Showing papers on "PI3K/AKT/mTOR pathway published in 1998"
TL;DR: The results show that PTEN may exert its role as a tumor suppressor by negatively regulating the PI3'K/PKB/Akt signaling pathway.
2,482 citations
TL;DR: It is demonstrated that the PI 3-kinase-Akt signaling pathway, in concert with FRAP/mTOR, induces the phosphorylation of 4E-BP1.
Abstract: Growth factors and hormones activate protein translation by phosphorylation and inactivation of the translational repressors, the eIF4E-binding proteins (4E-BPs), through a wortmannin- and rapamycin-sensitive signaling pathway. The mechanism by which signals emanating from extracellular signals lead to phosphorylation of 4E-BPs is not well understood. Here we demonstrate that the activity of the serine/threonine kinase Akt/PKB is required in a signaling cascade that leads to phosphorylation and inactivation of 4E-BP1. PI 3-kinase elicits the phosphorylation of 4E-BP1 in a wortmannin- and rapamycin-sensitive manner, whereas activated Akt-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. A dominant negative mutant of Akt blocks insulin-mediated phosphorylation of 4E-BP1, indicating that Akt is required for the in vivo phosphorylation of 4E-BP1. Importantly, an activated Akt induces phosphorylation of 4E-BP1 on the same sites that are phosphorylated upon serum stimulation. Similar to what has been observed with serum and growth factors, phosphorylation of 4E-BP1 by Akt inhibits the interaction between 4E-BP1 and eIF-4E. Furthermore, phosphorylation of 4E-BP1 by Akt requires the activity of FRAP/mTOR. FRAP/mTOR may lie downstream of Akt in this signaling cascade. These results demonstrate that the PI 3-kinase-Akt signaling pathway, in concert with FRAP/mTOR, induces the phosphorylation of 4E-BP1.
871 citations
TL;DR: It is shown that two C. elegans Akt/PKB homologs transduce insulin receptor-like signals that inhibit dauer arrest and that AKT-1 andAKT-2 signaling are indispensable for insulin receptors-like signaling in the nervous system and in tissues that are remodeled during dauer formation.
Abstract: A neurosecretory pathway regulates a reversible developmental arrest and metabolic shift at the Caenorhabditis elegans dauer larval stage. Defects in an insulin-like signaling pathway cause arrest at the dauer stage. We show here that two C. elegans Akt/PKB homologs, akt-1 and akt-2, transduce insulin receptor-like signals that inhibit dauer arrest and that AKT-1 and AKT-2 signaling are indispensable for insulin receptor-like signaling in C. elegans. A loss-of-function mutation in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling, which indicates that AKT-1 and AKT-2 function primarily to antagonize DAF-16. This is the first evidence that the major target of Akt/PKB signaling is a transcription factor. An activating mutation in akt-1, revealed by a genetic screen, as well as increased dosage of wild-type akt-1 relieves the requirement for signaling from AGE-1 PI3K, which acts downstream of the DAF-2 insulin/IGF-1 receptor homolog. This demonstrates that Akt/PKB activity is not necessarily dependent on AGE-1 PI3K activity. akt-1 and akt-2 are expressed in overlapping patterns in the nervous system and in tissues that are remodeled during dauer formation.
703 citations
TL;DR: In this article, the PTEN/MMAC1 mutation impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation.
Abstract: The PTEN/MMAC1 phosphatase is a tumor suppressor gene implicated in a wide range of human cancers. Here we provide biochemical and functional evidence that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive, dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1 expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its tumor suppressor function.
681 citations
TL;DR: It is shown that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells, and studies suggest that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway.
Abstract: PTEN/MMAC1/TEP1 is a tumor suppressor that possesses intrinsic phosphatase activity. Deletions or mutations of its encoding gene are associated with a variety of human cancers. However, very little is known about the molecular mechanisms by which this important tumor suppressor regulates cell growth. Here, we show that PTEN expression potently suppressed the growth and tumorigenicity of human glioblastoma U87MG cells. The growth suppression activity of PTEN was mediated by its ability to block cell cycle progression in the G1 phase. Such an arrest correlated with a significant increase of the cell cycle kinase inhibitor p27KIP1 and a concomitant decrease in the activities of the G1 cyclin-dependent kinases. PTEN expression also led to the inhibition of Akt/protein kinase B, a serine-threonine kinase activated by the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway. In addition, the effect of PTEN on p27KIP1 and the cell cycle can be mimicked by treatment of U87MG cells with LY294002, a selective inhibitor of PI 3-kinase. Taken together, our studies suggest that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI 3-kinase/Akt signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase inhibitor p27KIP1.
501 citations
TL;DR: The findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.
Abstract: The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin⋅FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.
497 citations
TL;DR: A role for PTEN is suggested in regulating the activity of the PI 3-kinase pathway in malignant human cells.
483 citations
TL;DR: A novel shear stress-stimulated signal transduction pathway, namely, activation of the serine/threonine kinase Akt, is defined, which may contribute to the profound changes in endothelial morphology and function byShear stress.
Abstract: Fluid shear stress alters the morphology and function of the endothelium by activating several kinases. Furthermore, shear stress potently inhibits apoptosis of endothelial cells. Since activation of Akt kinase has been shown to prevent cell death, we investigated the effects of shear stress on Akt phosphorylation. To test the hypothesis that shear stress interacts with the Akt kinase pathway, human umbilical venous endothelial cells were exposed to laminar shear stress (15 dyne/cm2). Western blotting with specific antibodies against the phosphorylated Akt demonstrated a time-dependent stimulation of Akt phosphorylation by shear stress with a maximal increase up to 6-fold after 1 hour of shear stress exposure. The stimulation of Akt phosphorylation by shear stress thereby seemed to be mediated by the phosphoinositide 3-OH kinase (PI3K), as evidenced by the significant inhibition of shear stress-induced Akt phosphorylation by the PI3K inhibitors wortmannin (20 nmol/L) and Ly294002 (10 micromol/L). In addition, pharmacological inhibition of P13K reduced the antiapoptotic effect of shear stress against growth factor depletion-induced apoptosis. Most important, overexpression of a dominant-negative Akt mutant significantly inhibited the apoptosis-suppressive effect of shear stress against serum depletion-induced apoptosis, thus indicating the direct involvement of shear stress-induced Akt phosphorylation for inhibition of endothelial cell apoptosis. These results define a novel shear stress-stimulated signal transduction pathway, namely, activation of the serine/threonine kinase Akt, which may contribute to the profound changes in endothelial morphology and function by shear stress.
427 citations
TL;DR: It is suggested that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKCλ pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.
Abstract: Phosphoinositide (PI) 3-kinase contributes to a wide variety of biological actions, including insulin stimulation of glucose transport in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, and atypical isoforms of protein kinase C (PKCzeta and PKClambda) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKClambda in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a dominant negative mutant of PI 3-kinase). Overexpression of kinase-deficient mutants of PKClambda (lambdaKD or lambdaDeltaNKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of insulin activation of PKClambda, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were inhibited by lambdaKD or lambdaDeltaNKD in a dose-dependent manner. The maximal inhibition of insulin-induced glucose uptake achieved by the dominant negative mutants of PKClambda was approximately 50 to 60%. These mutants did not inhibit insulin-induced activation of Akt. A PKClambda mutant that lacks the pseudosubstrate domain (lambdaDeltaPD) exhibited markedly increased kinase activity relative to that of the wild-type enzyme, and expression of lambdaDeltaPD in quiescent 3T3-L1 adipocytes resulted in the stimulation of glucose uptake and translocation of GLUT4 but not in the activation of Akt. Furthermore, overexpression of an Akt mutant in which the phosphorylation sites targeted by growth factors are replaced by alanine resulted in inhibition of insulin-induced activation of Akt but not of PKClambda. These results suggest that insulin-elicited signals that pass through PI 3-kinase subsequently diverge into at least two independent pathways, an Akt pathway and a PKClambda pathway, and that the latter pathway contributes, at least in part, to insulin stimulation of glucose uptake in 3T3-L1 adipocytes.
375 citations
TL;DR: The data show that C2-ceramide induces apoptosis in HMN1 motor neuron cells and decreases both basal and insulin- or serum-stimulated Akt kinase activity 65–70%.
355 citations
TL;DR: A mutant Akt in which the phosphorylation sites targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt.
Abstract: Akt is a pleckstrin homology (PH) domain-containing protein serine-threonine kinase whose kinase domain shares structural similarity with protein kinase C (PKC) isozymes and cyclic AMP-dependent protein kinase (PKA) (3). Thus, Akt has also been termed RAC-PK (protein kinase related to A and C kinases) (19) and PKB (protein kinase B) (7). Insulin and various other growth factors activate Akt, and this activation is inhibited by pharmacological blockers of phosphatidylinositol (PI) 3-kinase or by a dominant negative mutant of PI 3-kinase (4, 14, 25). Furthermore, Akt is activated by overexpression of a constitutively active mutant of PI 3-kinase in quiescent cells (11, 23). These observations indicate that Akt is a downstream effector of PI 3-kinase.
PI 3-kinase, which consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit (5), is implicated in various metabolic effects of insulin (18, 59). A dominant negative mutant of PI 3-kinase as well as various pharmacological inhibitors, such as wortmannin and LY294002, have been used to block specific signaling pathways that include this enzyme (6, 16, 31, 39, 61). The metabolic actions of insulin that are sensitive to either a dominant negative mutant or pharmacological inhibitors of PI 3-kinase include stimulation of glucose uptake, antilipolysis, activation of fatty acid synthase and glycogen synthase, and stimulation of amino acid transport and protein synthesis (6, 16, 34, 37, 47, 48, 54, 55). Moreover, regulation of the amounts of specific protein participants in metabolic pathways by insulin is mediated by this lipid kinase (52). Although these observations indicate that PI 3-kinase is a major regulator of the metabolic effects of insulin, the roles of the various downstream effectors of PI 3-kinase in each of these actions remain unclear.
The kinase activity of Akt fused with a viral Gag protein or tagged with a myristoylation signal sequence is higher than that of wild-type Akt (Akt-WT) (4, 26). Overexpression of these mutant Akt proteins induced activation of p70 S6 kinase (4, 26), which is also activated by insulin in a wortmannin-sensitive manner (42, 43). Expression of these active Akt mutants also promoted glucose uptake and translocation of GLUT4 glucose transporters in quiescent adipocytes (27, 53). These observations have suggested that Akt is a downstream effector of PI 3-kinase that mediates insulin-induced activation of p70 S6 kinase and glucose uptake. This proposal could be tested further by investigating the effects of specific inhibition of Akt activity on these actions of insulin. However, a mutant Akt that exerts dominant negative effects has not previously been described.
The mechanism by which Akt is activated in response to growth factor stimulation is not fully understood. PI 3,4-bisphosphate, one of the products of PI 3-kinase action, stimulates Akt activity in vitro (15, 24). Furthermore, Akt mutants with substitutions in or lacking the PH domain were not activated by this phospholipid (15, 24), suggesting that Akt is activated as a result of direct interaction of its PH domain with the lipid. On the other hand, other studies have suggested the importance of phosphorylation of Akt on serine and threonine residues in regulation of its activity. Akt is phosphorylated in vivo in response to various growth factors that stimulate Akt activity (4, 14, 26), and dephosphorylation of in vivo-activated Akt by a serine-threonine phosphatase abolished its enzymatic activity (26). Akt is phosphorylated on Thr308 and Ser473 in response to insulin in vivo, and Akt mutants in which either Thr308 or Ser473 was substituted were not activated (1). Moreover, a protein kinase which phosphorylates and activates Akt has been cloned and characterized (2, 44, 50, 51). These data have suggested that Akt is primarily activated as a result of its phosphorylation on serine and threonine residues by an upstream kinase.
We have now shown that when overexpressed in CHO cells or 3T3-L1 adipocytes, a mutant Akt in which growth factor-targeted serine and threonine phosphorylation sites are replaced with alanine exerted a dominant negative effect on endogenous Akt activity stimulated by insulin. With the use of this dominant negative Akt, we have investigated the roles of Akt in insulin-stimulated protein synthesis, p70 S6 kinase activation, and glucose transport in these cells.
Journal Article•
TL;DR: PTEN/MMAC1 is a tumor suppressor gene that is mutated in a variety of cancers and appears to suppress tumor growth by regulating phosphatidylinositol 3'-kinase signaling.
Abstract: PTEN/MMAC1 is a tumor suppressor gene that is mutated in a variety of cancers PTEN encodes a phosphatase that recognizes phosphoprotein substrates and the phospholipid, phosphatidylinositol-3,4,5-triphosphate PTEN inhibited cell growth and/or colony formation in all of the epithelial lines tested with one exception The decrease in cellular proliferation was associated with an induction of apoptosis and an inhibition of signaling through the phosphatidylinositol 3′-kinase pathway Akt/protein kinase B , a gene whose antiapoptotic function is regulated by phosphatidylinositol-3,4,5-triphosphate, was able to rescue cells from PTEN-dependent death PTEN, therefore, appears to suppress tumor growth by regulating phosphatidylinositol 3′-kinase signaling
TL;DR: It is shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and it is provided evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway.
Abstract: The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras–mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.
TL;DR: It is shown that the TOR signalling pathway phosphorylates the Ser/Thr kinase NPR1 and thereby inhibits the starvation‐induced turnover of TAT2, which controls growth by inhibiting a stationary phase (G0) programme.
Abstract: The Saccharomyces cerevisiae targets of rapamycin, TOR1 and TOR2, signal activation of cell growth in response to nutrient availability. Loss of TOR or rapamycin treatment causes yeast cells to arrest growth in early G1 and to express several other physiological properties of starved (G0) cells. As part of this starvation response, high affinity amino acid permeases such as the tryptophan permease TAT2 are targeted to the vacuole and degraded. Here we show that the TOR signalling pathway phosphorylates the Ser/Thr kinase NPR1 and thereby inhibits the starvation-induced turnover of TAT2. Overexpression of NPR1 inhibits growth and induces the degradation of TAT2, whereas loss of NPR1 confers resistance to rapamycin and to FK506, an inhibitor of amino acid import. NPR1 is controlled by TOR and the type 2A phosphatase-associated protein TAP42. First, overexpression of NPR1 is toxic only when TOR function is reduced. Secondly, NPR1 is rapidly dephosphorylated in the absence of TOR. Thirdly, NPR1 dephosphorylation does not occur in a rapamycin-resistant tap42 mutant. Thus, the TOR nutrient signalling pathway also controls growth by inhibiting a stationary phase (G0) programme. The control of NPR1 by TOR is analogous to the control of p70 s6 kinase and 4E-BP1 by mTOR in mammalian cells.
TL;DR: A transdominant negative form of Akt interferes with oncogenic transformation induced by the p3k oncogene, which codes for an activated form of PI 3-kinase, and is an essential mediator of p3K-induced oncogenicity.
Abstract: The serine-threonine kinase Akt is a downstream target of phosphoinositide 3-kinase (PI 3-kinase); it is activated by the phosphoinositide 3-phosphate-dependent kinases PDK1 and PDK2. Certain mutated forms of Akt induce oncogenic transformation in chicken embryo fibroblast cultures and hemangiosarcomas in young chickens. This ability to transform cells depends on localization of Akt at the plasma membrane and on the kinase activity of Akt. A transdominant negative form of Akt interferes with oncogenic transformation induced by the p3k oncogene, which codes for an activated form of PI 3-kinase. Akt is therefore an essential mediator of p3k-induced oncogenicity.
TL;DR: These findings demonstrate the existence of a new survival signaling pathway independent of PI3 kinase, Akt, and new transcription and which is evident in fibroblasts overexpressing the IGF-I receptor.
Abstract: Protein kinase B (PKB)/Akt is implicated in survival signaling in a wide variety of cells including fibroblasts and epithelial and neuronal cells. We and others have described a linear survival signaling cascade used by insulinlike growth factor I (IGF-I) that consists of the IGF-I receptor, phosphoinositide 3-kinase (PI3 kinase), Akt, and Bad. Activation of this pathway can be sufficient to protect cells from apoptosis. However, previous work had not determined whether this pathway is invariably necessary for protection from apoptosis or whether there are alternative survival signaling pathways. In this communication, we report the existence of two survival signaling pathways, one dependent on PI3 kinase and Akt and the other independent of these enzymes. We found that survival signaling initiated by IGF-I treatment of Rat-1 cells could be blocked by overexpression of a dominant negative kinase-deficient Akt (K179A) as well as by wortmannin. This demonstrates a survival signaling pathway dependent on PI3 kinase and Akt. However, when IGF-I receptors were overexpressed in a Rat-1 background (RIG cells), an alternative pathway became apparent, in which survival mediated by IGF-I was no longer sensitive to wortmannin or to overexpression of dominant negative Akt, even though Akt activation and Bad phosphorylation were still wortmannin sensitive. Experiments with inhibitors of RNA synthesis showed that transcriptional activation is dispensable for this alternative PI3 kinase/Akt-independent survival signaling. These findings demonstrate the existence of a new survival signaling pathway independent of PI3 kinase, Akt, and new transcription and which is evident in fibroblasts overexpressing the IGF-I receptor.
TL;DR: It is demonstrated that inhibition of Akt activity is mediated by the immune cell tyrosine-based inhibitory motif within FcγRIIB1 as well as SHIP, which suggests that PIP3 plays a greater role in Akt activation than PI 3,4-P2 in vivo.
TL;DR: It is shown that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation, and that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or BCl-xL or inhibition of Jun kinase activity.
Abstract: Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 Tts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-xL or inhibition of Jun kinase activity.
TL;DR: The PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-β-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.
Abstract: Insulin and insulin receptor substrate 1 (IRS-1) are capable of protecting liver cells from apoptosis induced by transforming growth factor-beta1 (TGF-beta). The Ras/mitogen-activated protein kinase (MAP kinase) and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways are both activated upon insulin stimulation and can protect against apoptosis under certain circumstances. We investigated which of these pathways is responsible for the protective effect of insulin on TGF-beta-induced apoptosis. An activated Ras, although elicited a strong mitogenic effect, could not protect Hep3B cells from TGF-beta-induced apoptosis. Furthermore, PD98059, a selective inhibitor of MEK, did not suppress the antiapoptotic effect of insulin. In contrast, the PI 3-kinase inhibitor, LY294002, efficiently blocked the effect of insulin. Protection against TGF-beta-induced apoptosis conferred by PI 3-kinase was further verified by stable transfection of an activated PI 3-kinase. Downstream targets of PI 3-kinase involved in this protection was further investigated. An activated Akt mimicked the antiapoptotic effect of insulin, whereas a dominant-negative Akt inhibited such effect. However, rapamycin, the p70S6 kinase inhibitor, had no effect on the protectivity of insulin against TGF-beta-induced apoptosis, suggesting that the antiapoptotic target of PI 3-kinase/Akt pathway is independent or lies upstream of the p70S6 kinase. The mechanism by which PI 3-kinase/Akt pathway interferes with the apoptotic signaling of TGF-beta was explored. Activation of PI 3-kinase did not lead to a suppression of Smad hetero-oligomerization or nuclear translocation but blocked TGF-beta-induced caspase-3-like activity. In summary, the PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-beta-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.
TL;DR: The rapamycin target protein functions as a protein kinase in a signal transduction pathway that regulates the synthesis of proteins required for cell-cycle progression in both lymphoid and nonlymphoid cells.
TL;DR: It is demonstrated that nitrosative and oxidative stressors trigger Ras-dependent and PI3K-regulated events in cells and define a biochemical pathway that is triggered by redox signaling.
TL;DR: It is shown that some cell lines derived from childhood tumors are highly sensitive to growth inhibition by rapamycin, whereas others have high intrinsic resistance (>1000-fold).
Abstract: Rapamycin is a potent cytostatic agent that arrests cells in the G1 phase of the cell cycle. The relationships between cellular sensitivity to rapamycin, drug accumulation, expression of mammalian target of rapamycin (mTOR), and inhibition of growth factor activation of ribosomal p70S6 kinase (p70(S6k)) and dephosphorylation of pH acid stable protein I (eukaryotic initiation factor 4E binding protein) were examined. We show that some cell lines derived from childhood tumors are highly sensitive to growth inhibition by rapamycin, whereas others have high intrinsic resistance (>1000-fold). Accumulation and retention of [14C]rapamycin were similar in sensitive and resistant cells, with all cells examined demonstrating a stable tight binding component. Western analysis showed levels of mTOR were similar in each cell line ( /=10-fold increased c-MYC compared with Rh30. These results suggest that the ability of rapamycin to inhibit c-MYC induction correlates with intrinsic sensitivity, whereas failure of rapamycin to inhibit induction or overexpression of c-MYC correlates with intrinsic and acquired resistance, respectively.
TL;DR: It is shown that NAC activates the Ras-extracellular signal-regulated kinase (ERK) pathway in PC12 cells, highlighting the role of Ras-ERK activation in the mechanism by which NAC prevents neuronal death after loss of trophic support.
Abstract: We have shown that N-acetylcysteine (NAC) promotes survival of sympathetic neurons and pheochromocytoma (PC12) cells in the absence of trophic factors. This action of NAC was not related to its antioxidant properties or ability to increase intracellular glutathione levels but was instead dependent on ongoing transcription and seemed attributable to the action of NAC as a reducing agent. Here, we investigate the mechanism by which NAC promotes neuronal survival. We show that NAC activates the Ras-extracellular signal-regulated kinase (ERK) pathway in PC12 cells. Ras activation by NAC seems necessary for survival in that it is unable to sustain serum-deprived PC12 MM17-26 cells constitutively expressing a dominant-negative form of Ras. Promotion of PC12 cell survival by NAC is totally blocked by PD98059, an inhibitor of the ERK-activating MAP kinase/ERK kinase, suggesting a required role for ERK activation in the NAC mechanism. In contrast, LY294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI3K) that partially block NGF-promoted PC12 cell survival, have no effect on prevention of death by NAC. We hypothesized previously that the ability of NAC to promote survival correlates with its antiproliferative properties. However, although NAC does not protect PC12 MM17-26 cells from loss of trophic support, it does inhibit their capacity to synthesize DNA. Thus, the antiproliferative effect of NAC does not require Ras activation, and inhibition of DNA synthesis is insufficient to mediate NAC-promoted survival. These findings highlight the role of Ras-ERK activation in the mechanism by which NAC prevents neuronal death after loss of trophic support.
TL;DR: It is shown that overexpression of EGFR and c‐erbB2 in cell lines increases cell migration, an important step in metastasis formation, and investigates the involvement of a number of signal transduction pathways known to be activated by the EGFR.
TL;DR: Both this novel pathway and the activation of PKB/Akt are inhibited by the PI3K inhibitor, wortmannin, and the calpain inhibitor, calpeptin, constituting the first evidence that PtdIns(3,4)P2 can stimulate in vitro the activity of the proto-oncogenic protein kinase PKB /Akt in vivo in the absence of PTDIns( 3,4,5)P3.
TL;DR: The role of calcium in the regulation of each kinase in Balb/c-3T3 fibroblasts and the relationship between signaling through these two kinases is examined to conclude Akt and p70S6k lie on separate signaling pathways.
TL;DR: Both PGF2α and 8,12-iso-iPF2α-III induce myocyte hypertrophy via discrete signaling pathways through JNK1 and c-Jun pathways and the hypertrophic effects of these two agonists on cardiomyocytes are additive.
TL;DR: This study is the first report to reveal a potential regulatory role of alpha4 and Tap42 to inibit the phosphatase activity of PP2A-C toward the physiologically relevant substrate in the mTOR signaling.
TL;DR: It is demonstrated that Akt is in the pathway controlling resumption of meiosis in the Xenopus oocyte and that regulation of the activity of a PDE3 is a step distal to the kinase activation.
TL;DR: The interpretation that increasing cAMP attenuates the effects of insulin on PHAS-I, p70S6K, and other downstream targets of the mTOR signaling pathway by inhibiting the phosphorylation and activation of mTOR is supported.