymorphonuclear leukocytes (PMNs) in lungs and in the mechanism of inflammatory lung vascular injury. We studied mice with deletion of the p110 catalytic subunit of PI3K-gamma (PI3K-gamma(-/-) mice). We measured lung tissue PMN sequestration, microvascular permeability, and edema formation after bacteremia induced by intraperitoneal Escherichia coli challenge. PMN infiltration into the lung interstitium in PI3K-gamma(-/-) mice as assessed morphometrically was increased 100% over that in control mice within 1 h after bacterial challenge. PI3K-gamma(-/-) mice also developed a greater increase in lung microvascular permeability after E. coli challenge, resulting in edema formation. The augmented lung tissue PMN sequestration in PI3K-gamma(-/-) mice was associated with increased expression of the PMN adhesive proteins CD47 and beta(3)-integrins. We observed increased association of CD47 and beta(3)-integrins with the extracellular matrix protein vitronectin in lungs of PI3K-gamma(-/-) mice after E. coli challenge. PMNs from these mice also showed increased beta(3)-integrin expression and augmented beta(3)-integrin-dependent PMN adhesion to vitronectin. These results point to a key role of PMN PI3K-gamma in negatively regulating CD47 and beta(3)-integrin expression in gram-negative sepsis. PI3K-gamma activation in PMNs induced by E. coli may modulate the extent of lung tissue PMN sequestration secondary to CD47 and beta(3)-integrin expression. Therefore, the level of PI3K-gamma activation may be an important determinant of PMN-dependent lung vascular injury.

Animals↗
PubMed2006

RNA interference-mediated depletion of phosphoinositide 3-kinase activates forkhead box class O transcription factors and induces cell cycle arrest and apoptosis in breast carcinoma cells.

Breast cancer is one of the most common malignancies affecting women in the Western world and one in seven women is predicted to develop invasive breast cancer in their lifetime. Breast cancer arises following the accumulation of a series of somatic changes often including deregulation of key signal transduction pathways. The phosphoinositide 3-kinase (PI3K) pathway has been shown to be activated in breast cancer and overexpression of PI3K is sufficient to confer a malignant phenotype. Activation of the PI3K pathway serves to repress forkhead box class O (FoxO) transcription factor-mediated growth arrest and apoptosis. In this study, we used small interfering RNA (siRNA) to knockdown PI3K in three breast cancer cell lines representing different stages of cancer development. Transfection of PI3K siRNA in breast cancer cells resulted in a significant decrease in cell viability and induction of apoptosis irrespective of their estrogen receptor alpha (ERalpha) or ErbB2 status. PI3K depletion also resulted in a significant G(1) phase cell cycle arrest in ERalpha-positive breast cancer cells. Further, our data showed that PI3K knockdown resulted in a significant activation of FoxO; interestingly, a simultaneous knockdown of FoxO1a rescued the cells from apoptosis. Furthermore, the downstream effects of FoxO activation were found to be inhibition of cyclin-dependent kinase 4, cyclin-dependent kinase 6, and cyclin D1, and accumulation of p27/Kip1. Thus, we suggest that (a) PI3K plays a critical role in breast cancer development and (b) gene therapeutic approaches aimed at PI3K or the pharmacologic inhibitors of PI3K could be developed for the management of breast cancer.

Apoptosis↗
PubMed1997

Angiotensin II activates phosphatidylinositol 3-kinase in vascular smooth muscle cells.

Phosphatidylinositol 3-kinase (PI3K) is an important component of the signal transduction systems activated by tyrosine kinase receptors. It has not been established, however, whether PI3K is also an essential mediator for G protein-coupled receptors. The potential involvement of PI3K in G protein-linked angiotensin II (Ang II)-dependent signaling was assessed in a primary cell culture system of porcine coronary artery smooth muscle cells (SMCs). Treatment of quiescent SMCs with Ang II (10(-5) to 10(-8) mol/L) resulted in a dose-dependent activation of PI3K when assayed in vivo and in vitro. The Ang II receptor antagonists losartan and PD123319 were used to establish that Ang II stimulates PI3K through the Ang II type-1 (AT1) receptor. Immunofluorescent microscopy revealed that Ang II (10(-6) mol/L) stimulated the translocation of p85, the regulatory subunit of PI3K, from the perinuclear region to distinct foci throughout the cell within 15 minutes. Western blot analysis of p85 subcellular distribution demonstrated that p85 concentrations were also increased within 15 minutes in the membrane fraction and concomitantly decreased in the cytoskeletal and nuclear fractions. These changes in PI3K location and activity were paralleled by increased tyrosine phosphorylation of p85. A potential correlation between angiotensin-mediated PI3K activation and SMC growth was found using LY294002, a specific inhibitor of PI3K, which blocked the increase in DNA and RNA synthesis as well as cellular hyperplasia generated by Ang II (10(-6) mol/L) stimulation of quiescent SMCs. These data indicate that PI3K may operate as a mediator of vascular SMC growth after stimulation with Ang II.

Angiotensin II↗
PubMed2006

Phosphatidylinositol 3-kinase is necessary for lens fiber cell differentiation and survival.

PURPOSE: To determine the mechanisms of action of phosphatidylinositol 3-kinase (PI3K) in lens cell differentiation and survival. METHODS: Primary quail lens cell cultures were treated at different stages of differentiation with the PI3K inhibitor LY294002, and expression of survival proteins and differentiation markers were determined by immunoblot analysis. The connection between PI3K regulation of lens differentiation and actin cytoskeleton reorganization was examined by fluorescent-phalloidin staining and Rac activity assay. Survival in the absence of PI3K signaling was examined by TUNEL and DAPI staining. Phosphorylation of the PI3K effector glycogen synthase kinase-3 (GSK3) in the absence of PI3K signaling was induced with lithium chloride. RESULTS: Exposure to LY294002 blocked lens epithelial cell differentiation initiation. This result was linked to attenuation of Rac activity and inhibition of actin filament reorganization from stress fibers to cortical fibers, which has been shown to signal lens differentiation initiation. The survival of lens epithelial cells in the absence of PI3K signaling correlated with induction of numerous survival factors, including Bcl-2. In contrast, inhibition of PI3K signaling in differentiating lens fiber cells induced apoptosis by blocking inactivation of GSK3, showing that PI3K/GSK3 signaling has a protective role in the late stages of differentiation as nuclei and organelles are lost. CONCLUSIONS: PI3K signaling regulates lens cell differentiation initiation through its ability to signal reorganization of the actin cytoskeleton from stress fibers to cortical fibers. In differentiating lens fiber cells, PI3K has a protective function, signaling survival through inactivation of its downstream effector GSK3.

Actins↗
PubMed2003

Novel pathways of F-actin polymerization in the human neutrophil.

Recently we demonstrated the existence of a phosphatidylinositol 3-kinase (PI3K)-independent F-actin polymerization during neutrophil pseudopod extension. Here we examine the use of the PI3K-dependent and PI3K-independent pathways of activation by the N-formyl peptide receptor and the chemokine receptors, and the priming of the 2 pathways by granulocyte-macrophage colony-stimulating factor (GM-CSF) and insulin. The inhibition of PI3K activity with wortmannin showed that rate of pseudopod extension stimulated with N-formyl-Met-Leu-Phe (fMLP was mostly dependent on PI3K, while the rate of interleukin-8 (IL-8)-stimulated pseudopod extension was less dependent on PI3K. The incubation of cells with either GM-CSF or insulin increased the rate of pseudopod extension by 50% when the cells were stimulated with IL-8 but not with fMLP. The stimulation with IL-8 phosphorylated the PI3K regulatory subunit. This phosphorylation was enhanced by GM-CSF, which increased PI3K activity and total phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) production. The effect of GM-CSF was blocked with wortmannin. In contrast, insulin did not increase p85 phosphorylation and did not enhance PI3K activity or PtdIns(3,4,5)P3 production. The effect of insulin was insensitive to wortmannin; however, it was blocked by an Src homology 2 (SH2)-binding peptide. These data indicate that priming of IL-8 activation with GM-CSF was mediated via the PI3Ks of class IA, while priming with insulin used a PI3K-independent pathway.

Actins↗
PubMed2003

Insulin activation of phosphatidylinositol 3-kinase in the hypothalamic arcuate nucleus: a key mediator of insulin-induced anorexia.

In peripheral tissues, insulin signaling involves activation of the insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase (PI3K) enzyme system. In the hypothalamus, insulin functions with leptin as an afferent adiposity signal important for the regulation of body fat stores and hepatic glucose metabolism. To test the hypothesis that hypothalamic insulin action involves intracellular PI3K signaling, we used histochemical and biochemical methods to determine the effect of insulin on hypothalamic IRS-PI3K activity. Here, we report that insulin induces tyrosine phosphorylation of the insulin receptor and IRS-1 and -2, increases binding of activated IRS-1 and -2 to the regulatory subunit of PI3K, and activates protein kinase B/Akt, a downstream target of PI3K. Using an immunohistochemical technique to detect PI 3,4,5-triphosphate, the main product of PI3K activity, we further demonstrate that in the arcuate nucleus, insulin-induced PI3K activity occurs preferentially within cells that contain IRS-2. Finally, we show that the food intake- lowering effects of insulin are reversed by intracerebroventricular infusion of either of two PI3K inhibitors at doses that have no independent feeding effects. These findings support the hypothesis that the IRS-PI3K pathway is a mediator of insulin action in the arcuate nucleus and, combined with recent evidence that leptin activates PI3K signaling in the hypothalamus, provide a plausible mechanism for neuronal cross-talk between insulin and leptin signaling.

Animals↗
PubMed1990

Phosphorylation of the PDGF receptor beta subunit creates a tight binding site for phosphatidylinositol 3 kinase.

The beta subunit of the platelet derived growth factor receptor (PDGFR) coprecipitates with a phosphatidyl-inositol 3 kinase activity (PI3K) following stimulation of cells by PDGF. Mutagenesis of a tyrosine (Y) phosphorylation site, Y751, in the PDGFR, greatly reduces PI3K, consistent with the possibility that phosphorylation of Y751 signals association of PI3K. To test this we have reconstituted the binding of the PDGFR beta subunit and PI3K in vitro. Binding is rapid, saturable and requires phosphorylation of the PDGFR at Y751, but does not require PDGF-dependent phosphorylation of PI3K. To test which portions of the PDGFR are important for binding, we used an antibody to a small region of the receptor that includes Y751. This antibody blocked in vitro binding of PI3K to the receptor, while an antiserum to the C-terminus of the receptor had no effect on binding of PI3K. In addition, we found that PDGF stimulation of a cell results in the association of essentially all the PI3K activity with cellular PDGFRs. These data suggest that PI3K is a specific ligand for PDGF receptors that are phosphorylated at Y751.

Amino Acid Sequence↗
PubMed2006

The N-terminus of phosphoinositide 3-kinase-C2beta regulates lipid kinase activity and binding to clathrin.

The class II phosphoinositide 3-kinase (PI3K)-C2beta is recruited to polypeptide growth factor receptors following ligand stimulation. In contrast to the class I A p85/p110 heterodimers, this interaction is dependent upon proline residues present within the N-terminal sequence of the 3-phosphoinositide kinase. However, the mechanism by which PI3K-C2beta catalytic activity is regulated currently remains unknown. In many tumours, increased expression of ErbB receptors confers a poor prognosis. We demonstrate that increased expression of EGFR enhanced its association with PI3K-C2beta following stimulation with EGF. Deletion of the first proline rich region within the N-terminus precluded recruitment of PI3K-C2beta to activated EGFR. Although deletion of the first proline rich motif also rendered the enzyme catalytically inactive, further deletions (residues 1-148 and 1-261) that removed the second and third proline rich motifs increased kinase activity. These data confirm a regulatory role for the N-terminus of class II PI3K enzymes suggesting that catalytic activity is regulated by factors that associate with this region during recruitment to activated growth factor receptors. Using an N-terminal PI3K-C2beta-GST fusion protein, clathrin heavy chain was affinity purified from A431 cell lysates. Association of PI3K-C2beta with clathrin was confirmed by co-immunoprecipitation from cell lysates while intracellular co-localisation of PI3K-C2beta and clathrin was confirmed by confocal microscopy. Our findings demonstrate for the first time that the PI3K-C2beta isoform associates with clathrin and thus provides a link between receptor mediated intracellular signalling and clathrin coated vesicle transport.

Binding Sites↗
PubMed2005

Suppression of the alpha-isoform of class II phosphoinositide 3-kinase gene expression leads to apoptotic cell death.

Phosphoinositide 3-kinases (PI3Ks) have known to be key enzymes activating intracellular signaling molecules when a number of growth factors bind to their cell surface receptors. PI3Ks are divided into three classes (I, II, and III) and enzymes of each class have different tissue-specificities and physiological functions. Class II PI3Ks consist of three isoforms (alpha,beta,gamma). Although the alpha-isoform (PI3K-C2alpha) is considered ubiquitous and preferentially activated by insulin rather than the beta-isoform, the physiological significance of PI3K-C2alpha is poorly understood. The present study aimed to determine whether PI3K-C2alpha is associated with the suppression of apoptotic cell death. Different sense- and antisense oligonucleotides (ODNs) were synthesized based on the sequence of C2 domain of PI3K-C2alpha gene. Transfection of CHO-IR cells with two different antisense ODNs clearly reduced the protein content as well as mRNA levels of PI3K-C2alpha whereas neither the nonspecific mock- nor sense ODNs affected. The decrease of PI3K-C2alpha gene expression was paralleled by cellular changes indicating apoptotic cell death such as nuclear condensation, formation of apoptotic bodies, and DNA fragmentation. PI3K-C2alpha mRNA levels were also reduced when cells were incubated in growth factor-deficient medium. Supplementing growth factors (serum or insulin) into medium lead to an increase of PI3K-C2alpha mRNA levels. This finding strongly suggests that PI3K-C2alpha is a crucial survival factor.

Animals↗
PubMed2004

Effects of phosphoinositide 3-kinase on endothelin-1-induced activation of voltage-independent Ca2+ channels and vasoconstriction.

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in rabbit basilar artery (BA) vascular smooth muscle cells (VSMCs). In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on ET-1-induced activation of these channels and BA contraction by using PI3K inhibitors, wortmannin and LY 249002. To determine which Ca(2+) channels are activated via PI3K, monitoring of intracellular Ca(2+) concentration was performed. Role of PI3K in ET-1-induced vasoconstriction was examined by tension study using rabbit BA rings. Only NSCC-1 was activated by ET-1 in wortmannin- or LY 294002-pretreated VSMCs. In contrast, addition of these drugs after ET-1 stimulation did not suppress Ca(2+) influx. Wortmannin inhibited the ET-1-induced contraction of rabbit BA rings that depends on the Ca(2+) influx through NSCC-2 and SOCC. The IC(50) values of wortmannin for the ET-1-induced Ca(2+) influx and vasoconstriction were similar to those for the ET-1-induced PI3K activation. These results indicate that (1) NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade; (2) PI3K is required for the activation of the Ca(2+) entry, but not for its maintenance; and (3) PI3K is involved in the ET-1-induced contraction of rabbit BA rings that depends on the extracellular Ca(2+) influx through SOCC and NSCC-2.

Acetamides↗
PubMed2003

Effect of ageing in the early biochemical signals elicited by PTH in intestinal cells.

In previous work, we have demonstrated that rPTH(1-34) increases cytoplasmic calcium concentration ([Ca(2+)](i)) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca(2+)](I) and the implication of Ca(2+) on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca(2+)](i) in a dose-dependent manner (1 pM-100 nM). In cells from aged rats, hormone concentrations higher than physiological (>/=1 nM) were required to observe significant increases in [Ca(2+)](i). Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca(2+)](i) biphasic response to PTH of young enterocytes while in old cells, no effects were observed. The voltage-dependent calcium-channel blocker (VDCC), nitrendipine, suppressed PTH-dependent changes of the sustained [Ca(2+)](i) phase in young and aged animals. In this study, we analysed, for the first time, alterations in phosphatidylinositol 3-kinase (PI3K) activity and response to PTH in rat enterocytes with ageing. Basal PI3K activity was significantly modified by ageing. Acute treatment with 10(-8) M PTH increased enzyme activity, with a maximun at 2 min (+3-fold) in young rats and only elevated by less than 1-fold basal PI3K activity in aged animals. Hormone-induced tyrosine phosphorylation of p85alpha, the regulatory subunit of PI3K, as well as the phosphorylation on Thr(308) of its downstream effector Akt/PKB was evident in enterocytes from 3-month-old rats, whereas it was greatly reduced in the cells from 24-month-old animals. Intracellular Ca(2+) chelation (BAPTA-AM, 5 microM) affected the tyrosine phosphorylation of p85alpha and inhibited PTH-dependent PI3K activation by 75% in young rats and completely abolished the enzyme activity in aged animals, demonstrating that Ca(2+) is required for full activation of PI3K in enterocytes stimulated with PTH. The Thr phosphorylation of PI3K downeffector, Akt/PKB, was also fully dependent on Ca(2+). Taken together, these results suggest that PTH regulation of enterocyte [Ca(2+)](i) involves Ca(2+) mobilization from IP(3)-sensitive stores and the influx of the cation from the extracellular milieu, the former pathway being blunted during ageing. The data also indicates a positive role for intracellular calcium in one of the early signals of PTH in rat enterocytes, the activation of PI3K, and that hormone regulation of PI3K activity and Akt/PKB phosphorylation on Thr(308) is impaired with ageing.

Aging↗
PubMed2003

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis.

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.

Apoptosis↗
PubMed2000

Targets of B-cell antigen receptor signaling: the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase-3 signaling pathway and the Rap1 GTPase.

In this review, we discuss the role of phosphatidylinositol 3-kinase (PI3K) and Rap 1 in B-cell receptor (BCR) signaling. PI3K produces lipids that recruit pleckstrin homology domain-containing proteins to the plasma membrane. Akt is a kinase that the BCR activates in this manner. Akt phosphorylates several transcription factors as well as proteins that regulate apoptosis and protein synthesis. Akt also regulates glycogen synthase kinase-3, a kinase whose substrates include the nuclear factor of activated T cells (NF-AT)cl and beta-catenin transcriptional activators. In addition to Akt, PI3K-derived lipids also regulate the activity and localization of other targets of BCR signaling. Thus, a key event in BCR signaling is the recruitment of PI3K to the plasma membrane where its substrates are located. This is mediated by binding of the Src homology (SH) 2 domains in PI3K to phosphotyrosine-containing sequences on membrane-associated docking proteins. The docking proteins that the BCR uses to recruit PI3K include CD19, Cbl, Gab1, and perhaps Gab2. We have shown that Gab1 colocalizes PI3K with SH2 domain-containing inositol phosphatase (SHIP) and SHP2, two enzymes that regulate PI3K-dependent signaling. In contrast to PI3K, little is known about the Rap1 GTPase. We showed that the BCR activates Rap1 via phospholipase C-dependent production of diacylglycerol. Since Rap1 is thought to regulate cell adhesion and cell polarity, it may be involved in B-cell migration.

Animals↗
PubMed2004

PIKE/nuclear PI 3-kinase signaling mediates the antiapoptotic actions of NGF in the nucleus.

PI 3-kinase (PI3K) occurs in the nuclei of a broad range of cell types, and various stimuli elicit PI3K nuclear translocation. However, little is known about the biological function of nuclear PI3K. Here we show that nuclear PI3K and its upstream regulator PIKE mediate the antiapoptotic activity of nerve growth factor (NGF) in the isolated nuclei. The nuclei from NGF-treated PC12 cells, EGF-treated HEK293 cells and HeLa cells are resistant to DNA fragmentation initiated by activated cell-free apoptosome. Nuclei from constitutively active PI3K adenovirus-infected cells display the same resistance as those treated by NGF, whereas PI3K inhibitors, dominant-negative PI3K or PIKE abolishes it. Knockdown of either PI3K or PIKE diminishes the antiapoptotic activity of NGF. PI (3,4,5)P3 alone mimics the antiapoptotic activity of NGF, for which nuclear Akt is required. These results demonstrate that PIKE/nuclear PI3K signaling through nuclear PI (3,4,5)P3 and Akt plays an essential role in promoting cell survival.

Adenoviridae↗
PubMed2006

Phosphatidylinositol 3-kinase mediates activation of ATM by high NaCl and by ionizing radiation: Role in osmoprotective transcriptional regulation.

High NaCl causes DNA double-strand breaks and activates the transcription factor, TonEBP/OREBP, resulting in increased transcription of several protective genes, including those involved in accumulation of compatible organic osmolytes. Several kinases are known to contribute to signaling activation of TonEBP/OREBP, including ATM, which is a member of the phosphatidylinositol 3-kinase (PI3K)-like kinase family and is activated by DNA double-strand breaks. The purpose of the present studies was to investigate a possible role of PI3K Class IA (PI3K-IA). We found that high NaCl increases PI3K-IA lipid kinase activity. Inhibiting PI3K-IA either by expressing a dominant negative of its regulatory subunit, p85, or by small interfering RNA-mediated knockdown of its catalytic subunit, p110alpha, reduces high NaCl-induced increases in TonEBP/OREBP transcriptional activity and transactivation, but not nuclear translocation of TonEBP/OREBP, or increases in its abundance. Further, suppression of PI3K-IA inhibits the activation of ATM that is caused by either ionizing radiation or high NaCl. High NaCl-induced increase in TonEBP/OREBP activity is reduced equally by inhibition of ATM or PI3K-IA, and the effects are not additive. The conclusions are as follows: (i) PI3K-IA activity is necessary for both high NaCl- and ionizing radiation-induced activation of ATM and (ii) high NaCl activates PI3K-IA, which, in turn, contributes to full activation of TonEBP/OREBP via ATM.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1992

Interkinase domain of kit contains the binding site for phosphatidylinositol 3' kinase.

Our previous analysis of the signal transduction pathway used by the c-kit-encoded receptor for the stem cell factor (SCF) indicated efficient coupling to the type I phosphatidylinositol 3' kinase (PI3K). In an attempt to localize the receptor's site of interaction with PI3K, we separately deleted either the noncatalytic 68-amino-acid-long interkinase domain or the carboxyl-terminal portion distal to the catalytic sequences. Loss of ligand-induced association of PI3K with the former deletion mutant and retention of the PI3K association by the carboxyl-terminally deleted receptor implied interactions of PI3K with the kinase insert. This was further supported by partial inhibition of the association by an anti-peptide antibody directed against the kinase insert and lack of effect of an antibody directed to the carboxyl tail of the SCF receptor. A bacterially expressed kinase insert domain was used as a fusion protein to directly test its presumed function as a PI3K association site. This protein bound PI3K from cell lysate as demonstrated by PI3K activity and by an associated phosphoprotein of 85 kDa. The association was dependent on phosphorylation of the tyrosine residues on the expressed kinase insert. On the basis of these observations, we conclude that the kinase insert domain of the SCF receptor selectively interacts with the p85 regulatory subunit of PI3K and that this association requires phosphorylation of tyrosine residues in the kinase insert region, with apparently no involvement of the bulk cytoplasmic structure or tyrosine kinase function of the receptor.

1-Phosphatidylinositol 4-Kinase↗
PubMed1994

A phosphatidylinositol 3-kinase is induced during soybean nodule organogenesis and is associated with membrane proliferation.

Phosphatidylinositol 3-kinase (PI3K) is an important component of various receptor tyrosine kinase complexes in mammalian cells and a key enzyme required for cell division and vacuolar protein sorting in yeast. To our knowledge, this enzyme has not been characterized in plants. We report the cloning and characterization of soybean PI3K cDNAs and present evidence for the induction of a distinctive form of this enzyme specific to nodule organogenesis. Expression of the root form of PI3K is repressed during nodule organogenesis and is reinduced in mature nodules. Primer-extension results showed that the gene encoding the nodule form of PI3K is highly expressed in young (12-15 day old) root nodules in parallel with membrane proliferation but is repressed in mature nodules. The root form of the PI3K cDNA (SPI3K-5) encodes a peptide of 814 amino acids and the nodule form (SPI3K-1) encodes a peptide of 812 amino acids. Both cDNAs share 98% sequence identity in the coding region but differ in the noncoding regions. The polypeptides encoded by soybean PI3K cDNAs show significant sequence homology (50-60% similarity and 20-40% identity) to both PI3Ks and phosphatidylinositol 4-kinases from mammalian and yeast cells. Escherichia coli expressed soybean PI3K phosphorylated phosphatidylinositol specifically at the D-3 position of the inositol ring to generate phosphatidylinositol 3-phosphate. The temporal increase of a specific PI3K activity during membrane proliferation in young nodules suggests that PI3K plays a pivotal role in development of the peribacteroid membrane forming a subcellular compartment.

Amino Acid Sequence↗
PubMed1998

Targeting of constitutively active phosphoinositide 3-kinase to GLUT4-containing vesicles in 3T3-L1 adipocytes.

Constitutive activation of phosphoinositide 3-kinase (PI3K) stimulates glucose transport and GLUT4 glucose transporter translocation to the plasma membrane in adipocytes. To determine whether a direct interaction of PI3K with GLUT4-containing vesicles (hereafter called GLUT4 vesicles) is important for the effect of insulin on GLUT4 translocation, we targeted constitutively active PI3K to GLUT4 vesicles. We fused the inter-Src homology region 2 of the regulatory p85alpha subunit of PI3K (iSH2) either to a C-terminal sequence of GLUT4 (G4c, amino acids 406-509) or to this region and the N-terminal tail of GLUT4 (G4n, amino acids 1-19), resulting in the fusion proteins iSH2-G4c and G4n-iSH2-G4c, respectively. Coexpression of the fusion proteins or untargeted iSH2 with the catalytic p110alpha subunit of PI3K (p110) in 3T3-L1 adipocytes by adenovirus-mediated gene transfer increased total PI3K activity in homogenates 5.0-6.7-fold over nontransduced cells or cells transduced with adenovirus encoding beta-galactosidase. In contrast, PI3K activity in GLUT4 vesicles increased 11-13-fold with expression of either targeted construct and p110 but only 2-fold with the untargeted iSH2 and p110, indicating successful targeting of PI3K to GLUT4 vesicles. Both targeted and nontargeted constructs stimulated DNA synthesis to levels greater than insulin, demonstrating that both types of constructs had biologic activity in intact cells. Despite this, untargeted iSH2/p110 coexpression was more effective in stimulating 2-deoxyglucose uptake (6-fold) than either iSH2-G4c/p110 or G4n-iSH2-G4c/p110 coexpression (both 2-fold). Only iSH2/p110 coexpression led to a significant GLUT4 translocation to the plasma membrane. Insulin-stimulated glucose transport was unaffected by any construct. Thus, a direct interaction between PI3K and GLUT4 vesicles is either not required or not sufficient for GLUT4 translocation and stimulation of glucose transport.

3T3 Cells↗