Substrate S6 Research Articles

Fibroblast Growth Factor Receptor 2 Homodimerization Rapidly Reduces Transcription of the Pluripotency Gene Nanog without Dissociation of Activating Transcription Factors

Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.

Abstract 2805: In vitro characterization of AMG 511, a potent and selective class I PI3K inhibitor for the treatment of cancer

Abstract The PI3K signaling pathway is frequently activated in cancer and has been implicated in many aspects of tumor growth and survival. Inhibition of this pathway represents a potential therapeutic path for the treatment of cancer. This study evaluated the in vitro characteristics of AMG 511, a potent and selective pan class I PI3K inhibitor exhibiting IC50 values of 8, 11, 2, and 6 nM against the PI3K β, α, β, and ≤ isoforms respectively. AMG 511 was shown to be inactive against members of the closely related phosphoinositide 3 kinase related kinases (PIKK) family of kinases and did not inhibit mTOR, hVPS34, PI4Kα or PI4Kα in-vitro (IC50 values > 1 μM). In addition, AMG 511 was inactive against a majority of protein kinases (372) in the human kinome as measured by in-vitro binding assays. AMG 511 inhibited PI3K pathway signaling in U87 MG glioblastoma cells as determined by dose-dependent reduction in AKT S473 phosphorylation (IC50 = 4 nM). AKT inhibition resulted in a concomitant reduction in PRAS40 phosphorylation (IC50 = 23 nM), a downstream effector of AKT. Reduced phosphorylation of mTORC1 substrates p70S6K (IC50 = 30 nM) and S6 (IC50 = 70 nM) but not 4EBP1 (T37/46), was also detected in U87 MG cells, suggesting that upstream blockade of PI3K pathway signaling with AMG 511 treatment leads to a selective reduction in downstream mTORC1 activity. Given the well documented role of mTORC1 in cap-dependent translation we profiled AMG 511 in a methionine-analog incorporation assay in U87 MG cells. However, no significant inhibition of bulk translation was observed following treatment with AMG 511 in U87 MG cells. Treatment of U87 MG cells with AMG 511 revealed a pronounced G1 arrest with a concurrent reduction in BrdU+ cells, detectable within 8 hours of treatment. This anti-proliferative effect was fully reversible by 18 hours following washout. In line with these anti-proliferative effects, reduced cyclin D1 levels and elevated p27 levels were detected within 4 hours of treatment. Minimal cell killing effects were detected with AMG 511 treatment in U87 MG cells as measured by induction of cleaved caspase-3 and DNA content < 2N. AMG 511 was profiled across a large panel of tumor cell lines encompassing several tumor types including breast and lung. A majority of the cell lines tested exhibited sensitivity to AMG 511 with a subset exhibiting evidence for cell death upon treatment with AMG 511. Breast cancer cell lines harboring activating mutations in PI3Kα, loss of PTEN, or amplification of Her2 tended to show greater sensitivity to AMG 511 treatment. In conclusion, AMG 511 is a potent and selective pan class I PI3K inhibitor, capable of inhibiting PI3K signaling and inducing robust anti-proliferative effects via a G1 arrest in many tumor cell lines, with evidence of cell killing in a subset of lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2805. doi:1538-7445.AM2012-2805

Abstract 2798: Cytotoxic and cystostatic properties of rapamycin are due to differential effects on the phosphorylation of mTORC1 substrates S6 kinase and 4E-BP1

Abstract The mammalian target of rapamycin (mTOR) has been widely implicated in signals that promote cell cycle progression and survival in cancer cells. Rapamycin, which inhibits mTOR with high specificity, has consequently attracted much attention as an anti-cancer therapy. Used clinically at nano-molar concentrations, rapamycin suppresses phosphorylation of mTOR complex 1 (mTORC1) substrate S6 kinase and has been considered a cytostatic drug. However, we have reported that higher (micro-molar) doses are required for complete cell cycle arrest. Such high doses suppress phosphorylation of another mTORC1 substrate, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). In this state, eIF4E is sequestered and unable to facilitate mRNA translation. Dual ablation of S6 kinase and eIF4e resulted in a larger increase in G1 content than by ablation of S6 kinase alone. This confirmed the complete cytostatic effect of high-dose rapamycin. We also demonstrated that the differential dose effects of rapamycin correlate with partial and complete dissociation of Raptor from mTORC1 at low and high doses, respectively. Therefore, high-dose rapamycin ensures complete non-functionality of mTORC1. Interestingly, in the absence of serum, high dose rapamycin induces apoptosis and dual inhibition of S6 kinase and eIF4e does not prevent it. We have established that the differential cytostatic and cytotoxic effects of rapamycin are due to intact TGFb signaling. In serum, TGFb is activated upon low dose rapamycin (suppression of of S6 kinase phosphorylation). The lack of TGFb in the absence of serum allows cells to progress through the cell cycle; at high rapamycin doses, the inactivity of eIF4e results in cell death. This study reveals the mechanism for both cytostatic and cytotoxic properties of rapmaycin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2798. doi:1538-7445.AM2012-2798

The non-proteinogenic amino acids l-methionine sulfoximine and dl-phosphinothricin activate mTOR

L-Methionine sulfoximine (MSO) and DL-Phosphinothricin (PPT), two non-proteinogenic amino acids known as inhibitors of Glutamine Synthetase, cause a dose-dependent increase in the phosphorylation of the mTOR substrate S6 kinase 1. The effect is particularly evident in glutamine-depleted cells, where mTOR activity is very low, but is detectable for PPT also in the presence of glutamine. The stimulation of mTOR activity by either MSO or PPT is strongly synergized by essential amino acids. Thus, the non-proteinogenic amino acids MSO and PPT are mTOR activators.

Phospholipase D Regulates Myogenic Differentiation through the Activation of Both mTORC1 and mTORC2 Complexes

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.

A functionalized single-walled carbon nanotube-induced autophagic cell death in human lung cells through Akt–TSC2-mTOR signaling

Nanoparticles are now emerging as a novel class of autophagy activators. Functionalized single-walled carbon nanotubes (f-SWCNTs) are valuable nanomaterials in many industries. This article is designed to assess the autophagic response for f-SWCNTs exposure in vitro and in vivo. A few types of f-SWCNTs were screened in human lung adenocarcinoma A549 cells for the autophagic response and related pathways in vitro. Formation of autophagosomes and LC3-II upregulation were confirmed on the basis of electron microscopy and LC3 western blotting for COOH-CNT, but not for PABS-CNT and PEG-CNT. MTT assay showed marked increase in cell viability, when COOH-CNT was added to cells in the presence of autophagy inhibitor 3MA, ATG6 or TSC2 siRNA. Consistent with the involvement of the Akt–TSC1/2–mTOR pathway, the phosphorylation levels of mTOR, mTOR's substrate S6 and Akt were shown significantly decreased in A549 cells on treatment with COOH-CNT using western blotting. What's more, autophagy inhibitor 3MA significantly reduced the lung edema in vivo. In a word, COOH-CNT induced autophagic cell death in A549 cells through the AKT–TSC2–mTOR pathway and caused acute lung injury in vivo. Inhibition of autophagy significantly reduced COOH-CNT-induced autophagic cell death and ameliorated acute lung injury in mice, suggesting a potential remedy to address the growing concerns on the safety of nanomaterials.

BH3 mimetics activate multiple pro-autophagic pathways

The BH3 mimetic ABT737 induces autophagy by competitively disrupting the inhibitory interaction between the BH3 domain of Beclin 1 and the anti-apoptotic proteins Bcl-2 and Bcl-X(L), thereby stimulating the Beclin 1-dependent allosteric activation of the pro-autophagic lipid kinase VPS34. Here, we examined whether ABT737 stimulates other pro-autophagic signal-transduction pathways. ABT737 caused the activating phosphorylation of AMP-dependent kinase (AMPK) and of the AMPK substrate acetyl CoA carboxylase, the activating phosphorylation of several subunits of the inhibitor of NF-κB (IκB) kinase (IKK) and the hyperphosphorylation of the IKK substrate IκB, inhibition of the activity of mammalian target of rapamycin (mTOR) and consequent dephosphorylation of the mTOR substrate S6 kinase. In addition, ABT737 treatment dephosphorylates (and hence likewise inhibits) p53, glycogen synthase kinase-3 and Akt. All these effects were shared by ABT737 and another structurally unrelated BH3 mimetic, HA14-1. Functional experiments revealed that pharmacological or genetic inhibition of IKK, Sirtuin and the p53-depleting ubiquitin ligase MDM2 prevented ABT737-induced autophagy. These results point to unexpected and pleiotropic pro-autophagic effects of BH3 mimetics involving the modulation of multiple signalling pathways.

Targeting of active mTOR inhibits primary leukemia T cells and synergizes with cytotoxic drugs and signaling inhibitors

Rationally designed therapies aim at the specific disruption of critical signaling pathways activated by malignant transformation or signals from the tumor microenvironment. Because mammalian target of rapamycin (mTOR) is an important signal integrator and a key translational regulator, we evaluated its potential involvement in T-cell acute lymphoblastic leukemia (T-ALL) and whether mTOR blockade synergizes with chemotherapeutic agents or other signaling antagonists to inhibit primary leukemia T cells. mTOR signaling status was assessed using biochemical, immunostaining, and molecular regulation studies and functional assays performed to assess the impact of mTOR blockade on T-ALL proliferation, survival, and cell cycle. We observed that mTOR signaling is highly activated in all T-ALL patients tested, with phosphorylation of its downstream substrates eIF4G and S6 ribosomal protein. mTOR activation was detected in vivo and was further increased in vitro by stimulation with interleukin-7, a potentially leukemogenic cytokine normally produced by the bone marrow microenvironment. In T-ALL cells, mTOR blockade was associated with accumulation of the cyclin-dependent kinase inhibitor p27(kip1), which preferentially adopted a nuclear localization. Functional studies using rapamycin or CCI-779 showed a dominant inhibitory effect of mTOR blockade on interleukin-7-induced proliferation, survival, and cell-cycle progression of T-ALL cells. Furthermore, mTOR blockade markedly potentiated the antileukemia effects of dexamethasone and doxorubicin, and showed highly synergistic interactions in combination with specific inhibitors of phosphatidylinositol 3-kinase/Akt and Janus kinase 3 signaling. This study shows activation of mTOR signaling in primary T-ALL cells evolving in the leukemic bone marrow, and supports the inclusion of mTOR antagonists in current therapeutic regimens for this cancer.

Ethyl Pyruvate Preserves IGF-I Sensitivity toward mTOR Substrates and Protein Synthesis in C2C12 Myotubes

Bacterial infection decreases skeletal muscle protein synthesis via inhibition of the mammalian target of rapamycin (mTOR), a key regulator of translation initiation. To better define the mechanism by which muscle mTOR activity is decreased, we used an in vitro model of C2C12 myotubes treated with endotoxin [lipopolysaccharide (LPS)]and interferon (IFN)-γ to determine whether stable lipophilic pyruvate derivatives restore mTOR signaling. Myotubes treated with a combination of LPS and IFNγ down-regulated the phosphorylation of the mTOR substrates S6 kinase-1 and 4E binding protein-1. The phosphorylation of ribosomal protein S6 was decreased, whereas phosphorylation of elongation factor-2 was enhanced; all results consistent with defects in both translation initiation and elongation. LPS/IFNγ decreased protein synthesis 60% in myotubes. Treatment with methyl or ethyl pyruvate partially protected against the LPS/IFNγ-induced fall in mTOR signaling. The protective effect of ethyl and methyl pyruvate could not be replicated by an equimolar amount of sodium pyruvate. Although LPS/IFNγ treated myotubes were initially IGF-I responsive, prolonged exposure (≥ 17 h) resulted in IGF-I resistance at the level of mTOR despite normal IGF-I receptor phosphorylation. Ethyl pyruvate treatment restored IGF-I sensitivity as evidenced by the left shift in the IGF-I dose-response curve and maintained IGF-I responsiveness for a prolonged period of time. Ethyl pyruvate also restored IGF-I-stimulated protein synthesis in LPS/IFNγ-treated myotubes. Cotreatment with N-acetyl cysteine or ascorbic acid also preserved IGF-I sensitivity and mTOR activity. The data suggest that the combination of LPS and IFNγ inhibits mTOR activity and that prolonged exposure induces IGF-I resistance in myotubes. Lipophilic pyruvate derivatives and antioxidants show promise at rescuing mTOR activity and muscle protein synthesis by maintaining IGF-I sensitivity in this model.

Regulation of mTORC1 and mTORC2 Complex Assembly by Phosphatidic Acid: Competition with Rapamycin

mTOR, the mammalian target of rapamycin, is a critical node for control of cell growth and survival and has widely been implicated in cancer survival signals. mTOR exists in two complexes: mTORC1 and mTORC2. Phospholipase D (PLD) and its metabolite phosphatidic acid (PA) have been implicated in the regulation of mTOR; however, their role has been controversial. We report here that suppression of PLD prevents phosphorylation of the mTORC1 substrate S6 kinase (S6K) at Thr389 and the mTORC2 substrate Akt at Ser473. Suppression of PLD also blocked insulin-stimulated Akt phosphorylation at Ser473 and the mTORC2-dependent phosphorylation of PRAS40. Importantly, PA was required for the association of mTOR with Raptor to form mTORC1 and that of mTOR with Rictor to form mTORC2. The effect of PA was competitive with rapamycin-with much higher concentrations of rapamycin needed to compete with the PA-mTORC2 interaction than with PA-mTORC1. Suppressing PA production substantially increased the sensitivity of mTORC2 to rapamycin. Data provided here demonstrate a PA requirement for the stabilization of both mTORC1 and mTORC2 complexes and reveal a mechanism for the inhibitory effect of rapamycin on mTOR. This study also suggests that by suppressing PLD activity, mTORC2 could be targeted therapeutically with rapamycin.

Enzastaurin‐induced apoptosis in glioma cells is caspase‐dependent and inhibited by BCL‐XL

The novel protein kinase C-beta inhibitor enzastaurin (ENZA) induced apoptosis in LNT-229 and T98G cells whereas A172 cells were resistant. Further, ENZA reduced proliferation in glioblastoma-initiating cells T 269 and T 323 but did not induce apoptosis. ENZA-induced apoptosis involved cleavage of caspases 3, 8, and 9 and led to mitochondrial cytochrome c release and was strongly suppressed by the broad spectrum caspase inhibitor zVAD-fmk but only slightly by the expression of the viral caspase 1/8 inhibitor cytokine response modifier-A. ENZA did not reduce the phosphorylation of protein kinase B (Akt), but of p70 S6 kinase and of its substrate S6 protein in T98G cells. Inhibition of the phosphatidylinositol 3 kinase signaling pathway did not restore sensitivity of A172 cells towards ENZA, and constitutively active Akt did not protect LNT-229 and T98G cells from ENZA-induced apoptosis. Dephosphorylation of glycogen synthase kinase 3beta, a biomarker of ENZA action, and cell death induction by ENZA were separately regulated. Inhibition or activation of Akt only weakly modulated ENZA-induced dephosphorylation of glycogen synthase kinase 3beta. In ENZA-resistant A172 cells, apoptosis ligand 2 (Apo2L.0)-induced cleavage of caspases 3, 8, and 9 was increased by ENZA, resulting in synergistic activity of ENZA and Apo2L.0.

MTORC1 signaling requires proteasomal function and the involvement ofCUL4-DDB1 ubiquitin E3 ligase

The mammalian target-of-rapamycin (mTOR) signaling pathway serves as a major regulator of cell growth, cell size, and metabolism. In vivo, mTOR exists in two complexes, both of which contain the catalytic subunit mTOR, the invariable subunit mLST8, and a complex specific subunit Raptor or Rictor, forming either the rapamycin-sensitive mTORC1 or rapamycin-insensitive mTORC2, respectively. The exact functions of Raptor or Rictor in these complexes are still unclear. Here we demonstrate that mTORC1-mediated signaling events require the function of the 26S proteasome. Inhibition of the 26S proteasome by MG132 leads to the rapid inhibition of phosphorylation of the mTORC1 substrates S6 kinase and 4E-BP1. We have further discovered that the WD40 repeat proteins Raptor and mLST8 bind the CUL4-DDB1 ubiquitin E3 ligase. Loss of CUL4B or DDB1 specifically blocks the phosphorylation of S6 kinase at threonine 389 and 4E-BP1 at serine 65 and threonines 37 and 46, while loss of CUL4B enhances the phosphorylation of AKT at serine 473. These phosphorylation effects are identical to those resulting from the inactivation of Raptor. Our data suggest that the CUL4-DDB1 ubiquitin ligase interacts with Raptor and regulates the mTORC1-mediated signaling pathway through ubiquitin-dependent proteolysis.

Altered activation of AKT is required for the suppressive function of human CD4+CD25+ T regulatory cells

AbstractSuppression by T regulatory cells (Treg cells) is a major mechanism by which the immune system controls responses to self and nonharmful foreign proteins. Although there are many different types of Treg cells, the best characterized are those that constitutively express cell-surface IL-2Rα (CD25). We investigated whether altered T-cell–receptor (TCR)–mediated signaling in pure populations of ex vivo human CD4+CD25+ Treg cells might underlie their unique phenotype, including hyporesponsiveness to TCR–mediated activation and lack of cytokine production. CD4+CD25+ Treg cells displayed a consistent defect in phosphorylation of AKT at serine 473 and reduced phosphorylation of the AKT substrates FOXO and S6. Restoration of AKT activity via lentiviral-mediated expression of an inducibly active form of the kinase revealed that reduced activity of this pathway was necessary for the suppressive function of CD4+CD25+ Treg cells. These data represent the first demonstration of a causal association between altered signaling and the function of CD4+CD25+ Treg cells. Moreover, we have created the first system allowing inducible abrogation of suppression through manipulation of the suppressor cells. This system will be a powerful tool to further study the mechanism(s) of suppression by CD4+CD25+ Treg cells.

Phospholipase D2‐derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR

The product of phospholipase D (PLD) enzymatic action in cell membranes, phosphatidic acid (PA), regulates kinases implicated in NADPH oxidase activation, as well as the mammalian target of rapamycin (mTOR) kinase. However, other protein targets for this lipid second messenger must exist in order to explain other key PA-mediated cellular functions. In this study, PA was found to specifically and saturably bind to and activate recombinant and immunoprecipitated endogenous ribosomal S6 kinase (S6K) with a stoichiometry of 94:1 lipid/protein. Polyphosphoinositides PI4-P and PI4,5P2 and cardiolipin could also bind to and activate S6K, albeit with different kinetics. Conversely, PA with at least one acyl side chain saturated (10:0) was ineffective in binding or activating the enzyme. Transfection of COS-7 cells with a wild-type myc-(pcDNA)-PLD2 construct resulted in high PLD activity, concomitantly with an increase in ribosomal p70S6K enzyme activity and phosphorylation in T389 and T421/S424 as well as phosphorylation of p70S6K's natural substrate S6 protein in S235/S236. Overexpression of a lipase inactive mutant (K758R), however, failed to induce an increase in both PLD and S6K activity or phosphorylation, indicating that the enzymatic activity of PLD2 (i.e., synthesis of PA) must be present to affect S6K. Neither inhibiting mTOR kinase activity with rapamycin nor silencing mTOR gene expression altered the augmentative effect of PLD2 exerted on p70S6K activity. This finding indicates that PA binds to and activates p70S6K, even in the absence of mTOR. Lastly, COS-7 transfection with PLD2 changed the pattern of subcellular expression, and a colocalization of S6K and PLD2 was observed by immunofluorescence microscopy. These results show for the first time a direct (mTOR-independent) participation of PLD in the p70S6K pathway and implicate PA as a nexus that brings together cell phospholipases and kinases.

Hypoxia-Induced Endothelial Proliferation Requires Both mTORC1 and mTORC2

A central regulator of cell growth that has been implicated in responses to stress such as hypoxia is mTOR (mammalian Target Of Rapamycin). We have shown previously that mTOR is required for angiogenesis in vitro and endothelial cell proliferation in response to hypoxia. Here we have investigated mTOR-associated signaling components under hypoxia and their effects on cell proliferation in rat aortic endothelial cells (RAECs). Hypoxia (1% O(2)) rapidly (>30 minutes) and in a concentration-dependent manner promoted rapamycin-sensitive and sustained phosphorylation of mTOR-Ser2448 followed by nuclear translocation in RAECs. Similarly, hypoxia induced phosphorylation of the mTORC2 substrate Akt-Ser473 (3 to 6 hours at 1% O(2)) and a brief phosphorylation peak of the mTORC1 substrate S6 kinase-Thr389 (10 to 60 minutes). Phosphorylation of Akt was inhibited by mTOR knockdown and partially with rapamycin. mTOR knockdown, rapamycin, or Akt inhibition specifically and significantly inhibited proliferation of serum-starved RAECs under hypoxia (P<0.05; n> or =4). Similarly, hypoxia induced Akt-dependent and rapamycin-sensitive proliferation in mouse embryonic fibroblasts. This response was partially blunted by hypoxia-inducible factor-1alpha knockdown and not affected by TSC2 knockout. Finally, mTORC2 inhibition by rictor silencing, especially (P<0.001; n=7), and mTORC1 inhibition by raptor silencing, partially (P<0.05; n=7), inhibited hypoxia-induced RAEC proliferation. Thus, mTOR mediates an early response to hypoxia via mTORC1 followed by mTORC2, promoting endothelial proliferation mainly via Akt signaling. mTORC1 and especially mTORC2 might therefore play important roles in diseases associated with hypoxia and altered angiogenesis.

Expression of an active LKB1 complex in cardiac myocytes results in decreased protein synthesis associated with phenylephrine-induced hypertrophy

AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte. Recently, LKB1 was identified as a kinase that regulates AMPK. Using immunoblot analysis, we confirmed high expression of LKB1 in isolated rat cardiac myocytes but show that, under basal conditions, LKB1 is primarily localized to the nucleus, where it is inactive. We examined the role of LKB1 in cardiac myocytes, using adenoviruses that express LKB1, and its binding partners Ste20-related adaptor protein (STRADalpha) and MO25alpha. Infection of neonatal rat cardiac myocytes with all three adenoviruses substantially increased LKB1/STRADalpha/MO25alpha expression, LKB1 activity, and AMPKalpha phosphorylation at its activating phosphorylation site (threonine-172). Since activation of AMPK can inhibit hypertrophic growth and since LKB1 is upstream of AMPK, we hypothesized that expression of an active LKB1 complex would also inhibit protein synthesis associated with hypertrophic growth. Expression of the LKB1/STRADalpha/MO25alpha complex in neonatal rat cardiac myocytes inhibited the increase in protein synthesis observed in cells treated with phenylephrine (measured via [(3)H]phenylalanine incorporation). This was associated with a decreased phosphorylation of p70S6 kinase and its substrate S6 ribosomal protein, key regulators of protein synthesis. In addition, we show that the pathological cardiac hypertrophy in transgenic mice with cardiac-specific expression of activated calcineurin is associated with a significant decrease in LKB1 expression. Together, our data show that increased LKB1 activity in the cardiac myocyte can decrease hypertrophy-induced protein synthesis and suggest that LKB1 activation may be a method for the prevention of pathological cardiac hypertrophy.

Effect of TRB3 on Insulin and Nutrient-stimulated Hepatic p70 S6 Kinase Activity

Insulin and nutrients activate hepatic p70 S6 kinase (S6K1) to regulate protein synthesis. Paradoxically, activation of S6K1 also leads to the development of insulin resistance. In this study, we investigated the effect of TRB3, which acts as an endogenous inhibitor of Akt, on S6K1 activity in vitro and in vivo. In cultured cells, overexpression of TRB3 completely inhibited insulin-stimulated S6K1 activation by mammalian target of rapamycin, whereas knockdown of endogenous TRB3 increased both basal and insulin-stimulated activity. In C57BL/6 mice, adenoviral overexpression of TRB3 inhibited insulin-stimulated activation of hepatic S6K1. In contrast, overexpression of TRB3 did not inhibit nutrient-stimulated S6K1 activity. We also investigated the effect of starvation, feeding, or insulin treatment on TRB3 levels and S6K1 activity in the liver of C57BL/6 and db/db mice. Both insulin and feeding activate S6K1 in db/db mice, but only insulin activates in the C57BL/6 strain. TRB3 levels were 3.5-fold higher in db/db mice than C57BL/6 mice and were unresponsive to feeding or insulin, whereas both treatments reduced TRB3 in C57BL/6 mice. Akt was activated by insulin alone in the C57BL/6 strain and but not in db/db mice. Both insulin and feeding activated mammalian target of rapamycin similarly in these mice; however, feeding was unable to activate the downstream target S6K1 in C57BL/6 mice. These results suggest that the nutrient excess in the hyperphagic, hyperinsulinemic db/db mouse primes the hepatocyte to respond to nutrients resulting in elevated S6K1 activity. The combination of elevated TRB3 and constitutive S6K1 activity results in decreased insulin signaling via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway.

Serotonin Activates S6 Kinase in a Rapamycin-Sensitive Manner inAplysiaSynaptosomes

The identification of tags that can specifically mark activated synapses is important for understanding how long-term synaptic changes can be restricted to specific synapses. The maintenance of synapse-specific facilitation in Aplysia sensory to motor neuron cultures can be blocked by inhibitors of translation and by the drug rapamycin, which specifically blocks a signaling pathway that regulates phosphorylation of translational regulators. One important target of rapamycin is the phosphorylation and subsequent activation of S6 kinase. To test whether S6 kinase is the target for the ability of rapamycin to block synapse-specific facilitation in Aplysia, we cloned Aplysia S6 kinase, its substrate S6, and the S6 kinase kinase phosphoinositide-dependent kinase 1 (PDK-1). Serotonin, which induces synapse-specific facilitation, increased phosphorylation of Aplysia S6 kinase at threonine 399 in a rapamycin-sensitive manner in Aplysia synaptosomes. The phosphorylation of threonine 399 by 5-HT was independent of phosphoinositide-3 kinase, dependent on PKA and PKC, and occluded by the phosphatase inhibitor calyculin-A. 5-HT also increased S6 kinase activity and led to increased phosphorylation of S6 in synaptosomes. 5-HT increased levels of S6 in synaptosomes because of a rapamycin-sensitive increase in translation-stabilization of S6. Aplysia PDK-1 bound to and phosphorylated Aplysia S6 kinase but only modulated phosphorylation of threonine 399 indirectly. These results suggest a mechanism by which the levels of translation factors can be increased specifically at activated synapses generating a long-lasting synaptic tag.