Showing papers on "PI3K/AKT/mTOR pathway published in 1999"
TL;DR: It is demonstrated that Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors, which triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.
6,481 citations
TL;DR: The mechanisms by which survival factors regulate the PI3K/c-Akt cascade, the evidence that activation of the PI 3K/ c-AKT pathway promotes cell survival, and the current spectrum of c- akt targets and their roles in mediating c- Akt-dependent cell survival are reviewed.
Abstract: The programmed cell death that occurs as part of normal mammalian development was first observed nearly a century ago (Collin 1906). It has since been established that approximately half of all neurons in the neuroaxis and >99.9% of the total number of cells generated during the course of a human lifetime go on to die through a process of apoptosis (for review, see Datta and Greenberg 1998; Vaux and Korsmeyer 1999). The induction of developmental cell death is a highly regulated process and can be suppressed by a variety of extracellular stimuli. The purification in the 1950s of the nerve growth factor (NGF), which promotes the survival of sympathetic neurons, set the stage for the discovery that peptide trophic factors promote the survival of a wide variety of cell types in vitro and in vivo (Levi-Montalcini 1987). The profound biological consequences of growth factor (GF) suppression of apoptosis are exemplified by the critical role of target-derived neurotrophins in the survival of neurons and the maintenance of functional neuronal circuits. (Pettmann and Henderson 1998). Recently, the ability of trophic factors to promote survival have been attributed, at least in part, to the phosphatidylinositide 38-OH kinase (PI3K)/c-Akt kinase cascade. Several targets of the PI3K/c-Akt signaling pathway have been recently identified that may underlie the ability of this regulatory cascade to promote survival. These substrates include two components of the intrinsic cell death machinery, BAD and caspase 9, transcription factors of the forkhead family, and a kinase, IKK, that regulates the NF-kB transcription factor. This article reviews the mechanisms by which survival factors regulate the PI3K/c-Akt cascade, the evidence that activation of the PI3K/c-Akt pathway promotes cell survival, and the current spectrum of c-Akt targets and their roles in mediating c-Akt-dependent cell survival.
4,260 citations
TL;DR: It is demonstrated that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increased NO production, and represents a novel Ca2+-independent regulatory mechanism for activation ofeNOS.
Abstract: Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) is a fundamental determinant of cardiovascular homesotasis: it regulates systemic blood pressure, vascular remodelling and angiogenesis. Physiologically, the most important stimulus for the continuous formation of NO is the viscous drag (shear stress) generated by the streaming blood on the endothelial layer. Although shear-stress-mediated phosphorylation of eNOS is thought to regulate enzyme activity, the mechanism of activation of eNOS is not yet known. Here we demonstrate that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increased NO production. Inhibition of the phosphatidylinositol-3-OH kinase/Akt pathway or mutation of the Akt site on eNOS protein (at serine 1177) attenuates the serine phosphorylation and prevents the activation of eNOS. Mimicking the phosphorylation of Ser 1177 directly enhances enzyme activity and alters the sensitivity of the enzyme to Ca2+, rendering its activity maximal at sub-physiological concentrations of Ca2+. Thus, phosphorylation of eNOS by Akt represents a novel Ca2+-independent regulatory mechanism for activation of eNOS.
3,530 citations
TL;DR: It is shown that the Akt serine–threonine kinase is involved in the activation of NF-κB by tumour necrosis factor (TNF), and that Akt is part of a signalling pathway that is necessary for inducing key immune and inflammatory responses.
Abstract: Activation of the nuclear transcription factor NF-kappaB by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and an IKB-kinase (IKK) complex composed of IKKalpha and IKKbeta. Here we show that the Akt serine-threonine kinase is involved in the activation of NF-kappaB by tumour necrosis factor (TNF). TNF activates phosphatidylinositol-3-OH kinase (PI(3)K) and its downstream target Akt (protein kinase B). Wortmannin (a PI(3)K inhibitor), dominant-negative PI(3)K or kinase-dead Akt inhibits TNF-mediated NF-kappaB activation. Constitutively active Akt induces NF-kappaB activity and this effect is blocked by dominant-negative NIK. Conversely, NIK activates NF-kappaB and this is blocked by kinase-dead Akt. Thus, both Akt and NIK are necessary for TNF activation of NF-kappaB. Akt mediates IKKalpha phosphorylation at threonine 23. Mutation of this amino acid blocks phosphorylation by Akt or TNF and activation of NF-kappaB. These findings indicate that Akt is part of a signalling pathway that is necessary for inducing key immune and inflammatory responses.
2,205 citations
TL;DR: A role for NF-κB in growth factor signalling is established and an anti-apoptotic Ras/PI(3)K/Akt/IKK/NF-κBs pathway is defined, thus linking anti-APoptotic signalling with transcription machinery.
Abstract: The mechanisms of cell proliferation and transformation are intrinsically linked to the process of apoptosis: the default of proliferating cells is to die unless specific survival signals are provided1,2. Platelet-derived growth factor (PDGF) is a principal survival factor that inhibits apoptosis and promotes proliferation1, but the mechanisms mediating its anti-apoptotic properties are not completely understood. Here we show that the transcription factor NF-κB3,4,5 is important in PDGF signalling. NF-κB transmits two signals: one is required for the induction of proto-oncogene c-myc and proliferation, and the second, an anti-apoptotic signal, counterbalances c-Myc cytotoxicity. We have traced a putative pathway whereby PDGF activates NF-κB through Ras and phospatidylinositol-3-kinase (PI(3)K) to the PKB/Akt protein kinase and the IκB kinase (IKK); NF-κB thus appears to be a target of the anti-apoptotic Ras/PI(3)K/Akt pathway6,7. We show that, upon PDGF stimulation, Akt transiently associates in vivo with IKK and induces IKK activation. These findings establish a role for NF-κB in growth factor signalling and define an anti-apoptotic Ras/PI(3)K/Akt/IKK/NF-κB pathway, thus linking anti-apoptotic signalling with transcription machinery.
1,819 citations
TL;DR: 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylated of the carboxy-terminal serum-sensitive sites, including those that interact with eIF4E.
Abstract: The multisubunit eukaryotic translation initiation factor (eIF) 4F recruits 40S ribosomal subunits to the 5′ end of mRNA. The eIF4F subunit eIF4E interacts directly with the mRNA 5′ cap structure. Assembly of the eIF4F complex is inhibited by a family of repressor polypeptides, the eIF4E-binding proteins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylation: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, whereas hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells, but is hyperphosphorylated on multiple sites following exposure to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is responsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions, a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were utilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping of the in vitro-labeled protein yielded two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo. Mass spectrometry analysis indicated that these peptides contain phosphorylated Thr-37 and Thr-46. Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR when 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was not associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-46 are detected in all phosphorylated in vivo 4E-BP1 isoforms, including those that interact with eIF4E. Finally, mutational analysis demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, our results suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal serum-sensitive sites.
1,227 citations
TL;DR: Observations provide a molecular basis for cross talk between two signaling pathways at the level of Raf and Akt in a human breast cancer cell line.
Abstract: Activation of the protein kinase Raf can lead to opposing cellular responses such as proliferation, growth arrest, apoptosis, or differentiation. Akt (protein kinase B), a member of a different signaling pathway that also regulates these responses, interacted with Raf and phosphorylated this protein at a highly conserved serine residue in its regulatory domain in vivo. This phosphorylation of Raf by Akt inhibited activation of the Raf-MEK-ERK signaling pathway and shifted the cellular response in a human breast cancer cell line from cell cycle arrest to proliferation. These observations provide a molecular basis for cross talk between two signaling pathways at the level of Raf and Akt.
1,089 citations
TL;DR: The serine/threonine kinase Akt, or protein kinase B (PKB), has recently been a focus of intense research and suggests that Akt/PKB is a critical factor in the genesis of cancer.
936 citations
TL;DR: It is shown that Akt can regulate signaling pathways that lead to induction of the NF-κB family of transcription factors in the Jurkat T-cell line, uncovering a previously unappreciated connection between Akt and NF-kkB induction that could have implications for the control of T- cell growth and survival.
826 citations
TL;DR: The studies suggest that PTEN regulates the phosphatidylinositol 3,4, 5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival.
Abstract: To investigate the molecular basis of PTEN-mediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten−/− ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G1/S transition was accompanied by down-regulation of p27KIP1, a major inhibitor for G1 cyclin-dependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosage-dependent increases in phosphorylation and activation of Akt/protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten−/− cell survival. Our studies suggest that PTEN regulates the phosphatidylinositol 3,4,5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival.
818 citations
TL;DR: The stage-specific inhibitory action of Akt correlated with its stage- specific ability to form a complex with Raf, suggesting the existence of differentially expressed mediators of an inhibitory Akt-Raf complex.
Abstract: Extracellular signals often result in simultaneous activation of both the Raf-MEK-ERK and PI3K-Akt pathways (where ERK is extracellular-regulated kinase, MEK is mitogen-activated protein kinase or ERK kinase, and PI3K is phosphatidylinositol 3-kinase). However, these two signaling pathways were shown to exert opposing effects on muscle cell hypertrophy. Furthermore, the PI3K-Akt pathway was shown to inhibit the Raf-MEK-ERK pathway; this cross-regulation depended on the differentiation state of the cell: Akt activation inhibited the Raf-MEK-ERK pathway in differentiated myotubes, but not in their myoblast precursors. The stage-specific inhibitory action of Akt correlated with its stage-specific ability to form a complex with Raf, suggesting the existence of differentially expressed mediators of an inhibitory Akt-Raf complex.
TL;DR: The results show that Akt promotes cell survival by intervening in the apoptosis cascade before cytochrome c release and caspase activation via a mechanism that is distinct from Bad phosphorylation.
Abstract: Growth factors signaling through the phosphoinositide 3-kinase/Akt pathway promote cell survival. The mechanism by which the serine/threonine kinase Akt prevents cell death remains unclear. We have previously shown that Akt inhibits the activity of DEVD-targeted caspases without changing the steady-state levels of Bcl-2 and Bcl-x(L). Here we show that Akt inhibits apoptosis and the processing of procaspases to their active forms by delaying mitochondrial changes in a caspase-independent manner. Akt activation is sufficient to inhibit the release of cytochrome c from mitochondria and the alterations in the inner mitochondrial membrane potential. However, Akt cannot inhibit apoptosis induced by microinjection of cytochrome c. We also demonstrated that Akt inhibits apoptosis and cytochrome c release induced by several proapoptotic Bcl-2 family members. Taken together, our results show that Akt promotes cell survival by intervening in the apoptosis cascade before cytochrome c release and caspase activation via a mechanism that is distinct from Bad phosphorylation.
TL;DR: It is shown that PTEN protein induces a G1 block when reconstituted in PTEN-null cells, suggesting a link between induction of a cell-cycle block by PTEN and its ability to dephosphorylate, in vivo, phosphatidylinositol 3,4,5-trisphosphate.
Abstract: PTEN/MMAC1 is a tumor suppressor gene located on chromosome 10q23. Inherited PTEN/MMAC1 mutations are associated with a cancer predisposition syndrome known as Cowden’s disease. Somatic mutation of PTEN has been found in a number of malignancies, including glioblastoma, melanoma, and carcinoma of the prostate and endometrium. The protein product (PTEN) encodes a dual-specificity protein phosphatase and in addition can dephosphorylate certain lipid substrates. Herein, we show that PTEN protein induces a G1 block when reconstituted in PTEN-null cells. A PTEN mutant associated with Cowden’s disease (PTEN;G129E) has protein phosphatase activity yet is defective in dephosphorylating inositol 1,3,4,5-tetrakisphosphate in vitro and fails to arrest cells in G1. These data suggest a link between induction of a cell-cycle block by PTEN and its ability to dephosphorylate, in vivo, phosphatidylinositol 3,4,5-trisphosphate. In keeping with this notion, PTEN can inhibit the phosphatidylinositol 3,4,5-trisphosphate-dependent Akt kinase, a downstream target of phosphatidylinositol 3-kinase, and constitutively active, but not wild-type, Akt overrides a PTEN G1 arrest. Finally, tumor cells lacking PTEN contain high levels of activated Akt, suggesting that PTEN is necessary for the appropriate regulation of the phosphatidylinositol 3-kinase/Akt pathway.
TL;DR: Results indicate that Pten is an essential mediator of the Fas response and a repressor of autoimmunity and thus implicate the PI 3-kinase/Akt pathway in Fas-mediated apoptosis.
Abstract: Inactivating mutations in the PTEN tumor suppressor gene, encoding a phosphatase, occur in three related human autosomal dominant disorders characterized by tumor susceptibility. Here it is shown that Pten heterozygous ( Pten +/−) mutants develop a lethal polyclonal autoimmune disorder with features reminiscent of those observed in Fas-deficient mutants. Fas-mediated apoptosis was impaired in Pten +/− mice, and T lymphocytes from these mice show reduced activation-induced cell death and increased proliferation upon activation. Phosphatidylinositol (PI) 3-kinase inhibitors restored Fas responsiveness in Pten +/− cells. These results indicate that Pten is an essential mediator of the Fas response and a repressor of autoimmunity and thus implicate the PI 3-kinase/Akt pathway in Fas-mediated apoptosis.
TL;DR: It is shown here that a loss-of-function mutation in pdk-1, the C. elegans homolog of the mammalian Akt/PKB kinase PDK1, results in constitutive arrest at the dauer stage and increased life span; these phenotypes are suppressed by a loss of function mutation in daf-16.
Abstract: An insulin receptor-like signaling pathway regulates Caenorhabditis elegans metabolism, development, and longevity. Inactivation of the insulin receptor homolog DAF-2, the AGE-1 PI3K, or the AKT-1 and AKT-2 kinases causes a developmental arrest at the dauer stage. A null mutation in the daf-16 Fork head transcription factor alleviates the requirement for signaling through this pathway. We show here that a loss-of-function mutation in pdk-1, the C. elegans homolog of the mammalian Akt/PKB kinase PDK1, results in constitutive arrest at the dauer stage and increased life span; these phenotypes are suppressed by a loss of function mutation in daf-16. An activating mutation in pdk-1 or overexpression of wild-type pdk-1 relieves the requirement for AGE-1 PI3K signaling. Therefore, pdk-1 activity is both necessary and sufficient to propagate AGE-1 PI3K signals in the DAF-2 insulin receptor-like signaling pathway. The activating mutation in pdk-1 requires akt-1 and akt-2 gene activity in order to suppress the dauer arrest phenotype of age-1. This indicates that the major function of C. elegans PDK1 is to transduce signals from AGE-1 to AKT-1 and AKT-2. The activating pdk-1 mutation is located in a conserved region of the kinase domain; the equivalent amino acid substitution in human PDK1 activates its kinase activity toward mammalian Akt/PKB.
TL;DR: Gene manipulation of the phosphoinositide-3-OH-kinase-dependent serine/threonine protein kinase Akt during development of the Drosophila imaginal disc affects cell and organ size in an autonomous manner and appears to stimulate intracellular pathways that specifically regulate cell and compartment size independently of cell proliferation in vivo.
Abstract: Organismal size is determined by a tightly regulated mechanism that coordinates cell growth, cell proliferation and cell death. The Drosophila insulin receptor/Chico/Dp110 pathway regulates cell and organismal size. Here we show that genetic manipulation of the phosphoinositide-3-OH-kinase-dependent serine/threonine protein kinase Akt (protein kinase B) during development of the Drosophila imaginal disc affects cell and organ size in an autonomous manner. Ectopic expression of Akt does not affect cell-fate determination, apoptosis or proliferation rates in imaginal discs. Thus, Akt appears to stimulate intracellular pathways that specifically regulate cell and compartment size independently of cell proliferation in vivo.
TL;DR: It is suggested that both PI3-K/Akt-dependent and -independent pathways contribute to the IL-3 activation of mcl-1 gene expression.
Abstract: mcl-1 is an immediate-early gene activated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) signaling pathways and plays an important role in the viability response of these cytokines In this study, we demonstrated that cytokine stimulation of mcl-1 mRNA and protein expression were attenuated by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-K) inhibitors Reporter gene assays further showed that the PI3-K/Akt signaling pathway was involved in IL-3 activation of mcl-1 gene transcription Analysis of the mcl-1 promoter revealed that both promoter elements, SIE at position -87 and CRE-2 at -70, contribute to IL-3 stimulation of mcl-1 gene expression Although either the SIE site or the CRE-2 site alone was sufficient to confer IL-3 inducibility on a heterologous promoter, only IL-3 activation of the CRE-2 reporter was mediated via the PI3-K/Akt pathway The SIE binding activity was constitutively high in cells deprived of or stimulated by IL-3 In contrast, the CRE-2 binding activity was low in cytokine-starved cells and was strongly induced within 1 h following cytokine treatment of cells In addition, cytokine induction of the CRE-2 but not of the SIE binding activity was dependent on activation of the PI3-K/Akt signaling pathway Lastly, we showed that CREB was one component of the CRE-2 binding complex and played a role in IL-3 activation of the mcl-1 reporter gene Taken together, our results suggest that both PI3-K/Akt-dependent and -independent pathways contribute to the IL-3 activation of mcl-1 gene expression Activation of mcl-1 by the PI3-K/Akt-dependent pathway is through a transcription factor complex containing CREB
TL;DR: It appears that the B-Raf/MEK/Erk pathway confers protection against apoptosis at the level of cytosolic caspase activation, downstream of the release of cytochromec from mitochondria.
Abstract: Growth factor-dependent kinases, such as phosphatidylinositol 3-kinase (PI 3-kinase) and Raf kinases, have been implicated in the suppression of apoptosis. We have recently established Rat-1 fibroblast cell lines overexpressing B-Raf, leading to activation of the MEK/Erk mitogen-activated protein kinase pathway. Overexpression of B-Raf confers resistance to apoptosis induced by growth factor withdrawal or PI 3-kinase inhibition. This is accompanied by constitutive activation of Erk without effects on the PI 3-kinase/Akt pathway. The activity of MEK is essential for cell survival mediated by B-Raf overexpression, since either treatment with the specific MEK inhibitor PD98059 or expression of a dominant inhibitory MEK mutant blocks the antiapoptotic activity of B-Raf. Activation of MEK is not only necessary but also sufficient for cell survival because overexpression of constitutively activated MEK, Ras, or Raf-1, like B-Raf, prevents apoptosis after growth factor deprivation. Overexpression of B-Raf did not interfere with the release of cytochrome c from mitochondria after growth factor deprivation. However, the addition of cytochrome c to cytosols of cells overexpressing B-Raf failed to induce caspase activation. It thus appears that the B-Raf/MEK/Erk pathway confers protection against apoptosis at the level of cytosolic caspase activation, downstream of the release of cytochrome c from mitochondria.
TL;DR: It is demonstrated that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.
Abstract: The PTEN/MMAC1/TEP (PTEN) tumor suppressor gene at 10q23.3 is mutated in multiple types of sporadic tumors including breast cancers and also in the germline of patients with the Cowden's breast cancer predisposition syndrome. The PTEN gene encodes a multifunctional phosphatase capable of dephosphorylating the same sites in membrane phosphatidylinositols phosphorylated by phosphatidylinositol 3'-kinase (PI3K). We demonstrate herein that loss of PTEN function in breast cancer cells results in an increase in basal levels of phosphorylation of multiple components of the P13K signaling cascade as well as an increase in duration of ligand-induced signaling through the P13K cascade. These alterations are reversed by wild-type but not phosphatase inactive PTEN. In the presence of high concentrations of serum, enforced expression of PTEN induces a predominant G1 arrest consistent with the capacity of PTEN to evoke increases in the expression of the p27Kip1 cyclin dependent kinase inhibitor. In the presence of low concentrations of serum, enforced PTEN expression results in a marked increase in cellular apoptosis, a finding which is consistent with the capacity of PTEN to alter the phosphorylation, and presumably function, of the AKT, BAD, p70S6 kinase and GSK3 alpha apoptosis regulators. Under anchorage-independent conditions, PTEN also induces anoikis, a form of apoptosis that occurs when cells are dissociated from the extracellular matrix, which is enhanced in conjunction with low serum culture conditions. Together, these data suggest that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli such as integrin ligation, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.
Journal Article•
TL;DR: This work reports that rapamycin, a specific inhibitor of mTOR kinase, induces G1 cell cycle arrest and apoptosis in two rhabdomyosarcoma cell lines (Rh1 and Rh30) under conditions of autocrine cell growth, and is the first to indicate thatRapamycin-induced apoptosis is mediated by inhibition of m TOR.
Abstract: The mammalian target of rapamycin (mTOR) has been shown to link growth factor signaling and posttranscriptional control of translation of proteins that are frequently involved in cell cycle progression. However, the role of this pathway in cell survival has not been demonstrated. Here, we report that rapamycin, a specific inhibitor of mTOR kinase, induces G 1 cell cycle arrest and apoptosis in two rhabdomyosarcoma cell lines (Rh1 and Rh30) under conditions of autocrine cell growth. To examine the kinetics of rapamycin action, we next determined the rapamycin sensitivity of rhabdomyosarcoma cells exposed briefly (1 h) or continuously (6 days). Results demonstrate that Rh1 and Rh30 cells were equally sensitive to rapamycin-induced growth arrest and apoptosis under either condition. Apoptosis was detected between 24 and 144 h of exposure to rapamycin. Both cell lines have mutant p53; hence, rapamycin-induced apoptosis appears to be a p53-independent process. To determine whether induction of apoptosis by rapamycin was specifically due to inhibition of mTOR signaling, we engineered Rh1 and Rh30 clones to stably express a mutant form of mTOR that was resistant to rapamycin (Ser 2035 →Ile; designated mTOR-rr). Rh1 and Rh30 mTOR-rr clones were highly resistant (>3000-fold) to both growth inhibition and apoptosis induced by rapamycin. These results are the first to indicate that rapamycin-induced apoptosis is mediated by inhibition of mTOR. Exogenous insulin-like growth factor (IGF)-I protected both Rh1 and Rh30 from apoptosis, without reactivating ribosomal p70 S6 kinase (p70 S6K ) downstream of mTOR. However, in rapamycin-treated cultures, the response to IGF-I differed between the cell lines: Rh1 cells proliferated normally, whereas Rh30 cells remained arrested in G 1 phase but viable. Rapamycin is known to inhibit synthesis of specific proteins but did not inhibit synthesis or alter the levels of mTOR. To examine the rate at which the mTOR pathway recovered, the ability of IGF-I to stimulate p70 S6K activity was followed in cells treated for 1 h with rapamycin and then allowed to recover in medium containing ≥100-fold excess of FK506 (to prevent rapamycin from rebinding to its cytosolic receptor FKBP-12). Our results indicate that, in Rh1 cells, rapamycin dissociates relatively slowly from FKBP-12, with a t 1/2 of ∼17.5 h. in the presence of FK506, whereas there was no recovery of p70 S6K activity in the absence of this competitor. This was of interest because rapamycin was relatively unstable under conditions of cell culture having a biological t 1/2 of ∼9.9 h. These results help to explain why cells are sensitive following short exposures to rapamycin and may be useful in guiding the use of rapamycin analogues that are entering clinical trials as novel antitumor agents.
TL;DR: The findings imply that theGLUT1 gene responds to insulin at the transcriptional level and that Akt mediates a step in the activation of GLUT1gene expression in this system.
TL;DR: It is shown that cells overproducing a mutant form of ezrin in which Tyr-353 was changed to a phenylalanine (Y353F) undergo apoptosis when assayed for tubulogenesis, and a novel function is established for eZrin in determining survival of epithelial cells by activating the PI 3-kinase/Akt pathway.
Abstract: ERM (Ezrin–Radixin–Moesin) proteins function as plasma membrane–actin cytoskeleton linkers and participate in the formation of specialized domains of the plasma membrane. We have investigated ezrin function in tubulogenesis of a kidney-derived epithelial cell line, LLC-PK1. Here we show that cells overproducing a mutant form of ezrin in which Tyr-353 was changed to a phenylalanine (Y353F) undergo apoptosis when assayed for tubulogenesis. While investigating the mechanism responsible for this apoptosis, we found that ezrin interacts with p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Two distinct sites of ezrin are involved in this interaction, the amino-terminal domain containing the first 309 aa and the phosphorylated Tyr-353 residue, which binds to the carboxyl-terminal SH2 domain of p85. Cells producing Y353F ezrin are defective in activation of the protein kinase Akt, a downstream target of PI 3-kinase that protects cells against apoptosis. Furthermore, the apoptotic phenotype of these cells is rescued by production of a constitutively activated form of PI 3-kinase. Taken together, these results establish a novel function for ezrin in determining survival of epithelial cells by activating the PI 3-kinase/Akt pathway.
TL;DR: The identification of particular downstream targets of PKB has provided new insights into the possible mechanism of PI3K/PKB-mediated tumorigenicity and a subfamily of Forkhead transcription factors was identified as additional targets for PI3k/P KB signaling.
Abstract: The proto-oncogene protein kinase B (PKB), also known as c-Akt, is a central player in a signaling pathway of which many components have been linked to tumorigenesis. Active forms of PKB as well as of its upstream activator phosphatidylinositol 3-kinase (PI3K) have been found to be responsible for the transforming activities of certain viruses, and the negative regulator of this pathway, PTEN, is a tumor suppressor. The identification of particular downstream targets of PKB has provided us with new insights into the possible mechanism of PI3K/PKB-mediated tumorigenicity. Recently a subfamily of Forkhead transcription factors was identified as additional targets for PI3K/PKB signaling. This review discusses the studies that have led to this conclusion and the possible implications of this finding for our understanding of how PI3K/PKB activity could lead to oncogenesis.
TL;DR: It is demonstrated that epidermal growth factor stimulation of T47D breast adenocarcinoma and embryonic kidney epithelial cells protects these cells from Fas-induced apoptosis, and indicates that EGF receptor stimulation of epithelial Cells has a significant survival function against death receptor- induced apoptosis mediated by Akt.
Journal Article•
TL;DR: The inverse correlation between PTEN protein level and Akt phosphorylation was found in a panel of breast cancer cell lines, and PTEN appears to suppress breast cancer growth through down-regulating PI3K signaling, which leads to the blockage of cell cycle progression and the induction of cell death in a sequential manner.
Abstract: PTEN/MMAC1/TEP1, a tumor suppressor gene, is frequently mutated in a variety of human cancers. Germ-line mutations of phosphatase and tensin homolog, deleted on chromosome ten (PTEN) are found in two inherited hamartoma tumor syndromes: Cowden syndrome, which has a high risk of breast, thyroid, and other cancers; and Bannayan-Zonana syndrome, a related disorder. PTEN encodes a phosphatase that recognizes both protein substrates and phosphatidylinositol-3,4,5-triphosphate. The lipid phosphatase activity of PTEN seems to be important for growth suppression through inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. We established clones with stable PTEN expression controlled by a tetracycline-inducible system to examine the consequences of increased levels of wild-type and mutant PTEN expression in a well-characterized breast cancer line, MCF-7. When we overexpressed PTEN in MCF-7, growth suppression was observed, but only if PTEN phosphatase activity is preserved. The initial growth suppression was attributable to G1 cell cycle arrest, whereas subsequent growth suppression was attributable to a combination of G1 arrest and cell death. Of note, the decrease in Akt phosphorylation preceded the onset-of suppression of cell growth. Treatment of MCF-7 cells with wortmannin, a PI3K inhibitor, caused cell growth inhibition in a way similar to the effects of overexpression of PTEN in this cell. In general, the inverse correlation between PTEN protein level and Akt phosphorylation was found in a panel of breast cancer cell lines. Therefore, PTEN appears to suppress breast cancer growth through down-regulating PI3K signaling, which leads to the blockage of cell cycle progression and the induction of cell death, in a sequential manner.
Journal Article•
TL;DR: Constitutive signaling through the PI3K pathway is required to prevent cell death in LNCaP, whereas activation of the Erk/MAPK and androgen response pathways is not obligatory for cell survival.
Abstract: Intracellular signaling pathways that mediate survival of prostate carcinoma (PCa) cells are poorly understood. We examined the potential role of the phosphatidylinositol 3′ kinase (PI3K) pathway as a mediator of cell survival in LNCaP human PCa cells, which express a variety of properties characteristic of human prostate cancer. LNCaP cell cultures rapidly became apoptotic when treated with the specific PI3K inhibitors, wortmannin and LY294002. In contrast, apoptosis was not induced when the cells were treated with: (a) rapamycin, an inhibitor of the ribosomal S6 kinase pp70S6K, which acts downstream of PI3K; (b) PD98059, a specific inhibitor of the extracellular signal-regulated kinase/mitogen-activated protein kinase (Erk/MAPK) kinase (MEK); or (c) the antiandrogen, Casodex; or when the cells were cultured under androgen-depleted conditions. Apoptosis induced by PI3K inhibition was attenuated by: (a) dihydrotestosterone; or (b) the ErbB1 activating ligands [epidermal growth factor (EGF), transforming growth factor α, or heparin-binding EGF-like growth factor]. In response to ErbB1 activation by ligand, the p85 regulatory subunit of PI3K associated specifically with ErbB3 but not detectably with ErbB1. The anti-apoptotic effect of ErbB1 activation was significantly reduced when cells were treated simultaneously with wortmannin and PD98059. These data indicate that survival signals can be evoked in LNCaP cells by several distinct pathways and can be triggered by nuclear and cell-surface receptors. Constitutive signaling through the PI3K pathway is required to prevent cell death in LNCaP, whereas activation of the Erk/MAPK and androgen response pathways is not obligatory for cell survival. These results also show that survival signals, as distinguished from mitogenic signals, can be evoked in PCa cells by ErbB1 ligands known to be synthesized within the human prostate.
TL;DR: It is reported that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis.
Abstract: The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.
TL;DR: It is concluded that Ang II-initiated activation of p70(S6K) requires both ERK cascade and PI3K/Akt cascade that bifurcate at the point of EGF receptor-dependent Ras activation.
TL;DR: Data suggest that M-CSF may induce cell survival through Akt-induced suppression of caspase-9 activation, and appears to be the primary regulator of survival in 3T3-FMS cells.