Protein synthesis inhibition promotes nitric oxide generation and activation of CGKII-dependent downstream signaling pathways in the retina

Nitric oxide is an important neuromodulator in the CNS, and its production within neurons is modulated by NMDA receptors and requires a fine-tuned availability of L-arginine. We have previously shown that globally inhibiting protein synthesis mobilizes intracellular L-arginine “pools” in retinal neurons, which concomitantly enhances neuronal nitric oxide synthase-mediated nitric oxide production. Activation of NMDA receptors also induces local inhibition of protein synthesis and L-arginine intracellular accumulation through calcium influx and stimulation of eucariotic elongation factor type 2 kinase. We hypothesized that protein synthesis inhibition might also increase intracellular L-arginine availability to induce nitric oxide-dependent activation of downstream signaling pathways. Here we show that nitric oxide produced by inhibiting protein synthesis (using cycloheximide or anisomycin) is readily coupled to AKT activation in a soluble guanylyl cyclase and cGKII-dependent manner. Knockdown of cGKII prevents cycloheximide or anisomycin-induced AKT activation and its nuclear accumulation. Moreover, in retinas from cGKII knockout mice, cycloheximide was unable to enhance AKT phosphorylation. Indeed, cycloheximide also produces an increase of ERK phosphorylation which is abrogated by a nitric oxide synthase inhibitor. In summary, we show that inhibition of protein synthesis is a previously unanticipated driving force for nitric oxide generation and activation of downstream signaling pathways including AKT and ERK in cultured retinal cells. These results may be important for the regulation of synaptic signaling and neuronal development by NMDA receptors as well as for solving conflicting data observed when using protein synthesis inhibitors for studying neuronal survival during development as well in behavior and memory studies.     

Inhibition of protein synthesis by activation of NMDA receptors in cultured retinal cells: a new mechanism for the regulation of nitric oxide production

The synthesis of nitric oxide (NO) is limited by the intracellular availability of L-arginine. Here we show that stimulation of NMDA receptors promotes an increase of intracellular L-arginine which supports an increase in the production of NO. Although L-[3H]arginine uptake measured in cultured chick retina cells incubated in the presence of cycloheximide (CHX, a protein synthesis inhibitor) was inhibited approximately 75% at equilibrium, quantitative thin-layer chromatography analysis showed that free intracellular L-[3H]arginine was six times higher in CHX-treated than in control cultures. Extracellular L-[3H]citrulline levels increased threefold in CHX-treated groups, an effect blocked by NG-nitro-L-arginine, a NO synthase (NOS) inhibitor. NMDA promoted a 40% increase of free intracellular L-[3H]arginine in control cultures, an effect blocked by the NMDA antagonist 2-amino 5-phosphonovaleric acid. In parallel, NMDA promoted a reduction of 40-50% in the incorporation of 35[S]methionine or L-[3H]arginine into proteins. Western blot analysis revealed that NMDA stimulates the phosphorylation of eukaryotic elongation factor 2 (eEF2, a factor involved in protein translation), an effect inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801). In conclusion, we have shown that the stimulation of NMDA receptors promotes an inhibition of protein synthesis and a consequent increase of an intracellular L-arginine pool available for the synthesis of NO. This effect seems to be mediated by activation of eEF2 kinase, a calcium/calmodulin-dependent enzyme which specifically phosphorylates and blocks eEF2. The results raise the possibility that NMDA receptor activation stimulates two different calmodulin-dependent enzymes (eEF2 kinase and NOS) reinforcing local NO production by increasing precursor availability together with NOS catalytic activity.     

Glutamate and nitric oxide modulate ERK and CREB phosphorylation in the avian retina: evidence for direct signaling from neurons to Müller glial cells

Glutamate signaling in the mature retinal tissue is very important for accurate sensory decoding by retinal neurons and orchestrates the fine-tuned output from the retina to higher-order centers at the cerebral cortex. In this study, we show that glutamate induces a rapid extracellular-regulated kinase and cAMP-responsive element binding protein (CREB) phosphorylation in cultured developing retinal neurons. This process is reliant on α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors and nitric oxide (NO) signaling and independent of NMDA receptors activation, as it is blocked by α-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate antagonists as well as inhibiting NO synthase with NG-nitro- l -arginine methyl ester but not by the NMDA channel blocker dizocilpine maleate. The effect of NO on extracellular-regulated kinase and CREB is mediated by the classical NO/soluble guanylyl cyclase/protein kinase G pathways as it is inhibited by the soluble guanylyl cyclase blocker 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one and the protein kinase G inhibitor KT5823, respectively. Immunocytochemical data suggest that increased CREB phosphorylation in response to glutamate occurs in glial cell nuclei. We also have supporting evidence suggesting that neuronally produced NO directly reaches the glial cells and stimulates CREB phosphorylation. Hence, the results indicate the importance of neuronal–glial communication and glutamate/NO/CREB linkage during retinal development.     

Nitric oxide-induced blockade of NMDA receptors.

We studied the effects of nitric oxide (NO)-producing agents on N-methyl-D-aspartate (NMDA) receptor activation in cultured neurons. 3-Morpholino-sydnonimine (SIN-1) blocked both NMDA-induced currents and the associated increase in intracellular Ca2+. The actions of SIN-1 were reversible and suppressed by hemoglobin. A degraded SIN-1 solution that did not release NO was unable to block NMDA receptors. This showed that the SIN-1 effects were due to NO and not to another breakdown product. Similar results were obtained with 1-nitrosopyrrolidine (an NO-containing drug) and with NO released from NaNO2. Pretreatment with hemoglobin potentiated NMDA-induced effects, demonstrating that endogenous NO modulates NMDA receptors. Since NMDA receptor activation induces NO synthesis, these results suggest a feedback inhibition of NMDA receptors by NO under physiological condition.     

Nitric oxide mediates NMDA-induced persistent inhibition of protein synthesis through dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 and eukaryotic initiation factor 4G proteolysis

Cerebral ischaemia causes long-lasting protein synthesis inhibition that is believed to contribute to brain damage. Energy depletion promotes translation inhibition during ischaemia, and the phosphorylation of eIF (eukaryotic initiation factor) 2alpha is involved in the translation inhibition induced by early ischaemia/reperfusion. However, the molecular mechanisms underlying prolonged translation down-regulation remain elusive. NMDA (N-methyl-D-aspartate) excitotoxicity is also involved in ischaemic damage, as exposure to NMDA impairs translation and promotes the synthesis of NO (nitric oxide), which can also inhibit translation. In the present study, we investigated whether NO was involved in NMDA-induced protein synthesis inhibition in neurons and studied the underlying molecular mechanisms. NMDA and the NO donor DEA/NO (diethylamine-nitric oxide sodium complex) both inhibited protein synthesis and this effect persisted after a 30 min exposure. Treatments with NMDA or NO promoted calpain-dependent eIF4G cleavage and 4E-BP1 (eIF4E-binding protein 1) dephosphorylation and also abolished the formation of eIF4E-eIF4G complexes; however, they did not induce eIF2alpha phosphorylation. Although NOS (NO synthase) inhibitors did not prevent protein synthesis inhibition during 30 min of NMDA exposure, they did abrogate the persistent inhibition of translation observed after NMDA removal. NOS inhibitors also prevented NMDA-induced eIF4G degradation, 4E-BP1 dephosphorylation, decreased eIF4E-eIF4G-binding and cell death. Although the calpain inhibitor calpeptin blocked NMDA-induced eIF4G degradation, it did not prevent 4E-BP1 dephosphorylation, which precludes eIF4E availability, and thus translation inhibition was maintained. The present study suggests that eIF4G integrity and hyperphosphorylated 4E-BP1 are needed to ensure appropriate translation in neurons. In conclusion, our data show that NO mediates NMDA-induced persistent translation inhibition and suggest that deficient eIF4F activity contributes to this process.     

Dopamine D(1) and D(3) receptors oppositely regulate NMDA- and cocaine-induced MAPK signaling via NMDA receptor phosphorylation

Development of drug addiction involves complex molecular changes in the CNS. The mitogen-activated protein kinase (MAPK) signaling pathway plays a key role in mediating neuronal activation induced by dopamine, glutamate, and drugs of abuse. We previously showed that dopamine D(1) and D(3) receptors play different roles in regulating cocaine-induced MAPK activation. Although there are functional and physical interactions between dopamine and glutamate receptors, little is known regarding the involvement of D(1) and D(3) receptors in modulating glutamate-induced MAPK activation and underlying mechanisms. In this study, we show that D(1) and D(3) receptors play opposite roles in regulating N-methyl-d-aspartate (NMDA) -induced activation of extracellular signal-regulated kinase (ERK) in the caudate putamen (CPu). D(3) receptors also inhibit NMDA-induced activation of the c-Jun N-terminal kinase and p38 kinase in the CPu. NMDA-induced activation of the NMDA-receptor R1 subunit (NR1), Ca(2+)/calmodulin-dependent protein kinase II and the cAMP-response element binding protein (CREB), and cocaine-induced CREB activation in the CPu are also oppositely regulated by dopamine D(1) and D(3) receptors. Finally, the blockade of NMDA-receptor reduces cocaine-induced ERK activation, and inhibits phosphorylation of NR1, Ca(2+)/calmodulin-dependent protein kinase II, and CREB, while inhibiting ERK activation attenuates cocaine-induced CREB phosphorylation in the CPu. These results suggest that dopamine D(1) and D(3) receptors oppositely regulate NMDA- and cocaine-induced MAPK signaling via phosphorylation of NR1.     

Myocardial ischemia and increased heart work modulate the phosphorylation state of eukaryotic elongation factor-2

Protein synthesis, in particular peptide chain elongation, is an energy-consuming biosynthetic process. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in cellular energy homeostasis. Therefore, we tested the hypothesis that, as in liver, it could mediate the inhibition of protein synthesis by oxygen deprivation in heart by modulating the phosphorylation of eukaryotic elongation factor-2 (eEF2), which becomes inactive in its phosphorylated form. In anoxic cardiomyocytes, AMPK activation was associated with an inhibition of protein synthesis and an increase in phosphorylation of eEF2. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), did not mimic the effect of oxygen deprivation to inhibit protein synthesis in cardiomyocytes or lead to eEF2 phosphorylation in perfused hearts, suggesting that AMPK activation did not inhibit mTOR/p70 ribosomal protein S6 kinase (p70S6K) signaling. Human recombinant eEF2 kinase (eEF2K) was phosphorylated by AMPK in a time- and AMP-dependent fashion, and phosphorylation led to eEF2K activation, similar to that observed in extracts from ischemic hearts. In contrast, increasing the workload resulted in a dephosphorylation of eEF2, which was rapamycin-insensitive, thus excluding a role for mTOR in this effect. eEF2K activity was unchanged by increasing the workload, suggesting that the decrease in eEF2 phosphorylation could result from the activation of an eEF2 phosphatase.     

Arginine Availability Controls the N-Methyl-d-Aspartate-Induced Nitric Oxide Synthesis: Involvement of a Glial-Neuronal Arginine Transfer

Abstract: The neuronal nitric oxide (NO) synthase generates NO from arginine. NO mediates its physiological effects mainly by stimulating the synthesis of cyclic GMP. We have investigated the role of the arginine availability on the NMDA-induced cyclic GMP accumulation in immature rat brain slices. The effect of NMDA was blocked by the inhibitor of the NO synthase, N ^G-nitro- l -arginine, and by the antagonist of ionotropic non-NMDA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). This inhibition was not due to a direct interaction of CNQX with the NMDA receptor, and it was overcome by the presence of exogenously applied arginine. CNQX also blocked the NMDA-evoked release of [³H]arginine from cerebellar slices. Moreover, the arginine uptake inhibitor l -lysine reduced the cyclic GMP response to NMDA significantly. Therefore, the extracellular arginine availability, which is dependent on the activation of ionotropic non-NMDA receptors, determines the rate of the NO biosynthesis by the neuronal NO synthase. Together with the reported release of arginine from glial cells upon activation of glial ionotropic non-NMDA receptors and the predominant glial localization of arginine, these data provide the first evidence of an essential role of the arginine transfer from glial cells to neurons for the biosynthesis of NO.     

Thioredoxin inhibits NMDA-induced neurotoxicity in the rat retina

Thioredoxin (TRX) plays a variety of redox-related roles in organisms. To investigate its function as an endogenous redox regulator in NMDA-induced retinal neurotoxicity, we injected NMDA with TRX, mutant TRX or saline into the vitreous cavity of rat eyes. Retinal ganglion cells were rescued by TRX, compared with saline, when evaluated by retrograde labeling analysis at 7 days after NMDA injection. TRX, but not its mutant form, prevented NMDA-induced apoptosis in the retina, as measured by terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling. The induction of caspase 3 and 9, but not caspase 8, by NMDA was significantly lower in TRX-treated eyes than in saline-treated eyes. NMDA-induced activation of the MAPKs, p38 kinase and c-Jun N-terminal kinase after 6 h and of the MAPK kinases (MKKs) MKK3/6 and MKK4 after 3 h was markedly suppressed in retinal ganglion cells by TRX but not by the mutant form. NMDA-induced increases in protein carbonylation, nitrosylation and lipid peroxidation were also suppressed in TRX-treated eyes. We concluded that the intravitreous injection of TRX effectively attenuated NMDA-induced retinal cell damage and that suppression of oxidative stress and inhibition of apoptotic signaling pathways were involved in this neuroprotection.     

The nitric oxide-cGKII system relays death and survival signals during embryonic retinal development via AKT-induced CREB1 activation

During early neurogenesis, retinal neuronal cells display a conserved differentiation program in vertebrates. Previous studies established that nitric oxide (NO) and cGMP accumulation regulate essential events in retinal physiology. Here we used pharmacological and genetic loss-of-function to investigate the effects of NO and its downstream signaling pathway in the survival of developing avian retinal neurons in vitro and in vivo. Six-day-old (E6) chick retinal cells displayed increased calcium influx and produced higher amounts of NO when compared with E8 cells. L-arginine (substrate for NO biosynthesis) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP; a nitrosothiol NO donor) promoted extensive cell death in E6 retinas, whereas in E8 both substances decreased apoptosis. The effect of NO at both periods was mediated by soluble guanylyl cyclase (sGC) and cGMP-dependent kinase (cGK) activation. In addition, shRNA-mediated cGKII knockdown prevented NO-induced cell death (E6) and cell survival (E8). This, NO-induced cell death or cell survival was not correlated with an early inhibition of retinal cell proliferation. E6 cells also responded differentially from E8 neurons regarding cyclic AMP-responsive element-binding protein (CREB) activation in the retina in vivo. NO strongly decreased nuclear phospho-CREB staining in E6 but it robustly enhanced CREB phosphorylation in the nuclei of E8 neurons, an effect that was completely abrogated by cGKII shRNAs at both embryonic stages. The ability of NO in regulating CREB differentially during retinal development relied on the capacity of cGKII in decreasing (E6) or increasing (E8) nuclear AKT (V-Akt murine thymoma viral oncogene) activation. Accordingly, inhibiting AKT prevented both cGKII shRNA-mediated CREB upregulation in E6 and SNAP-induced CREB activation in E8. Furthermore, shRNA-mediated in vivo cGKII or in vitro CREB1 knockdown confirmed that NO/cGKII dualistically regulated the downstream CREB1 pathway and caspase activation in the chick retina to modulate neuronal viability. These data demonstrate that NO-mediated cGKII signaling may function to control the viability of neuronal cells during early retinal development via AKT/CREB1 activity.     

Glutamate Receptor Agonists Stimulate Nitric Oxide Synthase in Primary Cultures of Cerebellar Granule Cells

The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca(2+)-dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor-mediated activation of nitric oxide (NO) synthase. The NMDA-induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled L-arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose-dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK-801 and by the inhibitors of NO synthase, NG-monomethyl-L-arginine (MeArg) and NG-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 microM) stimulated 45Ca2+ influx to the same extent as 100 microM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.     

Nitric oxide regulates AKT phosphorylation and nuclear translocation in cultured retinal cells

Previous studies have shown that nitric oxide (NO) inhibits apoptosis of retinal neurons in culture through the canonical cyclic GMP/protein kinase G (PKG)-dependent pathway, but also involving multiple kinase pathways, such as phosphatidylinositol 3′ kinase (PI3k) and AKT. NO and AKT exhibit survival-promoting properties and display important roles in both CNS development and plasticity. The purpose of this study was to evaluate the effects of exogenous NO, derived from the NO donor S-nitroso-N-acetylpenicillamin (SNAP), or endogenous NO, produced from l-arginine, on AKT phosphorylation in cultured chick retinal neurons. Our results demonstrate that SNAP or l-arginine enhances AKT phosphorylation on both serine-473 and threonine-308 residues in a concentration and time-dependent manner. This effect was mediated by the activation of soluble guanylyl cyclase and PKG, since it was blocked by the respective enzyme inhibitors ODQ or LY83583 and KT5823, as well as by transduction with shRNA lentiviruses coding PKGII shRNA, and mimicked by the respective enzyme activators YC-1 and 8-Bromo cyclic GMP, and also by the cyclic GMP phosphodiesterase inhibitor zaprinast. In addition, LY294002 or wortmannin suppressed the SNAP effect, indicating the involvement of phosphoinositide 3′ kinase. Moreover, the mTOR inhibitor KU0063794 blocked SNAP-induced AKT phosphorylation at both residues, suggesting the participation of the mTORC2 complex in the process. Glutamate and NMDA also promoted AKT phosphorylation and a nitric oxide synthase inhibitor abrogated these effects, revealing a mechanism involving the activation of NMDA receptors and NO production. We have also found that SNAP and l-arginine induced AKT translocation into the nucleus of retinal neurons as well as other neuronal cell lines. SNAP also protects retinal cells from death induced by hydrogen peroxide and this effect was blocked by the phosphoinositide 3′ kinase inhibitor LY294002. We therefore conclude that NO produced from endogenous or exogenous sources promotes AKT activation and its shuttling to the nucleus, probably participating in neuronal survival pathways important during CNS development.     

Insulin rapidly stimulates L-arginine transport in human aortic endothelial cells via Akt

Insulin stimulates endothelial NO synthesis, at least in part mediated by phosphorylation and activation of endothelial NO synthase at Ser1177 and Ser615 by Akt. We have previously demonstrated that insulin-stimulated NO synthesis is inhibited under high culture glucose conditions, without altering Ca(2+)-stimulated NO synthesis or insulin-stimulated phosphorylation of eNOS. This indicates that stimulation of endothelial NO synthase phosphorylation may be required, yet not sufficient, for insulin-stimulated nitric oxide synthesis. In the current study we investigated the role of supply of the eNOS substrate, L-arginine as a candidate parallel mechanism underlying insulin-stimulated NO synthesis in cultured human aortic endothelial cells. Insulin rapidly stimulated L-arginine transport, an effect abrogated by incubation with inhibitors of phosphatidylinositol-3'-kinase or infection with adenoviruses expressing a dominant negative mutant Akt. Furthermore, supplementation of endothelial cells with extracellular L-arginine enhanced insulin-stimulated NO synthesis, an effect reversed by co-incubation with the L-arginine transport inhibitor, L-lysine. Basal L-arginine transport was significantly increased under high glucose culture conditions, yet insulin-stimulated L-arginine transport remained unaltered. The increase in L-arginine transport elicited by high glucose was independent of the expression of the cationic amino acid transporters, hCAT1 and hCAT2 and not associated with any changes in the activity of ERK1/2, Akt or protein kinase C (PKC). We propose that rapid stimulation of L-arginine transport contributes to insulin-stimulated NO synthesis in human endothelial cells, yet attenuation of this is unlikely to underlie the inhibition of insulin-stimulated NO synthesis under high glucose conditions.     

The nNos/cGMP mediation of the depressor response to NMDA receptor stimulation in the caudal ventrolateral medulla

Using in vivo voltammetry to directly measure extracellular nitric oxide (NO) levels, our previous studies suggested that the neuronal NO synthase (nNOS) and cyclic guanosine monophosphate (cGMP) signal transducing systems are involved in the cardiovascular responses elicited by activation of N-methyl-D-aspartate (NMDA) receptors in the rostral ventrolateral medulla. In this study, we examined if the depressor responses elicited by activation of NMDA receptors in the caudal ventrolateral medulla (CVLM) also depend on the actions of nNOS and soluble guanylyl cyclase. In anesthetized cats, microinjection of NMDA into the CVLM produced hypotension and bradycardia associated with NO formation. These NMDA-induced responses were attenuated by prior injections of 2-amino-5-phosphonopentanoate (a NMDA receptor competitive antagonist), 7-nitroindazole (a nNOS inhibitor) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase). These findings suggest that NO is also involved in the NMDA-induced depressor responses of the CVLM.     

A role for polyamines in retinal ganglion cell excitotoxic death

Neuronal death due to excessive activation of N -methyl- d -aspartate (NMDA) receptors is a hallmark of neurodegenerative diseases. The polyamines: putrescine, spermine, and spermidine, bind to specific sites on the NMDA receptor and promote its activation, but their role in NMDA-induced neuronal death is ill defined. In this study, we characterized the role of polyamines in excitotoxic death of retinal ganglion cells (RGCs), a population of central neurons susceptible to NMDA-induced damage. Our data show that endogenous arginase I, the rate limiting enzyme for polyamine biosynthesis, is expressed in the intact, adult retina. Intraocular injection of NMDA visibly increased arginase I expression in Müller cells, the predominant glial cell-type in the mammalian retina. Inhibition of polyamine synthesis using di-fluoro-methyl-ornithine (DFMO) was markedly neuroprotective, while injection of exogenous polyamines in conjunction with NMDA exacerbated RGC death. Blockade of the polyamine binding sites on NMDA receptors using the non-competitive antagonist ifenprodil was neuroprotective, suggesting that polyamines contribute to excitotoxic death, at least partly, by binding to NMDA receptors. Importantly, we also demonstrate that NMDA leads to activation of both the Erk1/2 and PI3 K/Akt pathways, but only the PI3 K/Akt kinase was required for di-fluoro-methyl-ornithine-induced RGC survival. In summary, our study reveals that polyamines modulate neuronal death in the retina via different mechanisms that potentiate NMDA-triggered excitotoxicity.     

Hypoxia inhibits protein synthesis through a 4E-BP1 and elongation factor 2 kinase pathway controlled by mTOR and uncoupled in breast cancer cells

Hypoxia is a state of low oxygen availability that limits tumor growth. The mechanism of protein synthesis inhibition by hypoxia and its circumvention by transformation are not well understood. Hypoxic breast epithelial cells are shown to downregulate protein synthesis by inhibition of the kinase mTOR, which suppresses mRNA translation through a novel mechanism mitigated in transformed cells: disruption of proteasome-targeted degradation of eukaryotic elongation factor 2 (eEF2) kinase and activation of the regulatory protein 4E-BP1. In transformed breast epithelial cells under hypoxia, the mTOR and S6 kinases are constitutively activated and the mTOR negative regulator tuberous sclerosis complex 2 (TSC2) protein fails to function. Gene silencing of 4E-BP1 and eEF2 kinase or TSC2 confers resistance to hypoxia inhibition of protein synthesis in immortalized breast epithelial cells. Breast cancer cells therefore acquire resistance to hypoxia by uncoupling oxygen-responsive signaling pathways from mTOR function, eliminating inhibition of protein synthesis mediated by 4E-BP1 and eEF2.     

Roles of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling pathway in activity-dependent dendritic protein synthesis in hippocampal neurons

Local protein synthesis in neuronal dendrites is critical for synaptic plasticity. However, the signaling cascades that couple synaptic activation to dendritic protein synthesis remain elusive. The purpose of this study is to determine the role of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling in regulating dendritic protein synthesis in live neurons. We first characterized the involvement of various subtypes of glutamate receptors and the mTOR kinase in regulating dendritic synthesis of a green fluorescent protein (GFP) reporter controlled by alphaCaMKII 5' and 3' untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-d-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptors abolished glutamate-induced dendritic GFP synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activated GFP synthesis in dendrites. Inhibitions of the mTOR signaling, as well as its upstream activators, phosphatidylinositol 3-kinase and AKT, blocked NMDA receptor-dependent dendritic GFP synthesis. Conversely, activation of mTOR signaling stimulated dendritic GFP synthesis. In addition, we also found that inhibition of the mTOR kinase blocked dendritic synthesis of the endogenous alphaCaMKII and MAP2 proteins induced by tetanic stimulations in hippocampal slices. These results identify critical roles of NMDA receptors and the mTOR signaling pathway for control of synaptic activity-induced dendritic protein synthesis in hippocampal neurons.     

Eukaryotic Elongation Factor 2 Kinase Activity Is Controlled by Multiple Inputs from Oncogenic Signaling

Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways.     

Modulation of the L-arginine/nitric oxide signalling pathway in vascular endothelial cells

Nitric oxide (NO) is synthesized from L-arginine, and in endothelial cells influx of L-arginine is mediated predominantly via Na+-independent cationic amino acid transporters. Constitutive, Ca2+-calmodulin-sensitive eNOS (endothelial nitric oxide synthase) metabolizes L-arginine to NO and L-citrulline. eNOS is present in membrane caveolae and the cytosol and requires tetrahydrobiopterin, NADPH, FAD and FMN as additional cofactors for its activity. Supply of L-arginine for NO synthesis appears to be derived from a membrane-associated compartment distinct from the bulk intracellular amino acid pool, e.g. near invaginations of the plasma membrane referred to as 'lipid rafts' or caveolae. Co-localization of eNOS and the cationic amino acid transport system y+ in caveolae in part explains the 'arginine paradox', related to the phenomenon that in certain disease states eNOS requires an extracellular supply of L-arginine despite having sufficient intracellular L-arginine concentrations. Vasoactive agonists normally elevate [Ca2+]i (intracellular calcium concentration) in endothelial cells, thus stimulating NO production, whereas fluid shear stress, 17beta-oestradiol and insulin cause phosphorylation of the serine/threonine protein kinase Akt/protein kinase B in a phosphoinositide 3-kinase-dependent manner and activation of eNOS at basal [Ca2+]i levels. Adenosine causes an acute activation of p42/p44 mitogen-activated protein kinase and NO release, with membrane hyperpolarization leading to increased system y+ activity in fetal endothelial cells. In addition to acute stimulatory actions of D-glucose and insulin on L-arginine transport and NO synthesis, gestational diabetes, intrauterine growth retardation and pre-eclampsia induce phenotypic changes in the fetal vasculature, resulting in alterations in the L-arginine/NO signalling pathway and regulation of [Ca2+]i. These alterations may have significant implications for long-term programming of the fetal cardiovascular system.