Effects of PI3K catalytic subunit and Akt isoform deficiency on mTOR and p70S6K activation in myoblasts

The PI3K/Akt/mTOR signaling pathway is critical for cellular growth and survival in skeletal muscle, and is activated in response to growth factors such as insulin-like growth factor-I (IGF-I). We found that in C2C12 myoblasts, deficiency of PI3K p110 catalytic subunits or Akt isoforms had distinct effects on phosphorylation of mTOR and p70S6K. siRNA-mediated knockdown of PI3K p110α, p110β, and simultaneous knockdown of p110α and p110β resulted in increased basal and IGF-I-stimulated phosphorylation of mTOR S2448 and p70S6K T389; however, phosphorylation of S6 was reduced in p110β-deficient cells, possibly due to reductions in total S6 protein. We found that IGF-I-stimulated Akt1 activity was enhanced in Akt2- or Akt3-deficient cells, and that knockdown of individual Akt isoforms increased mTOR/p70S6K activation in an isoform-specific fashion. Conversely, levels of IGF-I-stimulated p70S6K phosphorylation in cells simultaneously deficient in both Akt1 and Akt3 were increased beyond those seen with loss of any single Akt isoform, suggesting an alternate, Akt-independent mechanism that activates mTOR/p70S6K. Our results collectively suggest that mTOR/p70S6K is activated in a PI3K/Akt-dependent manner, but that in the absence of p110α or Akt, alternate pathway(s) may mediate activation of mTOR/p70S6K in C2C12 myoblasts.     

The role of AMPK/mTOR/S6K1 signaling axis in mediating the physiological process of exercise-induced insulin sensitization in skeletal muscle of C57BL/6 mice

The crosstalk between mTORC1/S6K1 signaling and AMPK is emerging as a powerful and highly regulated way to gauge cellular energy and nutrient content. The aim of the current study was to determine the mechanism by which exercise training reverses lipid-induced insulin resistance and the role of AMPK/mTOR/S6K1 signaling axis in mediating this response in skeletal muscle. Our results showed that high-fat feeding resulted in decreased glucose tolerance, which was associated with decreased Akt expression and increased intramuscular triglyceride deposition in the skeletal muscle of C57BL/6 mice. Impairments in lipid metabolism were accompanied by increased total protein and phosphorylation of S6K1, SREBP-1c cleavage, and decreased AMPK phosphorylation. Exercise training reversed these impairments, resulting in improved serum lipid profiles and glucose tolerance. C2C12 myotubes were exposed to palmitate, resulting in an increased insulin-dependent Akt Ser473 phosphorylation, associated with a significant increase in the level of phosphorylation of S6K1 on T389. All these changes were reversed by activation of AMPK. Consistent with this, inhibition of AMPK by compound C induced an enhanced phosphorylation of both S6K1 and Akt, and silencing of S6K1 with siRNA showed no effect on Akt phosphorylation in both the absence and presence of palmitate cultured myotubes. In addition, compound C led to an elevated SREBP-1c cleavage but was blocked by S6K1 siRNA. In summary, exercise training inhibits SREBP-1c cleavage through AMPK/mTOR/S6K1 signaling, resulting in decreased intramyocellular lipid accumulation. Our results provide new insights into the mechanism by which AMPK/mTOR/S6K1 signaling axis mediates the physiological process of exercise-induced insulin sensitization.     

Regulation of mTOR and p70 S6 kinase by the muscarinic M4 receptor in PC12 cells

The G_i-coupled M₄ muscarinic acetylcholine receptor (mAChR) has recently been shown to stimulate the survival of PC12 cells through the PI3K/Akt/tuberin pathway. Since mTOR and p70S6K are critical components in activating translation which lie downstream of tuberin, we examined the ability of M₄ mAChR to regulate these targets in PC12 cells. Carbachol (CCh) dose-dependently stimulated both mTOR and p70S6K phosphorylations and these responses were abolished by pertussis toxin pretreatment, indicating the involvement of the G_i-coupled M₄ mAChR. Phosphorylations of both mTOR and p70S6K were effectively blocked upon inhibition of PI3K by wortmannin. As compared to similar responses elicited by the nerve growth factor (NGF), the M₄ mAChR-induced activation of Akt/tuberin/mTOR/p70S6K occurred in a relatively transient manner. Although inhibition of protein phosphatase 2A by okadaic acid augmented the transient effects of CCh on Akt/tuberin phosphorylations, it failed to significantly prolong these responses. The total protein level of PTEN (tumor suppressor gene phosphatase and tensin homologue deleted on chromosome ten) was attenuated upon NGF, but not CCh treatment. This indicates that downregulation of PTEN may help to sustain the phosphorylation of Akt/tuberin by NGF. Collectively, these findings suggest that PP2A and PTEN may be involved in fine tuning the regulation of Akt/tuberin/mTOR/p70S6K in PC12 cells by M₄ mAChR and TrkA, respectively.     

Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation.

In contrast to cell types in which exposure to hypoxia causes a general reduction of metabolic activity, a remarkable feature of pulmonary artery adventitial fibroblasts is their ability to proliferate in response to hypoxia. Previous studies have suggested that ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) are activated by hypoxia and play a role in a variety of cell responses. However, the pathways involved in mediating hypoxia-induced proliferation are largely unknown. Using pharmacological inhibitors, we established that PI3K-Akt, mTOR-p70 ribosomal protein S6 kinase (p70S6K), and EKR1/2 signaling pathways play a critical role in hypoxia-induced adventitial fibroblast proliferation. We found that exposure of serum-starved fibroblasts to 3% O2 resulted in a time-dependent activation of PI3K and transient phosphorylation of Akt. However, activation of PI3K was not required for activation of ERK1/2, implying a parallel involvement of these pathways in the proliferative response of fibroblasts to hypoxia. We found that hypoxia induced significant increases in mTOR, p70S6K, 4E-BP1, and S6 ribosomal protein phosphorylation, as well as dramatic increases in p70S6K activity. The activation of p70S6K/S6 pathway was sensitive to inhibition by rapamycin and LY294002, indicating that mTOR and PI3K/Akt are upstream signaling regulators. However, the magnitude of hypoxia-induced p70S6K activity and phosphorylation suggests involvement of additional signaling pathways. Thus our data demonstrate that hypoxia-induced adventitial fibroblast proliferation requires activation and interaction of PI3K, Akt, mTOR, p70S6K, and ERK1/2 and provide evidence for hypoxic regulation of protein translational pathways in cells exhibiting the capability to proliferate under hypoxic conditions.     

Zinc stimulates glucose consumption by modulating the insulin signaling pathway in L6 myotubes: essential roles of Akt–GLUT4, GSK3β and mTOR–S6K1

The present study was performed to evaluate the insulin-like effects of zinc in normal L6 myotubes as well as its ability to alleviate insulin resistance. Glucose consumption was measured in both normal and insulin-resistant L6 myotubes. Western blotting and immunofluorescence revealed that zinc exhibited insulin-like glucose transporting effects by activating key markers that are involved in the insulin signaling cascade (including Akt, GLUT4 and GSK3β), and downregulating members of the insulin signaling feedback cascade such as mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K1). In normal L6 myotubes, zinc enhanced glucose consumption via a mechanism that might involve the activation of Akt phosphorylation, glucose transporter 4 (GLUT4) translocation and GSK3β phosphorylation. In contrast, zinc exerted insulin-mimetic effects in insulin-resistant L6 myotubes by upregulating Akt phosphorylation, GLUT4 translocation and GSK3β phosphorylation, and downregulating the expression of mTOR and S6K1. In conclusion, zinc might enhance glucose consumption by modulating insulin signaling pathways including Akt–GLUT4, GSK3β, mTOR and S6K1.     

Rapamycin promotes vascular smooth muscle cell differentiation through insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt2 feedback signaling

The phenotypic plasticity of mature vascular smooth muscle cells (VSMCs) facilitates angiogenesis and wound healing, but VSCM dedifferentiation also contributes to vascular pathologies such as intimal hyperplasia. Insulin/insulin-like growth factor I (IGF-I) is unique among growth factors in promoting VSMC differentiation via preferential activation of phosphatidylinositol 3-kinase (PI3K) and Akt. We have previously reported that rapamycin promotes VSMC differentiation by inhibiting the mammalian target of rapamycin (mTOR) target S6K1. Here, we show that rapamycin activates Akt and induces contractile protein expression in human VSMC in an insulin-like growth factor I-dependent manner, by relieving S6K1-dependent negative regulation of insulin receptor substrate-1 (IRS-1). In skeletal muscle and adipocytes, rapamycin relieves mTOR/S6K1-dependent inhibitory phosphorylation of IRS-1, thus preventing IRS-1 degradation and enhancing PI3K activation. We report that this mechanism is functional in VSMCs and crucial for rapamycin-induced differentiation. Rapamycin inhibits S6K1-dependent IRS-1 serine phosphorylation, increases IRS-1 protein levels, and promotes association of tyrosine-phosphorylated IRS-1 with PI3K. A rapamycin-resistant S6K1 mutant prevents rapamycin-induced Akt activation and VSMC differentiation. Notably, we find that rapamycin selectively activates only the Akt2 isoform and that Akt2, but not Akt1, is sufficient to induce contractile protein expression. Akt2 is required for rapamycin-induced VSMC differentiation, whereas Akt1 appears to oppose contractile protein expression. The anti-restenotic effect of rapamycin in patients may be attributable to this unique pattern of PI3K effector regulation wherein anti-differentiation signals from S6K1 are inhibited, but pro-differentiation Akt2 activity is promoted through an IRS-1 feedback signaling mechanism.     

Extracellular ATP-induced proliferation of adventitial fibroblasts requires phosphoinositide 3-kinase, Akt, mammalian target of rapamycin, and p70 S6 kinase signaling pathways

Extracellular nucleotides are increasingly recognized as important regulators of growth in a variety of cell types. Recent studies have demonstrated that extracellular ATP is a potent inducer of fibroblast growth acting, at least in part, through an ERK1/2-dependent signaling pathway. However, the contributions of additional signaling pathways to extracellular ATP-mediated cell proliferation have not been defined. By using both pharmacologic and genetic approaches, we found that in addition to ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), and p70 S6K-dependent signaling pathways are required for ATP-induced proliferation of adventitial fibroblasts. We found that extracellular ATP acting in part through G(i) proteins increased PI3K activity in a time-dependent manner and transient phosphorylation of Akt. This PI3K pathway is not involved in ATP-induced activation of ERK1/2, implying activation of independent parallel signaling pathways by ATP. Extracellular ATP induced dramatic increases in mTOR and p70 S6K phosphorylation. This activation of the mTOR/p70 S6 kinase (p70 S6K) pathway in response to ATP is because of independent contributions of PI3K/Akt and ERK1/2 pathways, which converge on the level of p70 S6K. ATP-dependent activation of mTOR and p70 S6K also requires additional signaling inputs perhaps from pathways operating through Galpha or Gbetagamma subunits. Collectively, our data demonstrate that ATP-induced adventitial fibroblast proliferation requires activation and interaction of multiple signaling pathways such as PI3K, Akt, mTOR, p70 S6K, and ERK1/2 and provide evidence for purinergic regulation of the protein translational pathways related to cell proliferation.     

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.     

Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from AMPK alpha2 knockout (KO), AMPK gamma3 KO, and respective wild-type (WT) littermates (C57BL/6) were incubated in the presence of 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), insulin, or AICAR plus insulin. Phosphorylation of AMPK, Akt, and mTOR-associated signaling proteins were assessed. Insulin increased Akt Ser473 phosphorylation (P < 0.01), irrespective of genotype or presence of AICAR. AICAR increased phosphorylation of AMPK Thr172 (P < 0.01) in WT but not KO mice. Insulin stimulation increased phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46) (P < 0.01) in WT, AMPK alpha2 KO, and AMPK gamma3 KO mice. However, in WT mice, preincubation with AICAR completely inhibited insulin-induced phosphorylation of mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued in extensor digitorum longus muscle from either alpha2 or gamma3 AMPK KO mice, indicating functional alpha2 and gamma3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46). In conclusion, functional alpha2 and gamma3 AMPK subunits are required for AICAR-induced inhibitory effects on mTOR signaling.     

Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin

Phosphorylation of the ribosomal S6 subunit is tightly correlated with enhanced translation initiation of a subset of mRNAs that encodes components of the protein synthesis machinery, which is an important early event that controls mammalian cell growth and proliferation. The recently identified S6 kinase 2 (S6K2), together with its homologue S6K1, is likely responsible for the mitogen-stimulated phosphorylation of S6. Like S6K1, the activation of S6K2 requires signaling from both the phosphatidylinositol 3-kinase and the mammalian target of rapamycin (mTOR). Here we report the investigation of the mechanisms of S6K2 regulation by mTOR. We demonstrate that similar to S6K1 the serum activation of S6K2 in cells is dependent on mTOR kinase activity, amino acid sufficiency, and phosphatidic acid. Previously we have shown that mTOR is a cytoplasmic-nuclear shuttling protein. As a predominantly nuclear protein, S6K2 activation was facilitated by enhanced mTOR nuclear import with the tagging of an exogenous nuclear localization signal and diminished by enhanced mTOR nuclear export with the tagging of a nuclear export sequence. However, further increase of mTOR nuclear import by the tagging of four copies of nuclear localization signal resulted in its decreased ability to activate S6K2, suggesting that mTOR nuclear export may also be an integral part of the activation process. Consistently, the nuclear export inhibitor leptomycin B inhibited S6K2 activation. Taken together, our observations suggest a novel regulatory mechanism in which an optimal cytoplasmic-nuclear distribution or shuttling rate for mTOR is required for maximal activation of the nuclear S6K2.     

Carbachol induces p70S6K1 activation through an ERK-dependent but Akt-independent pathway in human colonic epithelial cells

Stimulation of human colonic epithelial T84 cells with the muscarinic receptor agonist carbachol, a stable analog of acetylcholine, induced Akt, p70S6K1 and ERK activation. Treatment of T84 cells with the selective inhibitor of EGF receptor (EGFR) tyrosine kinase AG1478 abrogated Akt phosphorylation on Ser⁴⁷³ induced by either carbachol or EGF, indicating that carbachol-induced Akt activation is mediated through EGFR transactivation. Surprisingly, AG1478 did not suppress p70S6K1 phosphorylation on Thr³⁸⁹ in response to carbachol, indicating the G protein-coupled receptor (GPCR) stimulation induces p70S6K1 activation, at least in part, via an Akt-independent pathway. In contrast, treatment with the selective MEK inhibitor U0126 (but not with the inactive analog U0124) inhibited carbachol-induced p70S6K1 activation, indicating that the MEK/ERK/RSK pathway plays a critical role in p70S6K1 activation in GPCR-stimulated T84 cells. These findings imply that GPCR activation induces p70S6K1 via ERK rather than through the canonical PI 3-kinase/Akt/TSC/mTORC1 pathway in T84 colon carcinoma cells.     

Thyroid hormone induces rapid activation of Akt/protein kinase B-mammalian target of rapamycin-p70S6K cascade through phosphatidylinositol 3-kinase in human fibroblasts

We have demonstrated that T3 increases the expression of ZAKI-4alpha, an endogenous calcineurin inhibitor. In this study we characterized a T3-dependent signaling cascade leading to ZAKI-4alpha expression in human skin fibroblasts. We found that T3-dependent increase in ZAKI-4alpha was greatly attenuated by rapamycin, a specific inhibitor of a protein kinase, mammalian target of rapamycin (mTOR), suggesting the requirement of mTOR activation by T3. Indeed, T3 activated mTOR rapidly through S2448 phosphorylation, leading to the phosphorylation of p70(S6K), a substrate of mTOR. This mTOR activation is mediated through phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) signaling cascade because T3 induced Akt/PKB phosphorylation more rapidly than that of mTOR, and these T3-dependent phosphorylations were blocked by both PI3K inhibitors and by expression of a dominant negative PI3K (Deltap85alpha). Furthermore, the association between thyroid hormone receptor beta1 (TRbeta1) and PI3K-regulatory subunit p85alpha, and the inhibition of T3-induced PI3K activation and mTOR phosphorylation by a dominant negative TR (G345R) demonstrated the involvement of TR in this T3 action. The liganded TR induces the activation of PI3K and Akt/PKB, leading to the nuclear translocation of the latter, which subsequently phosphorylates nuclear mTOR. The rapid activation of PI3K-Akt/PKB-mTOR-p70(S6K) cascade by T3 provides a new molecular mechanism for thyroid hormone action.     

Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells

Fluid shear stress generated by blood flow modulates endothelial cell function via specific intracellular signaling events. We showed previously that flow activated the phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) via Src kinase-dependent transactivation of vascular endothelial growth factor receptor 2 (VEGFR2). The scaffold protein Gab1 plays an important role in receptor tyrosine kinase-mediated signal transduction. We found here that laminar flow (shear stress = 12 dynes/cm2) rapidly stimulated Gab1 tyrosine phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial cells, which correlated with activation of Akt and eNOS. Gab1 phosphorylation as well as activation of Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and VEGFR2 kinase inhibitors SU1498 and VTI, suggesting that flow-mediated Gab1 phosphorylation is Src kinase-dependent and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally important, because flow stimulated the association of Gab1 with the PI3K subunit p85 in a time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites inhibited flow-induced activation of Akt and eNOS. Finally, knockdown of endogenous Gab1 by small interference RNA abrogated flow activation of Akt and eNOS. These data demonstrate a critical role of Gab1 in flow-stimulated PI3K/Akt/eNOS signal pathway in endothelial cells.     

EGF receptor transactivation mediates ANG II-stimulated mitogenesis in intestinal epithelial cells through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway

The role of epidermal growth factor receptor (EGFR) tyrosine kinase and its downstream targets in the regulation of the transition from the G0/G1 phase into DNA synthesis in response to ANG II has not been previously investigated in intestinal epithelial IEC-18 cells. ANG II induced a rapid and striking EGFR tyrosine phosphorylation, which was prevented by selective inhibitors of EGFR tyrosine kinase activity (e.g., AG-1478) or by broad-spectrum matrix metalloproteinase (MMP) inhibitor GM-6001. Pretreatment of these cells with either AG-1478 or GM-6001 reduced ANG II-stimulated DNA synthesis by approximately 50%. To elucidate the downstream targets of EGFR, we demonstrated that ANG II stimulated phosphorylation of Akt at Ser473, mTOR at Ser2448, p70S6K1 at Thr389, and S6 ribosomal protein at Ser(235/236). Pretreatment with AG-1478 inhibited Akt, p70S6K1, and S6 ribosomal protein phosphorylation. Inhibition of phosphatidylinositol (PI)3-kinase with LY-294002 or mTOR/p70S6K1 with rapamycin reduced [3H]thymidine incorporation by 50%, i.e., to levels comparable to those achieved by addition of either AG-1478 or GM-6001. Utilizing Akt small-interfering RNA targeted to Akt1 and Akt2, Akt protein knockdown dramatically inhibited p70S6K1 and S6 ribosomal protein phosphorylation. In contrast, AG-1478 or Akt gene silencing exerted no detectable inhibitory effect on ANG II-induced extracellular signal-regulated kinase 1/2 phosphorylation in IEC-18 cells. Taken together, our results demonstrate that EGFR transactivation mediates ANG II-stimulated mitogenesis through the PI3-kinase/Akt/mTOR/p70S6K1 signaling pathway in IEC-18 cells.     

Activation or inactivation of cardiac Akt/mTOR signaling diverges physiological from pathological hypertrophy

Cardiomyocyte hypertrophy differs according to the stress exerted on the myocardium. While pressure overload-induced cardiomyocyte hypertrophy is associated with depressed contractile function, physiological hypertrophy after exercise training associates with preserved or increased inotropy. We determined the activation state of myocardial Akt signaling with downstream substrates and fetal gene reactivation in exercise-induced physiological and pressure overload-induced pathological hypertrophies. C57BL/6J mice were either treadmill trained for 6 weeks, 5 days/week, at 85-90% of maximal oxygen uptake (VO(2max)), or underwent transverse aortic constriction (TAC) for 1 or 8 weeks. Total and phosphorylated protein levels were determined with SDS-PAGE, and fetal genes by real-time RT-PCR. In the physiologically hypertrophied heart after exercise training, total Akt protein level was unchanged, but Akt was chronically hyperphosphorylated at serine 473. This was accompanied by activation of the mammalian target of rapamycin (mTOR), measured as phosphorylation of its two substrates: the ribosomal protein S6 kinase-1 (S6K1) and the eukaryotic translation initiation factor-4E binding protein-1 (4E-BP1). Exercise training did not reactivate the fetal gene program (beta-myosin heavy chain, atrial natriuretic factor, skeletal muscle actin). In contrast, pressure overload after TAC reactivated fetal genes already after 1 week, and partially inactivated the Akt/mTOR pathway and downstream substrates after 8 weeks. In conclusion, changes in opposite directions of the myocardial Akt/mTOR signal pathway appears to distinguish between physiological and pathological hypertrophies; exercise training associating with activation and pressure overload associating with inactivation of the Akt/mTOR pathway.     

Different Patterns of Akt and ERK Feedback Activation in Response to Rapamycin,Active-Site mTOR Inhibitors and Metformin in Pancreatic Cancer Cells

The mTOR pathway is aberrantly stimulated in many cancer cells, including pancreatic ductal adenocarcinoma (PDAC), and thus it is a potential target for therapy. However, the mTORC1/S6K axis also mediates negative feedback loops that attenuate signaling via insulin/IGF receptor and other tyrosine kinase receptors. Suppression of these feed-back loops unleashes over-activation of upstream pathways that potentially counterbalance the antiproliferative effects of mTOR inhibitors. Here, we demonstrate that treatment of PANC-1 or MiaPaCa-2 pancreatic cancer cells with either rapamycin or active-site mTOR inhibitors suppressed S6K and S6 phosphorylation induced by insulin and the GPCR agonist neurotensin. Rapamycin caused a striking increase in Akt phosphorylation at Ser⁴⁷³ while the active-site inhibitors of mTOR (KU63794 and PP242) completely abrogated Akt phosphorylation at this site. Conversely, active-site inhibitors of mTOR cause a marked increase in ERK activation whereas rapamycin did not have any stimulatory effect on ERK activation. The results imply that first and second generation of mTOR inhibitors promote over-activation of different pro-oncogenic pathways in PDAC cells, suggesting that suppression of feed-back loops should be a major consideration in the use of these inhibitors for PDAC therapy. In contrast, metformin abolished mTORC1 activation without over-stimulating Akt phosphorylation on Ser⁴⁷³ and prevented mitogen-stimulated ERK activation in PDAC cells. Metformin induced a more pronounced inhibition of proliferation than either KU63794 or rapamycin while, the active-site mTOR inhibitor was more effective than rapamycin. Thus, the effects of metformin on Akt and ERK activation are strikingly different from allosteric or active-site mTOR inhibitors in PDAC cells, though all these agents potently inhibited the mTORC1/S6K axis.     

Differential regulation of mTOR-dependent S6 phosphorylation by muscarinic acetylcholine receptor subtypes

Muscarinic receptors subserve many functions in both peripheral and central nervous systems. Some of these processes depend on increases in protein synthesis, which may be achieved by activation of mammalian target of rapamycin (mTOR), a kinase that regulates protein translation capacity. Here, we examined the regulation of mTOR-dependent signaling pathways by muscarinic receptors in SK-N-SH human neuroblastoma cells, and in human embryonic kidney (HEK) cell lines transfected with individual muscarinic receptor subtypes. In SK-N-SH cells, the acetylcholine analog carbachol stimulated phosphorylation of the ribosomal S6 protein, a downstream target of mTOR. The sensitivity of the response to subtype-selective muscarinic receptor antagonists indicated that it was mediated by M3 receptors. Carbachol-evoked S6 phosphorylation was blocked by the mTOR inhibitor rapamycin, but was independent of phosphoinositide 3-kinase activation. The response was significantly reduced by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also inhibited carbachol-evoked S6 phosphorylation in HEK cells expressing M2 receptors, but was ineffective in M3 receptor-expressing HEK cells, although carbachol activated MAPK in both transfected lines. The p90 ribosomal S6 kinase has been implicated in mTOR regulation by phorbol esters, but was not activated by carbachol in any of the cell lines tested. The protein kinase C inhibitor bisindolylmaleimide I reduced carbachol-stimulated S6 phosphorylation in SK-N-SH cells, and in HEK cells expressing M3 receptors, but not in HEK cells expressing M2 receptors. The results demonstrate that multiple muscarinic receptor subtypes regulate mTOR, and that both MAPK-dependent and -independent mechanisms may mediate the response in a cell context-specific manner.     

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.     

PRR5, a novel component of mTOR complex 2, regulates platelet-derived growth factor receptor beta expression and signaling

The protein kinase mammalian target of rapamycin (mTOR) plays an important role in the coordinate regulation of cellular responses to nutritional and growth factor conditions. mTOR achieves these roles through interacting with raptor and rictor to form two distinct protein complexes, mTORC1 and mTORC2. Previous studies have been focused on mTORC1 to elucidate the central roles of the complex in mediating nutritional and growth factor signals to the protein synthesis machinery. Functions of mTORC2, relative to mTORC1, have remained little understood. Here we report identification of a novel component of mTORC2 named PRR5 (PRoline-Rich protein 5), a protein encoded by a gene located on a chromosomal region frequently deleted during breast and colorectal carcinogenesis (Johnstone, C. N., Castellvi-Bel, S., Chang, L. M., Sung, R. K., Bowser, M. J., Pique, J. M., Castells, A., and Rustgi, A. K. (2005) Genomics 85, 338-351). PRR5 interacts with rictor, but not raptor, and the interaction is independent of mTOR and not disturbed under conditions that disrupt the mTOR-rictor interaction. PRR5, unlike Sin1, another component of mTORC2, is not important for the mTOR-rictor interaction and mTOR activity toward Akt phosphorylation. Despite no significant effect of PRR5 on mTORC2-mediated Akt phosphorylation, PRR5 silencing inhibits Akt and S6K1 phosphorylation and reduces cell proliferation rates, a result consistent with PRR5 roles in cell growth and tumorigenesis. The inhibition of Akt and S6K1 phosphorylation by PRR5 knock down correlates with reduction in the expression level of platelet-derived growth factor receptor beta (PDGFRbeta). PRR5 silencing impairs PDGF-stimulated phosphorylation of S6K1 and Akt but moderately reduces epidermal growth factor- and insulin-stimulated phosphorylation. These findings propose a potential role of mTORC2 in the cross-talk with the cellular machinery that regulates PDGFRbeta expression and signaling.