Kegg Pathway: mTOR signaling pathway

Cancer Biol Ther. 2009 Dec 19; 8(24):
No M, Choi EJ, Kim IA

Several studies have indicated the potential value of targeting HER-2 signaling to enhance the anti-tumor activity of ionizing radiation. However, therapeutic resistance resulting from several factors, including activation of the downstream pathway, represents a major obstacle to treatment. Here, we investigated whether inhibitors targeting downstream of HER-2 signaling would radiosensitize SKBR3 breast cancer cells that exhibit overamplification of HER2. Selective inhibition of MEK-ERK signaling using pharmacologic inhibitors (PD98059, UO126) did not increase the radiosensitivity of SKBR3 cells. Selective inhibition of the PI3K-AKT-mTOR pathway using pharmacologic inhibitors (LY294002, AKT inhibitor VIII, Rapamycin) significantly attenuated expression of p-AKT and p-70S6K, respectively and radiosensitized SKBR3 cells. MCF-7 cells those did not overexpress HER-2, showed less radiosensitization compared to SKBR3 cells by inhibition of this pathway. Pre-treatment with these inhibitors also caused significant abrogation of typical G(2) arrest following ionizing radiation and induced marked prolongation of gammaH2AX foci indicating impairment of DNA damage repair. A dual inhibitor of Class I PI3K and mTOR, PI103 effectively radiosensitized SKBR3 cells and showed significant prolongation of gammaH2AX foci. Inhibition of PI3K-AKT signaling was associated with downregulation of DNA-PKs, respectively. While apoptosis was the major mode of cell death when the cells were pretreated with LY294002 or AKT inhibitor VIII, the cells were pretreated by rapamycin or PI103 showed mixed mode of cell death including autophagy. Our results suggest possible mechanisms to counteract the HER-2 prosurvival signaling implicated in radioresistance, and offer an alternative strategy to overcome resistance to HER-2 inhibitors combined with radiation.

Clin Transplant. 2009 Nov 16;
Ho CM, Huang SF, Hu RH, Ho MC, Wu YM, Lee PH

Sirolimus is one treatment option in transplant recipients with Kaposi's sarcoma (KS), which involves dysregulation of Akt-mammalian target of rapamycin (mTOR) signaling pathway. Signal modifications after sirolimus therapy in organ recipients with KS are largely unknown and not verified. We reported a case of KS found two yr after liver transplantation in which the immunosuppression was changed from tacrolimus, MMF, and steroid to sirolimus alone. In skin, which was found to have persistent KS after a two-month treatment of sirolimus and was removed completely one yr later, KS was no longer present. The patient went well without graft rejection. Tumor biopsies were performed before, two months, and one yr after the start of sirolimus. Immunohistochemical staining of vascular endothelial growth factor (VEGF), p-Akt, p-mTOR, p-p70 S6 kinase, and Western blot for p-tuberin/ tuberous sclerosis complex (TSC)2 was performed. VEGF was suppressed thoroughly in two-month use of sirolimus. In addition, p-Akt and p-mTOR, which were decreased at two months, could not be detected after one yr of treatment. Moreover, p-p70 S6 kinase, expressed strongly in overlying epidermis initially, was suppressed completely after two months of treatment. However, p-tuberin/TSC2, contrary to suggested theoretically, was not detected through all specimens, implying not to be a significant event. Suppressed expression of VEGF, p-Akt, and p-mTOR was the major event of signaling modification through the long-term use of sirolimus.

J Immunol. 2009 Dec 1; 183(11): 7388-7397
Salmond RJ, Emery J, Okkenhaug K, Zamoyska R

Ribosomal protein S6 (rpS6) is a key component of the translational machinery in eukaryotic cells and is essential for ribosome biogenesis. rpS6 is phosphorylated on evolutionarily conserved serine residues, and data indicate that rpS6 phosphorylation might regulate cell growth and protein synthesis. Studies in cell lines have shown an important role for the serine kinase mammalian target of rapamycin (mTOR) in rpS6 phosphorylation, further linking rpS6 to control of cellular metabolism. rpS6 is essential in T cells because its deletion in mouse double-positive thymocyte cells results in a complete block in T cell development; however, the signaling pathway leading to rpS6 phosphorylation downstream of TCR stimulation has yet to be fully characterized. We show that maximal TCR-induced rpS6 phosphorylation in CD8 T cells requires both Lck and Fyn activity and downstream activation of PI3K, mTOR, and MEK/ERK MAPK pathways. We demonstrate that there is cross-talk between the PI3K and MAPK pathways as well as PI3K-independent mTOR activity, which result in differential phosphorylation of specific rpS6 serine residues. These results place rpS6 phosphorylation as a point of convergence for multiple crucial signaling pathways downstream of TCR triggering.

J Hepatol. 2009 Oct 28;
Huynh H, Ngo VC, Koong HN, Poon D, Choo SP, Toh HC, Thng CH, Chow P, Ong HS, Chung A, Goh BC, Smith PD, Soo KC

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a particularly vascularized solid tumor where the Raf/MEK/ERK pathway is activated; suggesting that inhibition of this pathway may have therapeutic potential. METHODS: We treated patient-derived HCC xenografts with (i) sorafenib, (ii) AZD6244 (ARRY-142886), and (iii) sorafenib plus AZD6244. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant to both angiogenesis and MEK signaling. Apoptosis, microvessel density, and cell proliferation were analyzed by immunohistochemistry. RESULTS: We report here that sorafenib treatment resulted in suppression of tumor growth, reduction in cell proliferation, induction of apoptosis and inhibition of mTOR targets. Sorafenib-induced elevation of the insulin-like growth factor receptor 1 (IGF-1R), phospho-c-Raf Ser338, phospho-MEK Ser217/221 and phospho-ERK Thr202/Tyr204 was attenuated by co-treating cells with anti-human IGF-1R antibody or over-expression of activated mutant p70S6K. Pharmacological inhibition of the MEK/ERK pathway by AZD6244 enhanced the anti-tumor effect of sorafenib in both orthotopic and ectopic models of HCC. Such inhibition led to a further increase in pro-apoptotic Bim, apoptosis and a profound inhibition of cell proliferation. CONCLUSION: Our findings underscore the potential of a combined therapeutic approach with sorafenib and MEK inhibitors in the treatment of HCC.

J Biol Chem. 2009 Nov 11;
Ohira T, Arita M, Omori K, Recchiuti A, Van Dyke TE, Serhan CN

Resolvins are endogenous lipid mediators that actively regulate the resolution of acute inflammation. Resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is an endogenous anti-inflammatory and pro-resolving mediator derived from eicosapentaenoic acid that regulates leukocyte migration and enhances macrophage phagocytosis of apoptotic neutrophils to resolve inflammation. In the inflammatory milieu, RvE1 mediates counter-regulatory actions initiated via specific G-protein-coupled receptors (GPCR). Here, we identify RvE1 specific signaling pathways initiated by the RvE1 receptor ChemR23. RvE1 stimulated phosphorylation of Akt that was both ligand and receptor dependent. RvE1 regulated Akt phosphorylation in a time (0-15 min) and dose-dependent (0.01-100 nM) manner in human ChemR23-transfected CHO cells. RvE1 stimulated phosphorylation of both Akt and a 30-kDa protein, a downstream target of Akt, identified using a phospho-Akt substrate antibody. The 30-kDa protein was identified as ribosomal S6 protein, a translational regulator, and its phosphorylation was inhibited by a PI3-K inhibitor (wortmannin) and an ERK inhibitor (PD98059) but not by a p38-MAPK inhibitor (SB203580). Ribosomal S6 protein is a downstream target of the PI3-K/Akt signaling pathway as well as the Raf/ERK pathway. In ChemR23 expressing differentiated HL60 cells, RvE1 also stimulated the phosphorylation of ribosomal S6 protein. In addition, RvE1 enhanced phagocytosis of zymosan A by human macrophages that is inhibited by PD98059 and rapamycin (mTOR inhibitor). These results indicate that RvE1 initiates direct activation of ChemR23 and signals receptor dependent phosphorylation. These phosphorylation-signaling pathways identified for RvE1 receptor ligand interactions underscore the importance of endogenous pro-resolving agonists in resolving acute inflammation.

Am J Physiol Endocrinol Metab. 2009 Nov 10;
James LR, Le C, Scholey JW

Cells exposed to high glucose may undergo hypertrophy, proliferation, and apoptosis, but the role of hexosamine flux in mediating these effects has not been fully elucidated. Accordingly, we studied the effects of glucose and glucosamine on rat glomerular mesangial cells (MC) turnover. In comparison with physiologic glucose (5.6mM), treatment with high glucose (25mM) for 24 hours stimulated MC proliferation, an effect that was mimicked by exposure to low concentrations of glucosamine (0.05mM). The percentage of cells in G(0)/G(1) phase of the cell cycle was reduced with a concomitant increase of the number of cells in G2/M phase. Proliferating cell nuclear antigen (PCNA), phosphorylated mammalian target of rapamycin (phospho-mTOR [ser2448]) and total regulatory associated protein of mTOR (Raptor) were increased by high glucose and glucosamine treatment. Inhibition of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for hexosamine flux, with 6-diazo-5-oxonorleucine (DON, 10microM) and of mTOR with rapamycin both attenuated glucose-mediated MC proliferation. Higher glucosamine concentrations (0.25mM to 10mM) caused MC apoptosis after 48 hours and, in addition, GFAT overexpression also increased MC apoptosis (TUNEL-positive cells: 3.8+/-0.3 % versus 1.1+/-0.2% for empty vector; p<0.001). Hence, hexosamine flux is an important determinant of MC proliferation and apoptosis. The proliferative response to high glucose and hexosamine flux is rapamycin-sensitive, suggesting that this effect is associated with signaling through rapamycin-sensitive mTOR complex 1 (mTORC1).

Oncogene. 2009 Nov 9;
Xu X, Ehdaie B, Ohara N, Yoshino T, Deng CX

Mutations of SMAD4/DPC4 are found in about 60% of human invasive pancreatic ductal adenocarcinomas (PDACs); yet, the manner in which SMAD4 deficiency enhances tumorigenesis remains elusive. Using a Cre-LoxP approach, we generated a mutant mouse carrying a targeted deletion of Smad4 in the pancreas. We showed that the absence of Smad4 alone did not trigger pancreas tumor formation; however, it increased the expression of an inactivated form of Pten, suggesting a role of Pten in preventing Smad4-/- cells from undergoing malignancy. To investigate this, we disrupted both Pten and Smad4. We showed that Pten deficiency initiated widespread premalignant lesions, and a low tumor incidence that was significantly accelerated by Smad4-deficiency. The absence of Smad4 in a Pten-mutant background enhanced cell proliferation and triggered transdifferentiation from acinar, centroacinar and islet cells, accompanied by activation of Notch1 signaling. We showed that all tumors developed in the Smad4/Pten-mutant pancreas exhibited high levels of pAKT and mTOR, and that about 50 and 83% of human pancreatic cancers examined showed increased pAKT and pmTOR, respectively. Besides the similarity in gene expression, the pAKT and/or pmTOR-positive human PDACs and mouse pancreatic tumors also shared some histopathological similarities. These observations indicate that Smad4/Pten-mutant mice mimic the tumor progression of human pancreatic cancers that are driven by activation of the AKT-mTOR pathway, and uncovered a synergistic action of Smad4 and Pten in repressing pancreatic tumorigenesis.Oncogene advance online publication, 9 November 2009; doi:10.1038/onc.2009.375.

Target Oncol. 2009 Nov 7;
Capdevila J, Salazar R

Gastroenteropancreatic neuroendocrine tumors (GEPNETs) are rare neoplasms that require a multidisciplinary approach for an optimal management. The traditional cytotoxic agents are of limited efficacy in the treatment of these tumors. A better understanding of the molecular pathways that characterize tumor growth has provided novel targets in cancer treatment. Several proteins have been implicated as having a crucial role in GEPNETs. Several proangiogenic molecules are overexpressed in GEPNETs including vascular endothelial growth factor (VEGF) and its receptors, and related signaling pathway components such as epidermal growth factor receptor (EGFR), insulin growth factor-I receptor (IGF-IR) and PI3K-AKT-mTOR pathway. In this article we aim to review the recent development of the main molecules that target these proteins and have showed promising activity in the treatment of GEPNETs.

Mol Cell Endocrinol. 2009 Nov 5;
Saji M, Ringel MD

The Phosphoinositide 3 (OH) kinase (PI3K) signaling cascade is involved in regulating glucose uptake and metabolism, growth, motility, and other essential functions for cell survival. Unregulated activation of this pathway commonly occurs in cancer through a variety of mechanisms, including genetic mutations of kinases and regulatory proteins, epigenetic alterations that alter gene expression and translation, and posttranslational modifications. In thyroid cancer, constitutive activation of PI3K signaling has been shown to play a role in the genetic predisposition for thyroid neoplasia in Cowden's syndrome, and is recognized to be frequently overactivated in sporadic forms of thyroid cancer including those with aggressive clinical behaviors. In this review, the key signaling molecules in the PI3K signaling cascade, the abnormalities known to occur in thyroid cancer, and the potential for therapeutic targeting of PI3K pathway members will be discussed.

J Biol Chem. 2009 Nov 4;
Leee DY, Li YS, Chang SF, Zhou J, Ho HM, Chiu JJ, Chien S

Interstitial flow in and around bone tissue is oscillatory in nature and affects the mechanical microenvironment for bone cell growth and formation. We investigated the role of oscillatory shear stress (OSS) in modulating the proliferation of human osteoblast-like MG63 cells and its underlying mechanisms. Application of OSS (0.5+/-4 dynes/cm2) to MG63 cells induced sustained activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/p70S6K (p70S6 kinase) signaling cascades and hence cell proliferation, which was accompanied by increased expressions of cyclins A and D1, cyclin-dependent protein kinases (Cdk)-2, -4, and -6, and bone formation-related genes (c-fos, early growth response-1, and cyclooxygenase-2), and decreased expressions of p21CIP1 and p27KIP1. These OSS-induced activation of PI3K/Akt/mTOR/p70S6K and cell proliferation were inhibited by specific antibodies or small interference RNAs of alphav beta3 and beta1 integrins, and by dominant-negative mutants of Shc (Shc-SH2) and focal adhesion kinase (FAK) (FAK[F397Y]).Co-immunoprecipitation assay showed that OSS induces sustained increases in associations of Shc and FAK with alphav beta3 and beta1 integrins and PI3K subunit p85, which were abolished by transfecting the cells with FAK(F397Y) or Shc-SH2. OSS also induced sustained activation of extracellular signal-regulated kinase (ERK), which was inhibited by the specific PI3K inhibitor LY294002 and was required for OSS-induced activation of mTOR/p70S6K and proliferation in MG63 cells. Our findings provide insights into the mechanisms by which OSS induces osteoblast-like cell proliferation through activation of alphav beta3 and beta1 integrins and synergistic interactions of FAK and Shc with PI3K, leading to the modulation of downstream ERK and Akt/mTOR/p70S6K pathways.

Expert Opin Investig Drugs. 2009 Nov 4;
Trinh XB, van Dam PA, Dirix LY, Vermeulen PB, Tjalma WA

The AKT/mTOR signaling pathway is frequently overexpressed in human epithelial ovarian cancer and an attractive target for therapy. In vivo mouse models were confirmative for in vitro findings, where the administration of mTOR inhibitors in ovarian cancer xenografts showed antitumoral as well as antiangiogenic effects. Phase I - II trials are now ongoing with mTOR inhibitors in ovarian cancer patients, some in combination with conventional cytotoxic agents. If further development of mTOR inhibition in ovarian cancer is pursued, studying combinations of mTOR inhibitors with other new targeted therapies would be of interest. mTOR inhibitors in the adjuvant setting could have potential, since, for the moment, there is no standard maintenance therapy in ovarian cancer. A crucial challenge will be to identify strong predictive biomarkers. This review highlights the rationale for the use of mTOR inhibitors in ovarian cancer and summarizes the available preclinical findings.

Endocrinology. 2009 Nov 3;
Mayer CM, Belsham DD

Central insulin signaling is critical for the prevention of insulin resistance. Hyperinsulinemia contributes to insulin resistance, but it is not yet clear whether neurons are subject to cellular insulin resistance. We used an immortalized, hypothalamic, clonal cell line, mHypoE-46, which exemplifies neuronal function and expresses the components of the insulin signaling pathway, to determine how hyperinsulinemia modifies neuronal function. Western blot analysis indicated that prolonged insulin treatment of mHypoE-46 cells attenuated insulin signaling through phospho-Akt. To understand the mechanisms involved, time-course analysis was performed. Insulin exposure for 4 and 8 h phosphorylated Akt and p70-S6 kinase (S6K1), whereas 8 and 24 h treatment decreased insulin receptor (IR) and IR substrate 1 (IRS-1) protein levels. Insulin phosphorylation of S6K1 correlated with IRS-1 ser1101 phosphorylation and the mTOR-S6K1 pathway inhibitor rapamycin prevented IRS-1 serine phosphorylation. The proteasomal inhibitor epoxomicin and the lysosomal pathway inhibitor 3-methyladenine prevented the degradation of IRS-1 and IR by insulin, respectively, and pretreatment with rapamycin, epoxomicin, or 3-methyladenine prevented attenuation of insulin signaling by long-term insulin exposure. Thus, a sustained elevation of insulin levels diminishes neuronal insulin signaling through mTOR-S6K1-mediated IRS-1 serine phosphorylation, proteasomal degradation of IRS-1 and lysosomal degradation of the IR.

Oncogene. 2009 Nov 2;
Markova B, Albers C, Breitenbuecher F, Melo JV, Brümmendorf TH, Heidel F, Lipka D, Duyster J, Huber C, Fischer T

In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-gamma1 (PLC-gamma1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-gamma1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-gamma1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-gamma1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-gamma1 and Bcr-Abl inhibition. The pivotal role of PLC-gamma1 was further confirmed in a mouse leukemogenesis model.Oncogene advance online publication, 2 November 2009; doi:10.1038/onc.2009.374.

Biochem Pharmacol. 2009 Oct 29;
Arellano-Plancarte A, Hernandez-Aranda J, Catt KJ, Olivares-Reyes JA

To investigate the potential interactions between the angiotensin II (Ang II) and insulin signaling systems, regulation of IRS-1 phosphorylation and insulin-induced Akt activation by Ang II were examined in clone 9 (C9) hepatocytes. In these cells, Ang II specifically inhibited activation of insulin-induced Akt Thr(308) and its immediate downstream substrate GSK-3alpha/beta in a time-dependent fashion, with approximately 70% reduction at 15min. These inhibitory actions were associated with increased IRS-1 phosphorylation of Ser(636)/Ser(639) that was prevented by selective blockade of EGFR tyrosine kinase activity with AG1478. Previous studies have shown that insulin-induced phosphorylation of IRS-1 on Ser(636)/Ser(639) is mediated mainly by the PI3K/mTOR/S6K-1 sequence. Studies with specific inhibitors of PI3K (wortmannin) and mTOR (rapamycin) revealed that Ang II stimulates IRS-1 phosphorylation of Ser(636)/Ser(639) via the PI3K/mTOR/S6K-1 pathway. Both inhibitors blocked the effect of Ang II on insulin-induced activation of Akt. Studies using the specific MEK inhibitor, PD98059, revealed that ERK1/2 activation also mediates Ang II-induced S6K-1 and IRS-1 phosphorylation, and the impairment of Akt Thr(308) and GSK-3alpha/beta phosphorylation. Further studies with selective inhibitors showed that PI3K activation was upstream of ERK, suggesting a new mechanism for Ang II-induced impairment of insulin signaling. These findings indicate that Ang II has a significant role in the development of insulin resistance by a mechanism that involves EGFR transactivation and the PI3K/ERK1/2/mTOR-S6K-1 pathway.

Carcinogenesis. 2009 Oct 29;
Chen M, Cassidy A, Gu J, Delclos GL, Zhen F, Yang H, Hildebrandt M, Lin J, Ye Y, Chamberlain RM, Dinney CP, Wu X

Genetic variations in PI3K-AKT-mTOR pathway may affect critical cellular functions and increase an individual's cancer risk. We systematically evaluate 231 single nucleotide polymorphisms (SNPs) in 19 genes in the PI3K-AKT-mTOR signaling pathway as predictors of bladder cancer risk. In individual SNP analysis, 4 SNPs in RAPTOR remained significant after correcting for multiple testing: rs11653499 (OR: 1.79, 95%CI: 1.24-2.60, P = 0.002), rs7211818 (OR: 2.13, 95%CI: 1.35-3.36, P = 0.001), rs7212142 (OR: 1.57, 95%CI: 1.19-2.07, P = 0.002), and rs9674559 (OR: 2.05, 95%CI: 1.31-3.21, P = 0.002), among which rs7211818 and rs9674559 are within the same haplotype block. In haplotype analysis, compared to the most common haplotypes, haplotype containing the rs7212142 wild type allele showed a protective effect of bladder cancer (OR:0.83, 95%CI: 0.70-0.97). In contrast, the haplotype containing the rs7211818 variant allele showed a 1.32-fold elevated bladder cancer risk (95%CI: 1.09-1.60). In combined analysis of 3 independent significant RAPTOR SNPs (rs11653499, rs7211818, and rs7212142), a significant trend was observed for increased risk with an increase in the number of unfavorable genotypes (P for trend<0.001). Compared to the subjects without any of the unfavorable genotypes, those carrying all 3 unfavorable genotypes showed a 2.22 fold (95%CI: 1.33-3.71) fold increased bladder cancer risk. This is the first study to evaluate the role of germline genetic variations in PI3K-AKT-mTOR pathway as cancer susceptibility factors which will help us identify high risk individuals for bladder cancer.

J Biol Chem. 2009 Oct 28;
Foster KG, Acosta-Jaquez HA, Romeo Y, Ekim B, Soliman GA, Carriere A, Roux PP, Ballif BA, Fingar DC

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (Cluster 1: S696/T706 and Cluster 2: S855/S859/S863/S877) using tandem mass spectrometry and have generated phospho-specific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor S863 phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor S863 via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e.g. amino acids; EGF/MAPK signaling; cellular energy) also promote raptor S863 phosphorylation. Rheb overexpression increases phosphorylation on raptor S863 as well as on the five other identified sites (e.g. S859; S855; S877; S696; T706). Strikingly, raptor S863 phosphorylation is absolutely required for raptor S859 and S855 phosphorylation. These data suggest that mTORC1 activation leads to raptor multi-site phosphorylation and that raptor S863 phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e.g. on S859; S855). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multi-site raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues.

J Biol Chem. 2009 Oct 28;
Sunami T, Byrne N, Diehl RE, Funabashi K, Hall DL, Ikuta M, Patel SB, Shipman JM, Smith RF, Takahasi I, Zugay-Murphy J, Iwasawa Y, Lumb KJ, Munshi SK, Sharma S

p70 ribosomal S6 kinase (p70S6K) is a downstream effector of the mTOR signaling pathway involved in cell proliferation, cell growth, cell-cycle progression and glucose homeostasis. Multiple phosphorylation events within the catalytic, autoinhibitory and hydrophobic motif domains contribute to the regulation of p70S6K. We report the crystal structures of the kinase domain of p70S6K bound to staurosporine in both the unphosphorylated state and in the 3'-phosphoinositide-dependent kinase-1 (PDK1) phosphorylated state in which Thr 252 of the activation loop is phosphorylated. Unphosphorylated p70S6K1 exists in two crystal forms; one in which the p70S6K1 kinase domain exists as a monomer and the other as a domain-swapped dimer. The crystal structure of the partially activated kinase domain that is phosphorylated within the activation loop reveals conformational ordering of the activation loop that is consistent with a role in activation. The structures offer insights into the structural basis of the PDK1-induced activation of p70S6K and provide a platform for the rational structure-guided design of specific p70S6K inhibitors.

Pathogenetics. 2009 Oct 28; 2(1): 6
Boletta A

ABSTRACT: Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease characterized by the formation of renal cysts. This disease can be caused by mutations in two genes, PKD1 and PKD2, which encode polycystin-1 (PC-1) and -2 (PC-2), respectively. PC-1 is a large plasma membrane receptor involved in the regulation of several biological functions and signaling pathways, and PC-2 is a calcium channel of the TRP family. The two proteins associate in a complex to prevent cyst formation, but the precise mechanism(s) involved remain largely unknown. This review will focus on recent advances in our understanding of the functions of polycystins and their role in signal transduction. Increased activity of the mammalian target of rapamycin (mTOR) kinase has been observed in cysts found in ADPKD tissues. Rapamycin has been shown to have beneficial effects in rodent models of polycystic kidney disease, prompting the initiation of pilot clinical trials with human patients. Furthermore, a direct role for PC-1 in the regulation of cell growth (size) via mTOR has recently been demonstrated. Major advancements in the study of mTOR biology have highlighted that this kinase exists in association with two different complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The mTORC1 complex regulates cell growth (size), proliferation, translation and autophagy, and mTORC2 regulates the actin cytoskeleton and apoptosis. Interestingly, mTORC2 has been shown to contain the kinase responsible for the phosphorylation of Akt at Serine 473. Previous studies have shown that PC-1 controls the PI 3-kinase/Akt cascade to regulate apoptosis and the actin cytoskeleton, suggesting that this receptor might regulate mTOR at several levels. This review aims to discuss three different, inter-related themes emerging from the literature: (i) studies performed in our and other laboratories collectively suggest that PC-1 might be able to differentially regulate the two mTOR complexes; (ii) several studies point to genetic and functional cross-talk between the PKD and TSC genes, although the molecular details remain obscure; and (iii) studies performed in mammals and in the unicellular algae Chlamidomonas Reinhardtii might highlight a link between cilia, regulation of cell size and regulation of the cell cycle.

Int J Cancer. 2009 Oct 23;
Shigematsu H, Yoshida K, Sanada Y, Osada S, Takahashi T, Wada Y, Konishi K, Okada M, Fukushima M

We have previously reported the synergistic cytotoxic effects of Docetaxel (TXT) and S-1 in gastric cancer in vitro and in vivo and the combination regimen is now under Phase III clinical trail. In the present study, in order to elucidate whether the rapamycin, the inhibitor of the mTOR (mammalian target of rapamaycin), can enhance the potentiation of TXT and 5-fluorouracil (5-Fu) in gastric carcinoma cells. Rapamycin inhibited the growth of TMK-1, MKN-28, MKN-45 and MKN-74 cell lines by MTT assay and it demonstrated the cytostatic effects as G1 arrest shown by flowcytemetry. However, the cytotoxic effects of 5-Fu, TXT and cisplatin (CDDP) were enhanced by 2 to 4 times with the concomitant administration of rapamycin.In order to clarify the mechanism of the potentiation, the expression changes of the enzymes relating DNA metabolism and cell growth signal transduction pathways were examined by western blot analysis. Interestingly, the expression of thymidilate synthase (TS) were markedly decreased by the administration of rapamycin in TMK-1 cells in a time and dose dependent manner. Moreover, rapamycin decreased the phosphorylation of 4E-BP1, the phosphorylation of ERK1/2 and enhanced the phosphorylation of c-Jun NH2-terminal kinase (JNK) as well as the activation of caspase of apoptotic pathways in combination with TXT. These results strongly indicate that the mTOR inhibitor can enhance the potentiation of TXT and 5-Fu or S-1 and can serve as a new therapeutic tool for advanced and recurrent gastric cancer patients. (c) 2009 UICC.

Nutr Res. 2009 Sep; 29(9): 640-7
Ndong M, Kazami M, Suzuki T, Uehara M, Katsumata S, Inoue H, Kobayashi K, Tadokoro T, Suzuki K, Yamamoto Y

Iron deficiency (ID) is one of the most commonly known forms of nutritional deficiencies. Low body iron is thought to induce neurologic defects but may also play a protective role against cancer development by cell growth arrest. Thus, ID may affect cellular pathways controlling cell growth and proliferation, the mechanism of which is still not fully understood. The serine/threonine protein kinase Akt and its downstream target, the mammalian Target of Rapamycin (mTOR), is known to play a crucial role in the regulation of cell growth and survival. Therefore, we hypothesized that Akt/mTOR pathway could be influenced by ID. Three-week-old male Wistar-strain rats were divided into 3 groups and the 2 groups had free access to a control diet (C group) or an iron-deficient diet (D group). The third group (PF group) were pair-fed the control diet to the mean intake of the D group. After 4 weeks, rats were killed and their brains were sampled. In separate experiments, COS-1 cells were cultured with or without the iron chelator deferoxamine. Western blots of brain samples and COS-1 lysates were used to analyze the expression and phosphorylation state of Akt, TSC2, mTOR, and S6 kinase proteins implicated in the Akt/mTOR pathway. Using 2 different ID models, we show for the first time that iron deficiency depresses Akt activity in rats and in COS-1 cells, leading to a decrease in mTOR activity.