Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.     

Externally added aFGF mutants do not require extensive unfolding for transport to the cytosol and the nucleus in NIH/3T3 cells

Acidic fibroblast growth factor (aFGF) is transported to the cytosol and the nucleus when added to cells expressing FGF receptors, implying that aFGF must cross cellular membranes. Since protein translocation across membranes commonly requires extensive unfolding of the protein, we were interested in testing whether this is also necessary for membrane translocation of aFGF. We therefore constructed mutant growth factors with intramolecular disulfide bonds to prevent complete unfolding. Control experiments demonstrated that translocation of aFGF by the diphtheria toxin pathway, which requires extensive unfolding of the protein, was prevented by disulfide bond formation, indicating that the introduced disulfide bonds interfered with the unfolding of the growth factor. On the other hand, when the growth factor as such was added to cells expressing FGF receptors, the disulfide-bonded mutants were translocated to the cytosol and the nucleus equally well as wild-type aFGF. The possibility that the translocation of the mutants was due to reduction of the disulfide bonds prior to translocation was tested in experiments using an irreversibly cross-linked mutant. Also this mutant was transported to the cytosol and to the nucleus. The results suggest that extensive unfolding is not required for membrane translocation of aFGF.     

Domain Swapping Used To Investigate the Mechanism of Protein Kinase B Regulation by 3-Phosphoinositide-Dependent Protein Kinase 1 and Ser473 Kinase

Protein kinase B (PKB or Akt), a downstream effector of phosphoinositide 3-kinase (PI 3-kinase), has been implicated in insulin signaling and cell survival. PKB is regulated by phosphorylation on Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and on Ser473 by an unidentified kinase. We have used chimeric molecules of PKB to define different steps in the activation mechanism. A chimera which allows inducible membrane translocation by lipid second messengers that activate in vivo protein kinase C and not PKB was created. Following membrane attachment, the PKB fusion protein was rapidly activated and phosphorylated at the two key regulatory sites, Ser473 and Thr308, in the absence of further cell stimulation. This finding indicated that both PDK1 and the Ser473 kinase may be localized at the membrane of unstimulated cells, which was confirmed for PDK1 by immunofluorescence studies. Significantly, PI 3-kinase inhibitors prevent the phosphorylation of both regulatory sites of the membrane-targeted PKB chimera. Furthermore, we show that PKB activated at the membrane was rapidly dephosphorylated following inhibition of PI 3-kinase, with Ser473 being a better substrate for protein phosphatase. Overall, the results demonstrate that PKB is stringently regulated by signaling pathways that control both phosphorylation/activation and dephosphorylation/inactivation of this pivotal protein kinase.     

Role of Translation Initiation Factor 2B in Control of Cell Survival by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3β Signaling Pathway

The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway is an important mediator of growth factor-dependent survival of mammalian cells. A variety of targets of the Akt protein kinase have been implicated in cell survival, including the protein kinase glycogen synthase kinase 3beta (GSK-3beta). One of the targets of GSK-3beta is translation initiation factor 2B (eIF2B), linking global regulation of protein synthesis to PI 3-kinase/Akt signaling. Because of the central role of protein synthesis, we have investigated the involvement of eIF2B, which is inhibited as a result of GSK-3beta phosphorylation, in programmed cell death. We demonstrate that expression of eIF2B mutants lacking the GSK-3beta phosphorylation or priming sites is sufficient to protect both Rat-1 and PC12 cells from apoptosis induced by overexpression of GSK-3beta, inhibition of PI 3-kinase, or growth factor deprivation. Consistent with these effects on cell survival, expression of nonphosphorylatable eIF2B prevented inhibition of protein synthesis following treatment of cells with the PI 3-kinase inhibitor LY294002. Conversely, cycloheximide induced apoptosis of PC12 and Rat-1 cells, further indicating that protein synthesis was required for cell survival. Inhibition of translation resulting from treatment with cycloheximide led to the release of cytochrome c from mitochondria, similar to the effects of inhibition of PI 3-kinase. Expression of nonphosphorylatable eIF2B prevented cytochrome c release resulting from PI 3-kinase inhibition but did not affect cytochrome c release or apoptosis induced by cycloheximide. Regulation of translation resulting from phosphorylation of eIF2B by GSK-3beta thus appears to contribute to the control of cell survival by the PI 3-kinase/Akt signaling pathway, acting upstream of mitochondrial cytochrome c release.     

8p12 Stem Cell Myeloproliferative Disorder: the FOP-Fibroblast Growth Factor Receptor 1 Fusion Protein of the t(6;8) Translocation Induces Cell Survival Mediated by Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase/Akt/mTOR Pathways

The FOP-fibroblast growth factor receptor 1 (FGFR1) fusion protein is expressed as a consequence of a t(6;8) (q27;p12) translocation associated with a stem cell myeloproliferative disorder with lymphoma, myeloid hyperplasia and eosinophilia. In the present report, we show that the fusion of the leucine-rich N-terminal region of FOP to the catalytic domain of FGFR1 results in conversion of murine hematopoietic cell line Ba/F3 to factor-independent cell survival via an antiapoptotic effect. This survival effect is dependent upon the constitutive tyrosine phosphorylation of FOP-FGFR1. Phosphorylation of STAT1 and of STAT3, but not STAT5, is observed in cells expressing FOP-FGFR1. The survival function of FOP-FGFR1 is abrogated by mutation of the phospholipase C gamma binding site. Mitogen-activated protein kinase (MAPK) is also activated in FOP-FGFR1-expressing cells and confers cytokine-independent survival to hematopoietic cells. These results demonstrate that FOP-FGFR1 is capable of protecting cells from apoptosis by using the same effectors as the wild-type FGFR1. Furthermore, we show that FOP-FGFR1 phosphorylates phosphatidylinositol 3 (PI3)-kinase and AKT and that specific inhibitors of PI3-kinase impair its ability to promote cell survival. In addition, FOP-FGFR1-expressing cells show constitutive phosphorylation of the positive regulator of translation p70S6 kinase; this phosphorylation is inhibited by PI3-kinase and mTOR (mammalian target of rapamycin) inhibitors. These results indicate that translation control is important to mediate the cell survival effect induced by FOP-FGFR1. Finally, FOP-FGFR1 protects cells from apoptosis by survival signals including BCL2 overexpression and inactivation of caspase-9 activity. Elucidation of signaling events downstream of FOP-FGFR1 constitutive activation provides insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.     

Rac and phosphatidylinositol 3-kinase regulate the protein kinase B in Fc epsilon RI signaling in RBL 2H3 mast cells

FcepsilonRI signaling in rat basophilic leukemia cells depends on phosphatidylinositol 3-kinase (PI3-kinase) and the small GTPase Rac. Here, we studied the functional relationship among PI3-kinase, its effector protein kinase B (PKB), and Rac using inhibitors of PI3-kinase and toxins inhibiting Rac. Wortmannin, an inhibitor of PI3-kinase, blocked FcepsilonRI-mediated tyrosine phosphorylation of phospholipase Cgamma, inositol phosphate formation, calcium mobilization, and secretion of hexosaminidase. Similarly, Clostridium difficile toxin B, which inactivates all Rho GTPases including Rho, Rac and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac (possibly Cdc42) but not Rho, blocked these responses. Stimulation of the FcepsilonRI receptor induced a rapid increase in the GTP-bound form of Rac. Whereas toxin B inhibited the Rac activation, PI3-kinase inhibitors (wortmannin and LY294002) had no effect on activation of Rac. In line with this, wortmannin had no effect on tyrosine phosphorylation of the guanine nucleotide exchange factor Vav. Wortmannin, toxin B, and lethal toxin inhibited phosphorylation of PKB on Ser(473). Similarly, translocation of the pleckstrin homology domain of PKB tagged with the green fluorescent protein to the membrane, which was induced by activation of the FcepsilonRI receptor, was blocked by inhibitors of PI3-kinase and Rac inactivation. Our results indicate that in rat basophilic leukemia cells Rac and PI3-kinase regulate PKB and suggest that Rac is functionally located upstream and/or parallel of PI3-kinase/PKB in FcepsilonRI signaling.     

Syk activation initiates downstream signaling events during human polymorphonuclear leukocyte phagocytosis

We investigated the requirement for Syk activation to initiate downstream signaling events during polymorphonuclear leukocyte (PMN) phagocytosis of Ab-coated erythrocytes (EIgG). When PMN were challenged with EIgG, Syk phosphorylation increased in a time-dependent manner, paralleling the response of PMN phagocytosis. Pretreatment of PMN with piceatannol, a Syk-selective inhibitor, blocked EIgG phagocytosis and Syk phosphorylation. We found that piceatannol inhibited protein kinase Cdelta (PKCdelta) and Raf-1 translocation from cytosol to plasma membrane by >90%. Extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2) phosphorylation was similarly blocked. We also investigated phosphatidylinositide 3-kinase (PI 3-kinase) activity and Syk phosphorylation using piceatannol, wortmannin, and LY294002, inhibitors of PI 3-kinase. The phosphorylation of Syk preceded the activation of PI 3-kinase. Both wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol inhibited Syk. In contrast to piceatannol, wortmannin did not inhibit PKCdelta and Raf-1 translocation. To elucidate signaling downstream of Syk activation, we assessed whether the cell-permeable diacylglycerol analogue didecanoylglycerol could normalize PMN phagocytosis, PKCdelta and Raf-1 translocation, and ERK1 and ERK2 phosphorylation inhibited by piceatannol. The addition of didecanoylglycerol to the Syk-inhibited phagocytosing PMN normalized all three without a concomitant effect on PI 3-kinase activity and Syk phosphorylation. We conclude that Syk activation following Fcgamma receptor engagement initiates downstream signaling events leading to mitogen-activated protein kinase activation independent of PI 3-kinase activation.     

MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor

Although the significance of vascular endothelial growth factor (VEGF) and its receptors in angiogenesis is well established, the signal transduction cascades activated by VEGF and their involvement in mediating the mitogenic response of endothelial cells to VEGF are incompletely characterized. Here we demonstrate that VEGF activates mitogen-activated protein (MAP) kinases, including the extracellular signal-regulated protein kinase (ERK) and p38 MAP kinase, phosphatidylinositol 3-kinase (PI 3-kinase), and p70 S6 kinase in human umbilical vein endothelial cells (HUVEC). The activation of these enzymes was assayed by kinase phosphorylation and by kinase activity towards substrates. Studies with PI 3-kinase inhibitors revealed that activation of p70 S6 kinase was mediated by PI 3-kinase. Selective inhibition of ERK, PI 3-kinase, and p70 S6 kinase with the inhibitors PD098059, LY294002, and rapamycin, respectively, inhibited VEGF-stimulated HUVEC proliferation. In marked contrast, the p38 MAP kinase inhibitor SB203580 not only failed to inhibit but actually enhanced HUVEC proliferation; this effect was associated with the phosphorylation of Rb protein. Rb phosphorylation resulted from a decrease in the level of the cdk inhibitor p27KiP1. These results indicate that the activities of ERK, PI 3-kinase, and p70 S6 kinase are essential for VEGF-induced HUVEC proliferation. p38 MAP kinase suppresses endothelial cell proliferation by regulating cell-cycle progression.     

Regulation of protein kinase B

Protein kinase B (PKB) is a member of the second-messenger regulated subfamily of protein kinases implicated in signalling downstream of growth factor and insulin receptor tyrosine kinases and phosphatidylinositol 3-kinase (PI 3-kinase). PKB is activated by phosphorylation in response to mitogens and survival factors. Membrane recruitment driven by lipid second-messengers derived from PI 3-kinase leads to PKB phosphorylation and activation by upstream kinases (PDK1 and an as yet identified protein kinase). Prolonged stimulation with growth factors results in nuclear translocation, providing evidence that PKB activation at the plasma membrane precedes its nuclear translocation and supporting a role for PKB in signalling from receptor tyrosine kinases to the nucleus.     

Tight folding of acidic fibroblast growth factor prevents its translocation to the cytosol with diphtheria toxin as vector.

A fusion protein of acidic fibroblast growth factor and diphtheria toxin A-fragment was disulfide-linked to the toxin B-fragment. The complex bound specifically to diphtheria toxin receptors, and subsequent exposure to low pH induced the fusion protein to translocate to the cytosol. Heparin, inositol hexaphosphate and inorganic sulfate strongly increased the trypsin resistance of the growth factor part of the fusion protein, indicating tight folding, and they prevented translocation of the fusion protein to the cytosol. The data indicate that only a more disordered form of the growth factor is translocation competent.     

Targeting head and neck squamous cell carcinoma using a novel fusion toxin-diphtheria toxin/HN-1

The current treatment strategies, chemotherapy and radiation therapy being used for the management of cancer are deficient in targeted approach leading to treatment related toxicities and relapse. Contrarily, fusion toxins exhibit remarkable tumor specificity thus emerging as an alternative therapy for the treatment of cancer. Diphtheria toxin-HN-1 peptide (DT/HN-1) is a fusion toxin designed to target the head and neck squamous cell carcinoma (HNSCC). The aim of this study was to construct, characterize, and evaluate the cytotoxicity and specificity of DT/HN-1 fusion toxin against the HNSCC cells. The purified DT/HN-1 fusion toxin was characterized by SDS-PAGE and western blotting. Refolding of purified fusion toxins was monitored by fluorescence spectra and circular dichroism spectra. The activity of DT/HN-1 fusion toxin was demonstrated on various HNSCC cell lines by cell viability assay, cell proliferation assay, protein synthesis inhibition assay, apoptosis and cell cycle analysis. The fusion toxin DT/HN-1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells. The IC₅₀ of DT/HN-1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines. The percentage apoptotic cells in DT/HN-1 treated UMB-SCC-745 cells are 16% compared to 4% in untreated. To further demonstrate the specific toxicity of DT/HN-1 fusion toxin towards the HNSCC cells we constructed, characterized and evaluated the efficacy of DT protein. The DT protein coding for only a fragment of diphtheria toxin without its native receptor binding domain failed to exhibit any cytotoxicity on all the cell lines used in this study thus establishing the importance of a ligand in achieving targeted toxicity. To evaluate the translocation ability of HN-1 peptide, an additional construct DTΔT/HN-1 was constructed, characterized and evaluated for its cytotoxic activity. The fusion toxin DTΔT/HN-1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity on all the cell lines clearly indicating the inability of HN-1 peptide to translocate catalytic domain of the toxin into the cytosol.     

Differential effects of phosphatidylinositol 3-kinase inhibition on intracellular signals regulating GLUT4 translocation and glucose transport

Phosphatidylinositol (PI) 3-kinase is required for insulin-stimulated translocation of GLUT4 to the surface of muscle and fat cells. Recent evidence suggests that the full stimulation of glucose uptake by insulin also requires activation of GLUT4, possibly via a p38 mitogen-activated protein kinase (p38 MAPK)-dependent pathway. Here we used L6 myotubes expressing Myc-tagged GLUT4 to examine at what level the signals regulating GLUT4 translocation and activation bifurcate. We compared the sensitivity of each process, as well as of signals leading to GLUT4 translocation (Akt and atypical protein kinase C) to PI 3-kinase inhibition. Wortmannin inhibited insulin-stimulated glucose uptake with an IC(50) of 3 nm. In contrast, GLUT4myc appearance at the cell surface was less sensitive to inhibition (IC(50) = 43 nm). This dissociation between insulin-stimulated glucose uptake and GLUT4myc translocation was not observed with LY294002 (IC(50) = 8 and 10 microm, respectively). The sensitivity of insulin-stimulated activation of PKC zeta/lambda, Akt1, Akt2, and Akt3 to wortmannin (IC(50) = 24, 30, 35, and 60 nm, respectively) correlated closely with inhibition of GLUT4 translocation. In contrast, insulin-dependent p38 MAPK phosphorylation was efficiently reduced in cells pretreated with wortmannin, with an IC(50) of 7 nm. Insulin-dependent p38 alpha and p38 beta MAPK activities were also markedly reduced by wortmannin (IC(50) = 6 and 2 nm, respectively). LY294002 or transient expression of a dominant inhibitory PI 3-kinase construct (Delta p85), however, did not affect p38 MAPK phosphorylation. These results uncover a striking correlation between PI 3-kinase, Akt, PKC zeta/lambda, and GLUT4 translocation on one hand and their segregation from glucose uptake and p38 MAPK activation on the other, based on their wortmannin sensitivity. We propose that a distinct, high affinity target of wortmannin, other than PI 3-kinase, may be necessary for activation of p38 MAPK and GLUT4 in response to insulin.     

Involvement of phosphatidylinositol 3-kinase in nuclear translocation of protein kinase C zeta induced by C2-ceramide in rat hepatocytes

In this study we report that protein kinase C zeta (PKC zeta), one of the atypical isoforms of the PKC family located predominantly in cytosol, is redistributed by C2-ceramide treatment in isolated hepatocytes. PKC zeta increased in membrane and nuclear fractions after 30 min of treatment with C2-ceramide in a dose- and time-dependent manner. The action of C2-ceramide was inhibited by wortmannin and LY 294002, indicating that C2-ceramide-induced PKC zeta increase in both nucleus and membrane fractions is mediated by phosphatidylinositol 3-kinase (PI3-kinase) activation. In addition, a significant translocation of PI3-kinase to the nucleus was observed after C2-ceramide treatment.     

Cyclic Mechanical Stretch Decreases Cell Migration by Inhibiting Phosphatidylinositol 3-Kinase- and Focal Adhesion Kinase-mediated JNK1 Activation

Epithelial cell migration during wound healing requires coordinated signaling pathways that direct polarization of the leading and trailing ends of the cells, cytoskeletal organization, and remodeling of focal adhesions. These inherently mechanical processes are disrupted by cyclic stretch (CS), but the specific signaling molecules involved in this disruption are not well understood. In this study, we demonstrate that inhibition of phosphatidylinositol 3-kinase (PI3K) or expression of a dominant-negative form of PI3K caused inhibition of airway epithelial cell wound closure. CS caused a sustained decrease in activation of PI3K and inhibited wound healing. Expression of constitutively active PI3K stimulated translocation of Tiam1 to the membrane, increased Rac1 activity, and increased wound healing of airway epithelial cells. Increased Rac1 activity resulted in increased phosphorylation of JNK1. PI3K activation was not regulated by association with focal adhesion kinase. Restoration of efficient cell migration during CS required coexpression of constitutively active PI3K, focal adhesion kinase, and JIP3.     

The role of phosphatidylinositide-3-kinase in basal mitogen-activated protein kinase activity and cell survival

Phosphatidylinositide-3-OH-kinase (PI 3-kinase) is an upstream activator of p42/p44 mitogen-activated protein kinase (MAPK), but the role of PI 3-kinase-dependent MAPK remains obscure. Here we demonstrate that in a variety of different cell types, PI 3-kinase inhibition results in an inhibition of MAPK in unstimulated cells but does not interfere with growth factor-, or TPA-induced MAPK activity. Furthermore, inhibition of either PI 3-kinase or MEK/MAPK results in cell death in serum-starved cells. We concluded that basal, but not induced MAPK activity is mediated by PI 3-kinase and that this PI 3-kinase-mediated MEK/MAPK activity is essential for cell survival in quiescent cells.     

Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.     

Tumor necrosis factor induces phosphorylation and translocation of BAD through a phosphatidylinositide-3-OH kinase-dependent pathway.

Tumor necrosis factor (TNF) induced the phosphorylation of BAD at serine 136 in HeLa cells under conditions that are not cytotoxic. BAD phosphorylation by TNF was dependent on phosphatidylinositide-3-OH kinase (PI3K) and was accompanied by the translocation of BAD from the mitochondria to the cytosol. Blocking the phosphorylation of BAD and its translocation to the cytosol with the PI3K inhibitor wortmannin activated caspase-3 and markedly potentiated the cytotoxicity of TNF. Transient transfection with a PI3K dominant negative mutant or a dominant negative mutant of the serine-threonine kinase Akt, the downstream target of PI3K and the enzyme that phosphorylates BAD, similarly potentiated the cytotoxicity of TNF. By contrast, transfection with a constitutively active Akt mutant protected against the cytotoxicity of TNF in the presence of wortmannin. Phosphorylation of BAD prevents its interaction with the antiapoptotic protein Bcl-XL. Transfection with a Bcl-XL expression vector protected against the cytotoxicity of TNF in the presence of wortmannin. The mechanism by which the inhibition of the phosphorylation of BAD is likely linked to the induction of lethal mitochondrial damage in TNF-intoxicated cells is discussed.