Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70S6 kinase

The mammalian target of rapamycin (mTOR) coordinates cell growth with the growth factor and nutrient/energy status of the cell. The phosphatidylinositol 3-kinase-AKT pathway is centrally involved in the transmission of mitogenic signals to mTOR. Previous studies have shown that mTOR is a direct substrate for the AKT kinase and identified Ser-2448 as the AKT target site in mTOR. In this study, we demonstrate that rapamycin, a specific inhibitor of mTOR function, blocks serum-stimulated Ser-2448 phosphorylation and that this drug effect is not explained by the inhibition of AKT. Furthermore, the phosphorylation of Ser-2448 was dependent on mTOR kinase activity, suggesting that mTOR itself or a protein kinase downstream from mTOR was responsible for the modification of Ser-2448. Here we show that p70S6 kinase phosphorylates mTOR at Ser-2448 in vitro and that ectopic expression of rapamycin-resistant p70S6 kinase restores Ser-2448 phosphorylation in rapamycin-treated cells. In addition, we show that cellular amino acid status, which modulates p70S6 kinase (S6K1) activity via the TSC/Rheb pathway, regulates Ser-2448 phosphorylation. Finally, small interfering RNA-mediated depletion of p70S6 kinase reduces Ser-2448 phosphorylation in cells. Taken together, these results suggest that p70S6 kinase is a major effector of mTOR phosphorylation at Ser-2448 in response to both mitogen- and nutrient-derived stimuli.     

Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase

Elongation factor 2 kinase (eEF2k) phosphorylates and inactivates eEF2. Insulin induces dephosphorylation of eEF2 and inactivation of eEF2 kinase, and these effects are blocked by rapamycin, which inhibits the mammalian target of rapamycin, mTOR. However, the signalling mechanisms underlying these effects are unknown. Regulation of eEF2 phosphorylation and eEF2k activity is lost in cells in which phosphoinositide-dependent kinase 1 (PDK1) has been genetically knocked out. This is not due to loss of mTOR function since phosphorylation of another target of mTOR, initiation factor 4E-binding protein 1, is not defective. PDK1 is required for activation of members of the AGC kinase family; we show that two such kinases, p70 S6 kinase (regulated via mTOR) and p90(RSK1) (activated by Erk), phosphorylate eEF2k at a conserved serine and inhibit its activity. In response to insulin-like growth factor 1, which activates p70 S6 kinase but not Erk, regulation of eEF2 is blocked by rapamycin. In contrast, regulation of eEF2 by stimuli that activate Erk is insensitive to rapamycin, but blocked by inhibitors of MEK/Erk signalling, consistent with the involvement of p90(RSK1).     

Deoxyribose protects against rapamycin-induced cytotoxicity in colorectal cancer cells in vitro

Thymidine phosphorylase (TPase) is also known as the platelet-derived endothelial cell growth factor (PD-ECGF) and plays a role in angiogenesis. Deoxyribose (dR; a downstream TPase-product) addition to endothelial cells may stimulate FAK and p70/S6k signaling, which can be inhibited by rapamycin. Rapamycin is a specific mammalian target of the rapamycin (mTOR) inhibitor, a kinase that lies directly upstream of p70/S6k. This suggests a role for TPase in the mTOR/p70/S6k pathway. In order to study this in more detail, we exposed cells with and without TPase expression to dR and rapamycin and determined the effect on cell growth. We observed protection in cytotoxicity in Colo320 cells, but not Colo320 TP1 cells. This was in part mediated by activation of p70/S6k and inhibition of autophagy. Further studies are recommended to elucidate the mechanism behind the protective effect of dR.     

Coupling of Serotonin 5-HT1B Receptors to Activation of Mitogen-Activated Protein Kinase (ERK-2) and p70 S6 Kinase Signaling Systems

Abstract: Little is known about the coupling of serotonin 5-HT_1B receptors to cellular signals other than cyclic AMP. In the present studies, the activation by 5-HT_1B receptors of p70 S6 kinase and the mitogen-activated protein kinase (MAP kinase) ERK-2 was investigated. Studies were performed by using both nontransfected Chinese hamster ovary (CHO) cells, which express endogenous receptors at a very low density, and a stable transfected CHO cell line expressing 5-HT_1B receptors at 230 fmol/mg of membrane protein, a density similar to that expressed in cortex. In nontransfected cells, 5-HT was found to stimulate a greater than twofold increase in MAP kinase activity with an EC₅₀ of 20 n M . Reflecting increased density of receptors, 5-HT caused a greater than eightfold activation of ERK-2 in transfected cells with an EC₅₀ of 2 n M . 5-HT was found to also stimulate p70 S6 kinase in both nontransfected and transfected cells. The stimulation was sixfold in both types of cells, but the EC₅₀ for 5-HT was fourfold lower in transfected cells. The coupling of 5-HT_1B receptors to ERK-2 and to p70 S6 kinase was inhibited by pertussis toxin, inhibitors of phosphatidylinositol 3-kinase, and by the inhibitor of MAP kinase kinase PD098059. Activation of p70 S6 kinase, but not ERK-2, was also inhibited by rapamycin. These findings demonstrate that 5-HT_1B receptors couple to ERK-2 and p70 S6 kinase through overlapping, but nonidentical, pathways.     

A downstream kinase of the mammalian target of rapamycin, p70S6K1, regulates human double minute 2 protein phosphorylation and stability

Human double minute 2 (HDM2) is an oncoprotein overexpressed in many human cancers. HDM2 expression is regulated at multiple levels in cells. Phosphorylation of HDM2 plays an important role in its post-translational regulation. In this study, we have shown that the phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, have similar effects on the inhibition of HDM2 phosphorylation and protein turnover. Rapamycin inhibited p70S6K1, but not AKT activation, indicating that rapamycin affects HDM2 phosphorylation via an AKT-independent mechanism. Rapamycin also decreased HDM2 protein stability. Knockdown of p70S6K1 by a p70S6K1 siRNA resulted in the inhibition of HDM2 phosphorylation and a decrease in HDM2 protein turnover. Overexpression of p70S6K1 enhanced HDM2 phosphorylation and led to an increase in HDM2 protein turnover. Our results suggest that p70S6K1 regulates turnover of HDM2 protein for cancer development.     

Role of mTOR signaling in intestinal cell migration

An early signaling event activated by amino acids and growth factors in many cell types is the phosphorylation of the mammalian target of rapamycin (mTOR; FRAP), which is functionally linked to ribosomal protein s6 kinase (p70(s6k)), a kinase that plays a critical regulatory role in the translation of mRNAs and protein synthesis. We previously showed that intestinal cell migration, the initial event in epithelial restitution, is enhanced by l-arginine (ARG). In this study, we used amino acids as prototypic activators of mTOR and ARG, IGF-1, or serum as recognized stimulators of intestinal cell migration. We found that 1) protein synthesis is required for intestinal cell migration, 2) mTOR/p70(s6k) pathway inhibitors (rapamycin, wortmannin, and intracellular Ca(2+) chelation) inhibit cell migration, 3) ARG activates migration and mTOR/p70(s6k) (but not ERK-2) in migrating enterocytes, and 4) immunocytochemistry reveals abundant p70(s6k) staining in cytoplasm, whereas phospho-p70(s6k) is virtually all intranuclear in resting cells but redistributes to the periphery on activation by ARG. We conclude that mTOR/p70(s6k) signaling is essential to intestinal cell migration, is activated by ARG, involves both nuclear and cytoplasmic events, and may play a role in intestinal repair.     

mTORC1 inhibition increases neurotensin secretion and gene expression through activation of the MEK/ERK/c-Jun pathway in the human endocrine cell line BON

The mammalian target of rapamycin (mTOR) signaling exists in two complexes: mTORC1 and mTORC2. Neurotensin (NT), an intestinal hormone secreted by enteroendocrine (N) cells in the small bowel, has important physiological effects in the gastrointestinal tract. The human endocrine cell line BON abundantly expresses the NT gene and synthesizes and secretes NT in a manner analogous to that of N cells. Here, we demonstrate that the inhibition of mTORC1 by rapamycin (mTORC1 inhibitor), torin1 (both mTORC1 and mTORC2 inhibitor) or short hairpin RNA-mediated knockdown of mTOR, regulatory associated protein of mTOR (RAPTOR), and p70 S6 kinase (p70S6K) increased basal NT release via upregulating NT gene expression in BON cells. c-Jun activity was increased by rapamycin or torin1 or p70S6K knockdown. c-Jun overexpression dramatically increased NT promoter activity, which was blocked by PD98059, an mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, overexpression of MEK1 or extracellular signal-regulated kinase 1 (ERK1) increased c-Jun expression and NT promoter activity. More importantly, PD98059 blocked rapamycin- or torin1-enhanced NT secretion. Consistently, rapamycin and torin1 also increased NT gene expression in Hep3B cells, a human hepatoma cell line that, similar to BON, expresses high levels of NT. Phosphorylation of c-Jun and ERK1/2 was also increased by rapamycin and torin1 in Hep3B cells. Finally, we showed activation of mTOR in BON cells treated with amino acids, high glucose, or serum and, concurrently, the attenuation of ERK1/2 and c-Jun phosphorylation and NT secretion. Together, mTORC1, as a nutrient sensor, negatively regulates NT secretion via the MEK/ERK/c-Jun signaling pathway. Our results identify a physiological link between mTORC1 and MEK/ERK signaling in controlling intestinal hormone gene expression and secretion.     

Leukotriene D4 mediates survival and proliferation via separate but parallel pathways in the human intestinal epithelial cell line Int 407

We demonstrated previously that leukotriene D4 (LTD4) regulates proliferation of intestinal epithelial cells through a CysLT receptor by protein kinase C (PKC)epsilon-dependent stimulation of the mitogen-activated protein kinase ERK1/2. Our current study provides the first evidence that LTD4 can activate 90-kDa ribosomal S6 kinase (p90RSK) and cAMP-responsive element-binding protein (CREB) via pertussis-toxin-sensitive Gi protein pathways. Transfection and inhibitor experiments revealed that activation of p90RSK, but not CREB, is a PKCepsilon/Raf-1/ERK1/2-dependent process. LTD4-mediated CREB activation was not affected by expression of kinase-dead p90RSK but was abolished by transfection with the regulatory domain of PKCalpha (a specific dominant-inhibitor of PKCalpha). Kinase-negative mutants of p90RSK and CREB (K-p90RSK and K-CREB) blocked the LTD4-induced increase in cell number and DNA synthesis (thymidine incorporation). Compatible with these results, flow cytometry showed that LTD4 caused transition from the G0/G1 to the S+G2/M cell cycle phase, indicating increased proliferation. Similar treatment of cells transfected with K-p90RSK resulted in cell cycle arrest in the G0/G1 phase, consistent with a role of p90RSK in LTD4-induced proliferation. On the other hand, expression of K-CREB caused a substantial buildup in the sub-G0/G1 phase, suggesting a role for CREB in mediating LTD4-mediated survival in intestinal epithelial cells. Our results show that LTD4 regulates proliferation and survival via distinct intracellular signaling pathways in intestinal epithelial cells.     

Regulation of the rapamycin and FKBP-target 1/mammalian target of rapamycin and cap-dependent initiation of translation by the c-Abl protein-tyrosine kinase

The c-Abl protein-tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The rapamycin and FKBP-target 1 (RAFT1), also known as FKBP12-rapamycin-associated protein (FRAP, mTOR), regulates the p70S6 kinase (p70(S6k)) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). The present results demonstrate that c-Abl binds directly to RAFT1 and phosphorylates RAFT1 in vitro and in vivo. c-Abl inhibits autophosphorylation of RAFT1 and RAFT1-mediated phosphorylation p70(S6k). The functional significance of the c-Abl-RAFT1 interaction is further supported by the finding that eIF4E-dependent translation in mouse embryo fibroblasts from Abl(-/-) mice is significantly higher than that compared in wild-type cells. The results also demonstrate that exposure of cells to ionizing radiation is associated with c-Abl-mediated binding of 4E-BP1 to eIF4E and inhibition of translation. These findings with the c-Abl tyrosine kinase represent the first demonstration of a negative physiologic regulator of RAFT1-mediated 5' cap-dependent translation.     

Nutrient-dependent multimerization of the mammalian target of rapamycin through the N-terminal HEAT repeat region

The mammalian target of rapamycin (mTOR) plays a pivotal role in the regulation of cell growth in response to a variety of signals such as nutrients and growth factors. mTOR forms two distinct complexes in vivo. mTORC1 (mTOR complex 1) is rapamycin-sensitive and regulates the rate of protein synthesis in part by phosphorylating two well established effectors, S6K1 (p70 ribosomal S6 kinase 1) and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1). mTORC2 is rapamycin-insensitive and likely regulates actin organization and activates Akt/protein kinase B. Here, we show that mTOR forms a multimer via its N-terminal HEAT repeat region in mammalian cells. mTOR multimerization is promoted by amino acid sufficiency, although the state of multimerization does not directly correlate with the phosphorylation state of S6K1. mTOR multimerization was insensitive to rapamycin treatment but hindered by butanol treatment, which inhibits phosphatidic acid production by phospholipase D. We also found that mTOR forms a multimer in both mTORC1 and mTORC2. In addition, Saccharomyces cerevisiae TOR proteins Tor1p and Tor2p also exist as homomultimers. These results suggest that TOR multimerization is a conserved mechanism for TOR functioning.     

Rapamycin-insensitive regulation of 4e-BP1 in regenerating rat liver

In cultured cells, growth factor-induced phosphorylation of two translation modulators, p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), is blocked by nanomolar concentrations of the immunosuppressant rapamycin. Rapamycin also attenuates liver regeneration after partial hepatectomy, but it is not known if this growth-suppressive effect is due to dephosphorylation of p70 S6 kinase and/or 4E-BP1. We found that partial hepatectomy induced a transient increase in liver p70 S6 kinase activity and 4E-BP1 phosphorylation as compared with sham-operated rats. The amount of p70 S6 kinase protein in regenerating liver did not increase, but active kinase from partially hepatectomized animals was highly phosphorylated. Phosphorylated 4E-BP1 from regenerating liver was unable to form an inhibitory complex with initiation factor 4E. Rapamycin blocked the activation of p70 S6 kinase in response to partial hepatectomy in a dose-dependent manner, but 4E-BP1 phosphorylation was not inhibited. By contrast, functional phosphorylation of 4E-BP1 induced by injection of cycloheximide or growth factors was partially reversed by the drug. The mammalian target of rapamycin (mTOR) has been proposed to directly phosphorylate 4E-BP1. Western blot analysis using phospho-specific antibodies showed that phosphorylation of Thr-36/45 and Ser-64 increased in response to partial hepatectomy in a rapamycin-resistant manner. Thus, rapamycin inhibits p70 S6 kinase activation and liver regeneration, but not functional phosphorylation of 4E-BP1, in response to partial hepatectomy. These results indicate that the effect of rapamycin on 4E-BP1 function in vivo can be significantly different from its effect in cultured cells.     

Zinc stimulates the activity of the insulin- and nutrient-regulated protein kinase mTOR

Recent studies indicate that zinc activates p70 S6 kinase (p70(S6k)) by a mechanism involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt (protein kinase B). Here it is shown that phenanthroline, a zinc and heavy metal chelator, inhibited both amino acid- and insulin-stimulated phosphorylation of p70(S6k). Both amino acid and insulin activations of p70(S6k) involve a rapamycin-sensitive step that involves the mammalian target of rapamycin (mTOR, also known as FRAP and RAFT). However, in contrast to insulin, amino acids activate p70(S6k) by an unknown PI 3-kinase- and Akt-independent mechanism. Thus the effects of chelator on amino acid activation of p70(S6k) were surprising. For this reason, we tested the hypothesis that zinc directly regulates mTOR activity, independently of PI 3-kinase activation. In support of this, basal and amino acid stimulation of p70(S6k) phosphorylation was increased by zinc addition to the incubation media. Furthermore, the protein kinase activities of mTOR immunoprecipitated from rat brain lysates were stimulated two- to fivefold by 10-300 microM Zn2+ in the presence of an excess of either Mn2+ or Mg2+, whereas incubation with 1,10-phenanthroline had no effect. These findings indicate that Zn2+ regulates, but is not absolutely required for, mTOR protein kinase activity. Zinc also stimulated a recombinant human form of mTOR. The stimulatory effects of Zn2+ were maximal at approximately 100 microM but decreased and became inhibitory at higher physiologically irrelevant concentrations. Micromolar concentrations of other divalent cations, Ca2+, Fe2+, and Mn2+, had no effect on the protein kinase activity of mTOR in the presence of excess Mg2+. Our results and the results of others suggest that zinc acts at multiple steps in amino acid- and insulin cell-signaling pathways, including mTOR, and that the additive effects of Zn2+ on these steps may thereby promote insulin and nutritional signaling.     

Frap-dependent serine phosphorylation of IRS-1 inhibits IRS-1 tyrosine phosphorylation

We have previously shown that interferon-alpha (IFN alpha)-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) is impaired by serine phosphorylation of IRS-1 due to the reduced ability of serine phosphorylated IRS-1 to serve as a substrate for Janus kinase 1 (JAK1). Here we report that FKBP12-rapamycin-associated protein (FRAP) is a physiologic IRS-1 kinase that blocks IFN alpha signaling by serine phosphorylating IRS-1. We found that both FRAP and insulin-activated p70 S6 kinase (p70(s6k)) serine phosphorylated IRS-1 between residues 511 and 772 (IRS-1(511-772)). Importantly, only FRAP-dependent IRS-1(511-772) serine phosphorylation inhibited by 50% subsequent JAK1-dependent tyrosine phosphorylation of IRS-1. Furthermore, treatment of U266 cells with the FRAP inhibitor rapamycin increased IFN alpha-dependent tyrosine phosphorylation by twofold while reducing constitutive IRS-1 serine phosphorylation within S/T-P motifs by 80%. Taken together, these data indicate that FRAP, but not p70(s6k), is a likely physiologic IRS-1 serine kinase that negatively regulates JAK1-dependent IRS-1 tyrosine phosphorylation and suggests that FRAP may modulate IRS-dependent cytokine signaling.     

VIIa/tissue factor interaction results in a tissue factor cytoplasmic domain-independent activation of protein synthesis,p70, and p90 S6 kinase phosphorylation

FVIIa binding to tissue factor (TF) and subsequent signal transduction have now been implicated in a variety of pathophysiological processes, including cytokine production during sepsis, tumor angiogenesis and neoangiogenesis, and leukocyte diapedesis. The molecular details, however, by which FVIIa/TF affects gene expression and cellular physiology, remain obscure. Here we show that FVIIa induces a transient phosphorylation of p70/p85(S6K) and p90(RSK) in BHK cells stably transfected with either full-length TF or with a cytoplasmic domain-truncated TF but not in wild type BHK cells. Phosphorylation of these kinases was also observed in HaCaT cells, expressing endogenous TF. Phosphorylation of p70/p85(S6K) coincided with protein kinase B and GSK-3beta phosphorylation. Activation of p70/p85(S6K) was sensitive to inhibitors of phosphatidylinositol 3-kinase and to rapamycin, whereas phosphorylation of p90(RSK) was sensitive to PD98059. FVIIa stimulation of p70/p85(S6K) and p90(RSK) correlated with phosphorylation of the eukaryotic initiation factor eIF-4E, up-regulation of protein levels of eEF1alpha and eEF2, and enhanced [(35)S]methionine incorporation. These effects were not influenced by inhibitors of thrombin or FXa generation and were strictly dependent on the presence of the extracellular domain of TF, but they did not require the intracellular portion of TF. We propose that a TF cytoplasmic domain-independent stimulation of protein synthesis via activation of S6 kinase contributes to FVIIa effects in pathophysiology.     

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.