mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E

The mammalian target of rapamycin (mTOR) integrates nutrient and mitogen signals to regulate cell growth (increased cell mass and cell size) and cell division. The immunosuppressive drug rapamycin inhibits cell cycle progression via inhibition of mTOR; however, the signaling pathways by which mTOR regulates cell cycle progression have remained poorly defined. Here we demonstrate that restoration of mTOR signaling (by using a rapamycin-resistant mutant of mTOR) rescues rapamycin-inhibited G(1)-phase progression, and restoration of signaling along the mTOR-dependent S6K1 or 4E-BP1/eukaryotic translation initiation factor 4E (eIF4E) pathways provides partial rescue. Furthermore, interfering RNA-mediated reduction of S6K1 expression or overexpression of mTOR-insensitive 4E-BP1 isoforms that block eIF4E activity inhibit G(1)-phase progression individually and additively. Thus, the activities of both the S6K1 and 4E-BP1/eIF4E pathways are required for and independently mediate mTOR-dependent G(1)-phase progression. In addition, overexpression of constitutively active mutants of S6K1 or wild-type eIF4E accelerates serum-stimulated G(1)-phase progression, and stable expression of wild-type S6K1 confers a proliferative advantage in low-serum-containing media, suggesting that the activity of each of these pathways is limiting for cell proliferation. These data demonstrate that, as for the regulation of cell growth and cell size, the S6K1 and 4E-BP1/eIF4E pathways each represent critical mediators of mTOR-dependent cell cycle control.     

PLD2 forms a functional complex with mTOR/raptor to transduce mitogenic signals

Mammalian target-of-rapamycin (mTOR), which is a master controller of cell growth, senses a mitogenic signal in part through the lipid second messenger phosphatidic acid (PA), generated by phospholipase D (PLD). To understand further which isozymes of PLD are involved in this process, we compared the effect of PLD isozymes on mTOR activation. We found that PLD2 has an essential role in mitogen-induced mTOR activation as the siRNA-mediated knockdown of PLD2, not of PLD1, profoundly reduced the phosphorylations of S6K1 and 4EBP1, well-known mTOR effectors. Furthermore, exogenous PA-induced mTOR activation was abrogated by PLD2 knockdown, but not by PLD1 knockdown. This abrogation was found to be the result of complex formation between PLD2 and mTOR/raptor. PLD2 possesses a TOS-like motif (Phe-Glu-Val-Gln-Val, a.a. 265–269), through which it interacts with raptor independently of the other TOS motif-containing proteins, S6K1 and 4EBP1. PLD2-dependent mTOR activation appears to require PLD2 binding to mTOR/raptor with lipase activity, since lipase-inactive PLD2 cannot trigger mTOR activation despite its ability to interact with mTOR/raptor. Abrogation of mitogen-dependent mTOR activation by PLD2 knockdown was rescued only by wild type PLD2, but not by raptor binding-deficient and lipase-inactive PLD2. Our results demonstrate the importance of localized PA generation for the mitogen-induced activation of mTOR, which is achieved by a specific interaction between PLD2 and mTOR/raptor.     

Phospholipase D signaling in serotonin-induced mitogenesis of pulmonary artery smooth muscle cells

We have previously reported the participation of mitogen-activated protein, Rho, and phosphoinositide-3 (PI3) kinases in separate pathways in serotonin (5-HT)-induced proliferation of pulmonary artery smooth muscle cells (SMCs). In this study, we investigated the possible participation of phospholipase D (PLD) and phosphatidic acid (PA) in this growth process. 5-HT stimulated a time-dependent increase in [(3)H]phosphatidylbutanol and PA generation. Exposure of SMCs to 1-butanol or overexpression of an inactive mutant of human PLD1R898R blocked 5-HT-induced proliferation. Furthermore, 1-butanol inhibited 5-HT activation of S6K1 and S6 protein, downstream effectors of mammalian target of rapamycin (mTOR), by 80 and 72%, respectively, and partially blocked activation of extracellular signal-regulated kinase (ERK) by 30% but had no effect on other associated signaling pathways. Exogenous PA caused cellular proliferation and revitalized cyclin D1 expression by 5-HT of the 1-butanol-treated cells. PA also reproduced activations by 5-HT of mTOR, S6K1, and ERK. Transfection with inactive human PLD1 reduced 5-HT-induced activation of S6K1 by approximately 50%. Inhibition of 5-HT receptor 2A (R 2A) with ketaserin blocked PLD activation by 5-HT. Inhibition with PI3-kinase inhibitor failed to block either activation of PLD by 5-HT or PA-dependent S6K1 phosphorylation. Taken together, these results indicate that ligation of the 5-HTR 2A by 5-HT initiates PLD activation in SMCs, and that its product, PA, is an early signaling molecule in 5-HT-induced pulmonary artery SMC proliferation. Signaling by PA produces its downstream effects primarily through the mTOR/S6K1 pathway and to a lesser extent through the ERK pathway. Hydrolysis of cell membrane lipid may be important in vascular effects of 5-HT.     

Rapamycin inhibits cytoskeleton reorganization and cell motility by suppressing RhoA expression and activity

The mammalian target of rapamycin (mTOR) functions in cells at least as two complexes, mTORC1 and mTORC2. Intensive studies have focused on the roles of mTOR in the regulation of cell proliferation, growth, and survival. Recently we found that rapamycin inhibits type I insulin-like growth factor (IGF-1)-stimulated lamellipodia formation and cell motility, indicating involvement of mTOR in regulating cell motility. This study was set to further elucidate the underlying mechanism. Here we show that rapamycin inhibited protein synthesis and activities of small GTPases (RhoA, Cdc42, and Rac1), crucial regulatory proteins for cell migration. Disruption of mTORC1 or mTORC2 by down-regulation of raptor or rictor, respectively, inhibited the activities of these proteins. However, only disruption of mTORC1 mimicked the effect of rapamycin, inhibiting their protein expression. Ectopic expression of rapamycin-resistant and constitutively active S6K1 partially prevented rapamycin inhibition of RhoA, Rac1, and Cdc42 expression, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or down-regulation of S6K1 by RNA interference suppressed expression of the GTPases, suggesting that both mTORC1-mediated S6K1 and 4E-BP1 pathways are involved in protein synthesis of the GTPases. Expression of constitutively active RhoA, but not Cdc42 and Rac1, conferred resistance to rapamycin inhibition of IGF-1-stimulated lamellipodia formation and cell migration. The results suggest that rapamycin inhibits cell motility at least in part by down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1 and 4E-BP1-signaling pathways.     

Protein Kinase B Localization and Activation Differentially Affect S6 Kinase 1 Activity and Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 Phosphorylation

Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1) activation is mediated by protein kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. Here we set out to examine the importance of PKB signaling in S6K1 activation. In parallel, glycogen synthase kinase 3beta (GSK-3beta) inactivation and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation were monitored as markers of the rapamycin-insensitive and -sensitive branches of the PI3K signaling pathway, respectively. The results demonstrate that two activated PKBalpha mutants, whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3beta to the same extent as a highly active, constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation and GSK-3beta inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced 4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independently of its intracellular location.     

TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function

BACKGROUND: The mammalian target of rapamycin, mTOR, is a serine/threonine kinase that controls cell growth and proliferation via the translation regulators eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). We recently identified a TOR signaling (TOS) motif in the N terminus of S6K1 and the C terminus of 4E-BP1 and demonstrated that in S6K1, the TOS motif is necessary to facilitate mTOR signaling to phosphorylate and activate S6K1. However, it is unclear how the TOS motif in S6K1 and 4E-BP1 mediates mTOR signaling. RESULTS: Here, we show that a functional TOS motif is required for 4E-BP1 to bind to raptor (a recently identified mTOR-interacting protein), for 4E-BP1 to be efficiently phosphorylated in vitro by the mTOR/raptor complex, and for 4E-BP1 to be phosphorylated in vivo at all identified mTOR-regulated sites. mTOR/raptor-regulated phosphorylation is necessary for 4E-BP's efficient release from the translational initiation factor eIF4E. Consistently, overexpression of a mutant of 4E-BP1 containing a single amino acid change in the TOS motif (F114A) reduces cell size, demonstrating that mTOR-dependent regulation of cell growth by 4E-BP1 is dependent on a functional TOS motif. CONCLUSIONS: Our data demonstrate that the TOS motif functions as a docking site for the mTOR/raptor complex, which is required for multisite phosphorylation of 4E-BP1, eIF4E release from 4E-BP1, and cell growth.     

An upstream regulator and downstream target of phospholipase D1 activity during pheromone response in Saccharomyces cerevisiae

Phospholipase D1 (PLD1), which is the product of the SPO14 gene, has been shown to play a role in the process of polarized cell growth (PCG) during the pheromone response in Saccharomyces cerevisiae. PLD1 hydrolyzes phosphatidylcholine to produce phosphatidic acid (PA) and a free choline headgroup. This study investigated the interactions of PLD1 and PA with two proteins known to be involved in the cellular signaling leading to PCG in yeast, the small GTPase Cdc42p and the PAK family kinase Ste20p. Constitutively activated Cdc42p stimulates PLD1 activity. Protein-lipid binding blots confirmed the specific binding of Ste20p to the PLD1 product, PA. Finally, kinase activity assays provided evidence for the stimulation of Ste20p by PA. These findings highlight the important interactions among PLD1, Cdc42p and Ste20p during PCG in S. cerevisiae.     

S6K1 and 4E-BP1 are independent regulated and control cellular growth in bladder cancer

Aberrant activation and mutation status of proteins in the phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and the mitogen activated protein kinase (MAPK) signaling pathways have been linked to tumorigenesis in various tumors including urothelial carcinoma (UC). However, anti-tumor therapy with small molecule inhibitors against mTOR turned out to be less successful than expected. We characterized the molecular mechanism of this pathway in urothelial carcinoma by interfering with different molecular components using small chemical inhibitors and siRNA technology and analyzed effects on the molecular activation status, cell growth, proliferation and apoptosis. In a majority of tested cell lines constitutive activation of the PI3K was observed. Manipulation of mTOR or Akt expression or activity only regulated phosphorylation of S6K1 but not 4E-BP1. Instead, we provide evidence for an alternative mTOR independent but PI3K dependent regulation of 4E-BP1. Only the simultaneous inhibition of both S6K1 and 4E-BP1 suppressed cell growth efficiently. Crosstalk between PI3K and the MAPK signaling pathway is mediated via PI3K and indirect by S6K1 activity. Inhibition of MEK1/2 results in activation of Akt but not mTOR/S6K1 or 4E-BP1. Our data suggest that 4E-BP1 is a potential new target molecule and stratification marker for anti cancer therapy in UC and support the consideration of a multi-targeting approach against PI3K, mTORC1/2 and MAPK.     

Activation of AMPK and inactivation of Akt result in suppression of mTOR-mediated S6K1 and 4E-BP1 pathways leading to neuronal cell death in in vitro models of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. Dysregulation of mammalian target of rapamycin (mTOR) has been implicated in the pathogenesis of PD. However, the underlying mechanism is incompletely elucidated. Here, we show that PD mimetics (6-hydroxydopamine, N-methyl-4-phenylpyridine or rotenone) suppressed phosphorylation of mTOR, S6K1 and 4E-BP1, reduced cell viability, and activated caspase-3 and PARP in PC12 cells and primary neurons. Overexpression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 in PC12 cells partially prevented cell death in response to the PD toxins, revealing that mTOR-mediated S6K1 and 4E-BP1 pathways due to the PD toxins were inhibited, leading to neuronal cell death. Furthermore, we found that the inhibition of mTOR signaling contributing to neuronal cell death was attributed to suppression of Akt and activation of AMPK. This is supported by the findings that ectopic expression of constitutively active Akt or dominant negative AMPKα, or inhibition of AMPKα with compound C partially attenuated inhibition of phosphorylation of mTOR, S6K1 and 4E-BP1, activation of caspase-3, and neuronal cell death triggered by the PD toxins. The results indicate that PD stresses activate AMPK and inactivate Akt, causing neuronal cell death via inhibiting mTOR-mediated S6K1 and 4E-BP1 pathways. Our findings suggest that proper co-manipulation of AMPK/Akt/mTOR signaling may be a potential strategy for prevention and treatment of PD.     

Ca(2+)- and phospholipase D-dependent and -independent pathways activate mTOR signaling

The mammalian target of rapamycin (mTOR) promotes increased protein synthesis required for cell growth. It has been suggested that phosphatidic acid, produced upon activation of phospholipase D (PLD), is a common mediator of growth factor activation of mTOR signaling. We used Rat-1 fibroblasts expressing the alpha(1A) adrenergic receptor to study if this G(q)-coupled receptor uses PLD to regulate mTOR signaling. Phenylephrine (PE) stimulation of the alpha(1A) adrenergic receptor induced mTOR autophosphorylation at Ser2481 and phosphorylation of two mTOR effectors, 4E-BP1 and p70 S6 kinase. These PE-induced phosphorylations were greatly reduced in cells depleted of intracellular Ca(2+). PE activation of PLD was also inhibited in Ca(2+)-depleted cells. Incubation of cells with 1-butanol to inhibit PLD signaling attenuated PE-induced phosphorylation of mTOR, 4E-BP1 and p70 S6 kinase. By contrast, platelet-derived growth factor (PDGF)-induced phosphorylation of these proteins was not blocked by Ca(2+) depletion or 1-butanol treatment. These results suggest that the alpha(1A) adrenergic receptor promotes mTOR signaling via a pathway that requires an increase in intracellular Ca(2+) and activation of PLD. The PDGF receptor, by contrast, appears to activate mTOR by a distinct pathway that does not require Ca(2+) or PLD.     

Identification of a conserved motif required for mTOR signaling

BACKGROUND: The mammalian target of rapamycin (mTOR) controls the translation machinery via activation of S6 kinases 1 and 2 (S6K1/2) and inhibition of the eukaryotic initiation factor 4E (eIF4E) binding proteins 1, 2, and 3 (4E-BP1/2/3). S6K1 and 4E-BP1 are regulated by nutrient-sensing and mitogen-activated pathways. The molecular basis of mTOR regulation of S6K1 and 4E-BP1 remains controversial. RESULTS: We have identified a conserved TOR signaling (TOS) motif in the N terminus of all known S6 kinases and in the C terminus of the 4E-BPs that is crucial for phosphorylation and regulation S6K1 and 4E-BP1 activities. Deletion or mutations within the TOS motif significantly inhibit S6K1 activation and the phosphorylation of its hydrophobic motif, Thr389. In addition, this sequence is required to suppress an inhibitory activity mediated by the S6K1 C terminus. The TOS motif is essential for S6K1 activation by mTOR, as mutations in this motif mimic the effect of rapamycin on S6K1 phosphorylation, and render S6K1 insensitive to changes in amino acids. Furthermore, only overexpression of S6K1 with an intact TOS motif prevents 4E-BP1 phosphorylation by a common mTOR-regulated modulator of S6K1 and 4E-BP1. CONCLUSIONS: S6K1 and 4E-BP1 contain a conserved five amino acid sequence (TOS motif) that is crucial for their regulation by the mTOR pathway. mTOR seems to regulate S6K1 by two distinct mechanisms. The TOS motif appears to function as a docking site for either mTOR itself or a common upstream activator of S6K1 and 4E-BP1.     

Tuberous sclerosis complex gene products,Tuberin and Hamartin,control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb

BACKGROUND: Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear. RESULTS: We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. CONCLUSIONS: We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells.     

Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin on initiation factor 4E-binding proteins

Signaling through the mammalian target of rapamycin (mTOR) controls cell size and growth as well as other functions, and it is a potential therapeutic target for graft rejection, certain cancers, and disorders characterized by inappropriate cell or tissue growth. mTOR signaling is positively regulated by hormones or growth factors and amino acids. mTOR signaling regulates the phosphorylation of several proteins, the best characterized being ones that control mRNA translation. Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) undergoes phosphorylation at multiple sites. Here we show that amino acids regulate the N-terminal phosphorylation sites in 4E-BP1 through the RAIP motif in a rapamycin-insensitive manner. Several criteria indicate this reflects a rapamycin-insensitive output from mTOR. In contrast, the insulin-stimulated phosphorylation of the C-terminal site Ser64/65 is generally sensitive to rapamycin, as is phosphorylation of another well-characterized target for mTOR signaling, S6K1. Our data imply that it is unlikely that mTOR directly phosphorylates Thr69/70 in 4E-BP1. Although 4E-BP1 and S6K1 bind the mTOR partner, raptor, our data indicate that the outputs from mTOR to 4E-BP1 and S6K1 are distinct. In cells, efficient phosphorylation of 4E-BP1 requires it to be able to bind to eIF4E, whereas phosphorylation of 4E-BP1 by mTOR in vitro shows no such preference. These data have important implications for understanding signaling downstream of mTOR and the development of new strategies to impair mTOR signaling.     

Arf6 promotes cell proliferation via the PLD-mTORC1 and p38MAPK pathways

The small G-protein ADP-ribosylation factor 6 (Arf6) belongs to the Ras GTPases superfamily and is mostly known for its actin remodeling functions and involvement in the processes of plasma membrane reorganization and vesicular transport. The majority of data indicates that Arf6 contributes to cancer progression through activation of cell motility and invasion. Alternatively, we found that the expression of a wild-type or a constitutively active Arf6 does not influence tumor cell motility and invasion but instead significantly stimulates cell proliferation and activates phospholipase D (PLD). Conversely the expression of a mutant Arf6 (Arf6N48I), that is, unable to interact with PLD has no effect on proliferation but promotes motility, invasion, and matrix degradation by uPA extracellular proteinase. Studying the mechanisms of Arf6-dependent stimulation of cell proliferation, we found some signaling pathways contributing to Arf6 promitogenic activity. Namely, we showed that Arf6 in a PLD-mTORC1-dependent manner activates S6K1 kinase, a well-known regulator of mitogen-stimulated translation initiation. Furthermore, we demonstrated an Arf6-dependent phosphorylation of mTORC1 downstream targets, 4E-BP1 and ribosomal S6 protein, confirming an existence of Arf6-PLD-mTORC1-S6K1/4E-BP1 signaling pathway and also demonstrated its impact on proliferation stimulation. Next, we found that Arf6 activation potentiates Erk1/2 and p38MAP kinases phosphorylation. Surprisingly, p38 opposite to Erk1/2 significantly contributes to Arf6-dependent proliferation increase promoting S6 ribosomal protein phosphorylation at Ser235/236 residues. Therefore, we demonstrated Arf6 proliferation stimulating activity and revealed PLD-mTORC1 and p38MAP kinase as Arf6 partners mediating promitogenic activity. These results highlight a new aspect of Arf6 functioning in cancer cell biology.     

Activation of phospholipase D1 by Cdc42 requires the Rho insert region

Members of the Rho subfamily of GTP-binding proteins are implicated in the regulation of phospholipase D (PLD). In the present study, we demonstrate a physical association between a Rho family member, Cdc42, and PLD1. Binding of Cdc42 to PLD1 and subsequent activation are GTP-dependent. Although binding of Cdc42 to PLD1 does not require geranylgeranylation, activation of PLD1 is dependent on this lipid modification of Cdc42. Specific point mutations in the switch I region of Cdc42 abolish binding to and, therefore, activation of PLD1 by Cdc42. Deletion of the Rho insert region, which consists of residues 120-139, from Cdc42 does not interfere with binding to PLD1 but inhibits Cdc42 stimulated PLD1 activity. Interestingly, deletion of the insert region from Cdc42 also inhibits activation of PLD1 by Arf and protein kinase C. With the lack of specific inhibitors of PLD activity, the insert deletion mutant of Cdc42 (designated (DeltaL8)Cdc42) is a novel reagent for in vitro studies of PLD1 regulation, as well as for in vivo studies of Cdc42-mediated signaling pathways leading to PLD1 activation. Because the insert region is required for the transforming activity of Cdc42, regulation of PLD1 by this region on Cdc42 is of major interest.     

Mammalian target of rapamycin-independent S6K1 and 4E-BP1 phosphorylation during contraction in rat skeletal muscle

Muscle protein synthesis rates decrease during contraction/exercise, but rapidly increase post-exercise. Previous studies mainly focused on signaling pathways that control protein synthesis during post-exercise recovery, such as mTOR and its downstream targets S6K1 and 4E-BP1. In this study, we investigated the effect of high-frequency electrical stimulation on the phosphorylation state of signaling components controlling protein synthesis in rat skeletal muscle. Electrical stimulation increased S6K1 Thr389 phosphorylation, which was unaffected by Torin1, a selective mTOR inhibitor, suggesting that S6K1 phosphorylation by contraction was mTOR-independent. Phosphorylation of eIF4B Ser422 was also increased during electrical stimulation, which was abrogated by inhibition of MEK/ERK/RSK1 activation. Moreover, although phosphorylation of conventional mTOR sites in 4E-BP1 decreased during contraction, mTOR-independent phosphorylation was also apparent, which was associated with the release of 4E-BP1 from eIF4E. The results indicate mTOR-independent phosphorylation of S6K1 and 4E-BP1 and suggest MEK/ERK/RSK1-dependent phosphorylation of eIF4B during skeletal muscle contraction. These phosphorylation events would keep the translation initiation machinery “primed” in an active state so that protein synthesis could quickly resume post-exercise.     

Mammalian Target of Rapamycin (mTOR) and S6 Kinase Down-regulate Phospholipase D2 Basal Expression and Function

The mammalian target of rapamycin (mTOR) and S6 kinase (S6K) pathway is essential for cell differentiation, growth, and survival. Phospholipase D2 (PLD2) plays a key role in mTOR/S6K mitogenic signaling. However, the impact of PLD on mTOR/S6K gene expression is not known. Here we show that interleukin-8 (IL-8) increases mRNA expression levels for PLD2, mTOR, and S6K, with PLD2 preceding mTOR/S6K in time. Silencing of PLD2 gene expression abrogated IL-8-induced mTOR/S6K mRNA expression, whereas silencing of mTOR or S6K gene expression resulted in large (>3-fold and >5-fold, respectively) increased levels of PLD2 RNA, which was paralleled by increases in protein expression and lipase activity. Treatment of cells with 0.5 nm rapamycin induced a similar trend. These results suggest that, under basal conditions, PLD2 expression and concomitant activity is negatively regulated by the mTOR/S6K signaling pathway. Down-regulation of PLD2 was confirmed in differentiated HL-60 leukocytes overexpressing an mTOR-wild type, but not an mTOR kinase-dead construct. At the cellular level, overexpression of mTOR-wild type resulted in lower basal cell migration, which was reversed by treatment with IL-8. We propose that IL-8 reverses an mTOR/S6K-led down-regulation of PLD2 expression and enables PLD2 to fully function as a facilitator for cell migration.     

Clofibrate treatment promotes branched-chain amino acid catabolism and decreases the phosphorylation state of mTOR, eIF4E-BP1, and S6K1 in rat liver

Leucine stimulates protein synthesis by modulating the mammalian target of rapamycin (mTOR) signaling pathway. We hypothesized that promotion of the branched-chain amino acid (BCAA) catabolism might influence the leucine-induced protein synthesis. Clofibric acid (an active metabolite of clofibrate) is known to promote the BCAA catabolism by activation of branched-chain alpha-keto acid dehydrogenase complex (BCKDC), the rate-limiting enzyme of the BCAA catabolism. In the present study, we examined the phosphorylation state of mTOR, eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), and ribosomal protein S6 kinase 1 (S6K1) in liver of rats with or without activation of the BCKDC by clofibrate treatment. Clofibrate-treated rats were prepared by oral administration of clofibrate 5 h before sacrifice. In order to stimulate phosphorylation of components in the mTOR signaling pathway, rats were orally administered with leucine 1 h before sacrifice. Clofibrate treatment almost fully activated hepatic BCKDC and significantly decreased the plasma leucine concentration in rats without leucine administration, resulting in decreased mTOR and 4E-BP1 phosphorylation. Similarly, in rats administered with leucine, clofibrate treatment attenuated the predicted increase in plasma leucine concentration as well as the phosphorylation of mTOR, 4E-BP1, and S6K1. These results suggest that BCAA catabolism enhanced by clofibrate treatment has significant influences on the leucine-induced activation of translation initiation processes.     

Modulation of the mammalian target of rapamycin pathway by diacylglycerol kinase-produced phosphatidic acid

The protein known as mammalian target of rapamycin (mTOR) regulates cell growth by integrating different stimuli, such as available nutrients and mitogenic factors. The lipid messenger phosphatidic acid (PA) binds and positively regulates the mitogenic response of mTOR. PA generator enzymes are consequently potential regulators of mTOR. Here we explored the contribution to this pathway of the enzyme diacylglycerol kinase (DGK), which produces PA through phosphorylation of diacylglycerol. We found that overexpression of the DGKzeta, but not of the alpha isoform, in serum-deprived HEK293 cells induced mTOR-dependent phosphorylation of p70S6 kinase (p70S6K). After serum addition, p70S6K phosphorylation was higher and more resistant to rapamycin treatment in cells overexpressing DGKzeta. The effect of this DGK isoform on p70S6K hyperphosphorylation required the mTOR PA binding region. Down-regulation of endogenous DGKzeta by small interfering RNA in HEK293 cells diminished serum-induced p70S6K phosphorylation, highlighting the role of this isoform in the mTOR pathway. Our results confirm a role for PA in mTOR regulation and describe a novel pathway in which DGKzeta-derived PA acts as a mediator of mTOR signaling.     

Strain-induced vascular endothelial cell proliferation requires PI3K-dependent mTOR-4E-BP1 signal pathway

The aim of this study was to determine whether the phosphatidylinositol 3-kinase (PI3K)-dependent mammalian target of rapamycin (mTOR)-eukaryotic initiation factor 4E binding protein 1 (4E-BP1) signal pathway and S6 kinase (S6K), the major element of the mTOR pathway, play a role in the enhanced vascular endothelial cell (EC) proliferation induced by cyclic strain. Bovine aortic ECs were subjected to an average of 10% strain at a rate of 60 cycles/min for < or =24 h. Cyclic strain-induced EC proliferation was reduced by pretreatment with rapamycin but not the MEK1 inhibitor PD-98059. The PI3K inhibitors wortmannin and LY-294002 also attenuated strain-induced EC proliferation and strain-induced activation of S6K. Rapamycin but not PD-98059 prevented strain-induced S6K activation, and PD-98059 but not rapamycin prevented strain-induced activation of extracellular signal-regulated kinases 1 and 2. Cyclic strain also activated 4E-BP1, which could be inhibited by PI3K inhibitors. These data suggest that the PI3K-dependent S6K-mTOR-4E-BP1 signal pathway may be critically involved in strain-induced bovine aortic EC proliferation.