An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis

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Inactivation of Rheb by PRAK-mediated phosphorylation is essential for energy-depletion-induced suppression of mTORC1

Cell growth can be suppressed by stressful environments, but the role of stress pathways in this process is largely unknown. Here we show that a cascade of p38β mitogen-activated protein kinase (MAPK) and p38-regulated/activated kinase (PRAK) plays a role in energy-starvation-induced suppression of mammalian target of rapamycin (mTOR), and that energy starvation activates the p38β-PRAK cascade. Depletion of p38β or PRAK diminishes the suppression of mTOR complex 1 (mTORC1) and reduction of cell size induced by energy starvation. We show that p38β-PRAK operates independently of the known mTORC1 inactivation pathways--phosphorylation of tuberous sclerosis protein 2 (TSC2) and Raptor by AMP-activated protein kinase (AMPK)--and surprisingly, that PRAK directly regulates Ras homologue enriched in brain (Rheb), a key component of the mTORC1 pathway, by phosphorylation. Phosphorylation of Rheb at Ser 130 by PRAK impairs the nucleotide-binding ability of Rheb and inhibits Rheb-mediated mTORC1 activation. The direct regulation of Rheb by PRAK integrates a stress pathway with the mTORC1 pathway in response to energy depletion.     

Rheb mediates neuronal-activity-induced mitochondrial energetics through mTORC1-independent PDH activation

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Rheb1 is required for mTORC1 and myelination in postnatal brain development

mTor kinase is involved in cell growth, proliferation, and differentiation. The roles of mTor activators, Rheb1 and Rheb2, have not been established in?vivo. Here, we report that Rheb1, but not Rheb2, is critical for embryonic survival and mTORC1 signaling. Embryonic deletion of Rheb1 in neural progenitor cells?abolishes mTORC1 signaling in developing brain and increases mTORC2 signaling. Remarkably, embryonic and early postnatal brain development appears grossly normal in these Rheb1f/f,Nes-cre mice with the notable exception of deficits of myelination. Conditional expression of Rheb1 transgene in neural progenitors increases mTORC1 activity and promotes myelination in the brain. In addition the Rheb1 transgene rescues mTORC1 signaling and hypomyelination in the Rheb1f/f,Nes-cre mice. Our study demonstrates that Rheb1 is essential for mTORC1 signaling and myelination in the brain, and suggests that mTORC1 signaling plays a role in selective cellular adaptations, rather than general cellular viability.     

Reassessment of the role of FKBP38 in the Rheb/mTORC1 pathway

The small G-protein Rheb regulates cell growth via the mTORC1 complex by incompletely understood mechanisms. Recent studies document that Rheb activates mTORC1 via direct, GTP-dependent interaction with the peptidyl-prolyl-cis/trans-isomerase FKBP38, which is proposed to act as an inhibitor of mTORC1. We have conducted a comprehensive biochemical characterization of the Rheb/FKBP38 interaction. Using three different in vitro assays we did not detect an interaction between Rheb and FKBP38. Cell biological experiments illustrate that FKBP38 plays only a very minor, if any, role in mTORC1 activation. Our data document that FKBP38 is not the long-sought Rheb effector linking Rheb to mTORC1 activation.

Structured summary

MINT-6946532: Ral (uniprotkb:P11233) binds (MI:0407) to Ha-Ras (uniprotkb:P01112) by pull down (MI:0096)MINT-6946500: RAF (uniprotkb:P04049) binds (MI:0407) to RHEB2 (uniprotkb:Q15382) by pull down (MI:0096)MINT-6946517: RAF (uniprotkb:P04049) binds (MI:0407) to Ha-Ras (uniprotkb:P01112) by pull down (MI:0096)     

Caspase-2 Maintains Bone Homeostasis by Inducing Apoptosis of Oxidatively-Damaged Osteoclasts

Osteoporosis is a silent disease, characterized by a porous bone micro-structure that enhances risk for fractures and associated disabilities. Senile, or age-related osteoporosis (SO), affects both men and women, resulting in increased morbidity and mortality. However, cellular and molecular mechanisms underlying senile osteoporosis are not fully known. Recent studies implicate the accumulation of reactive oxygen species (ROS) and increased oxidative stress as key factors in SO. Herein, we show that loss of caspase-2, a cysteine aspartate protease involved in oxidative stress-induced apoptosis, results in total body and femoral bone loss in aged mice (20% decrease in bone mineral density), and an increase in bone fragility (30% decrease in fracture strength). Importantly, we demonstrate that genetic ablation or selective inhibition of caspase-2 using zVDVAD-fmk results in increased numbers of bone-resorbing osteoclasts and enhanced tartrate-resistant acid phosphatase (TRAP) activity. Conversely, transfection of osteoclast precursors with wild type caspase-2 but not an enzymatic mutant, results in a decrease in TRAP activity. We demonstrate that caspase-2 expression is induced in osteoclasts treated with oxidants such as hydrogen peroxide and that loss of caspase-2 enhances resistance to oxidants, as measured by TRAP activity, and decreases oxidative stress-induced apoptosis of osteoclasts. Moreover, oxidative stress, quantified by assessment of the lipid peroxidation marker, 4-HNE, is increased in Casp2^-/- bone, perhaps due to a decrease in antioxidant enzymes such as SOD2. Taken together, our data point to a critical and novel role for caspase-2 in maintaining bone homeostasis by modulating ROS levels and osteoclast apoptosis during conditions of enhanced oxidative stress that occur during aging.     

TOR Complex 2-Ypk1 Signaling Maintains Sphingolipid Homeostasis by Sensing and Regulating ROS Accumulation

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组蛋白去乙酰化酶3通过抑制AICD维持T细胞自稳

为探讨组蛋白去乙酰化酶3(HDAC3)在T细胞自稳中的作用,采用LoxP-cd4cre酶系统在胸腺CD4⁺CD8⁺双阳性T细胞(DP)中敲除hdac3基因．hdac3基因敲除小鼠不影响T细胞在胸腺中的发育,但导致外周T细胞显著降低,而且,hdac3基因敲除的外周T细胞主要以活化/效应/记忆表型为主．机制分析表明,hdac3基因敲除的外周T细胞凋亡增加并伴随细胞增殖加速,同时,Fas 和Fas配体阳性细胞比率以及Fas配体的表达显著增加．体外TCR活化不影响正常外周T细胞的凋亡,但导致hdac3基因敲除的外周T细胞凋亡显著增加．实验结果表明,HDAC3通过抑制活化诱导的细胞凋亡维持外周T细胞自稳．     

Hydrolysis of GTP by the alpha-chain of Gs and other GTP binding proteins

The functions of G proteins--like those of bacterial elongation factor (EF) Tu and the 21 kDa ras proteins (p21ras)--depend upon their abilities to bind and hydrolyze GTP and to assume different conformations in GTP- and GDP-bound states. Similarities in function and amino acid sequence indicate that EF-Tu, p21ras, and G protein alpha-chains evolved from a primordial GTP-binding protein. Proteins in all three families appear to share common mechanisms for GTP-dependent conformational change and hydrolysis of bound GTP. Biochemical and molecular genetic studies of the alpha-chain of Gs (alpha s) point to key regions that are involved in GTP-dependent conformational change and in hydrolysis of GTP. Tumorigenic mutations of alpha s in human pituitary tumors inhibit the protein's GTPase activity and cause constitutive elevation of adenylyl cyclase activity. One such mutation replaces a Gln residue in alpha s that corresponds to Gln-61 of p21ras; mutational replacements of this residue in both proteins inhibit their GTPase activities. A second class of GTPase inhibiting mutations in alpha s occurs in the codon for an Arg residue whose covalent modification by cholera toxin also inhibits GTP hydrolysis by alpha s. This Arg residue is located in a domain of alpha s not represented in EF-Tu or p21ras. We propose that this domain constitutes an intrinsic activator of GTP hydrolysis, and that it performs a function analogous to that performed for EF-Tu by the programmed ribosome and for p21ras by the recently discovered GTPase-activating protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyr39 of Ran Preserves the Ran·GTP Gradient by Inhibiting GTP Hydrolysis

Ran is a member of the superfamily of small GTPases, which cycle between a GTP-bound “on” and a GDP-bound “off” state. Ran regulates nuclear transport. In order to maintain a gradient of excess Ran·GTP within the nucleoplasm and excess Ran·GDP within the cytoplasm, the hydrolysis of Ran·GTP in the nucleoplasm should be prevented, whereas in the cytoplasm, hydrolysis is catalyzed by Ran·GAP (GTPase-activating protein). In this article, we investigate the GTPase reaction of Ran in complex with its binding protein Ran-binding protein 1 by time-resolved Fourier transform infrared spectroscopy: We show that the slowdown of the intrinsic hydrolysis of RanGTP is accomplished by tyrosine 39, which is probably misplacing the attacking water. We monitored the interaction of Ran with RanGAP, which reveals two reactions steps. By isotopic labeling of Ran and RanGAP, we were able to assign the first step to a small conformational change within the catalytic site. The following bond breakage is the rate-limiting step of hydrolysis. An intermediate of protein-bound phosphate as found for Ras or Rap systems is kinetically unresolved. This demonstrates that despite the structural similarity among the G-domain of the GTPases, different reaction mechanisms are utilized.     

Specific Activation of mTORC1 by Rheb G-protein in Vitro Involves Enhanced Recruitment of Its Substrate Protein

Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.Rheb defines a unique member of the Ras superfamily G-proteins (1). We have shown that Rheb proteins are conserved and are found from yeast to human (2). Although yeast and fruit fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or simply Rheb) and Rheb2 (RhebL1) (2). Structurally, these proteins contain G1-G5 boxes, short stretches of amino acids that define the function of the Ras superfamily G-proteins including guanine nucleotide binding (1, 3, 4). Rheb proteins have a conserved arginine at residue 15 that corresponds to residue 12 of Ras (1). The effector domain required for the binding with downstream effectors encompasses the G2 box and its adjacent sequences (1, 5). Structural analysis by x-ray crystallography further shows that the effector domain is exposed to solvent, is located close to the phosphates of GTP especially at residues 35–38, and undergoes conformational change during GTP/GDP exchange (6). In addition, all Rheb proteins end with the CAAX (C is cysteine, A is an aliphatic amino acid, and X is the C-terminal amino acid) motif that signals farnesylation. In fact, we as well as others have shown that these proteins are farnesylated (7–9).Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling pathway that plays central roles in regulating protein synthesis and growth in response to nutrient, energy, and growth conditions (10–14). Rheb is down-regulated by a TSC1·TSC2 complex that acts as a GTPase-activating protein for Rheb (15–19). Recent studies established that the GAP domain of TSC2 defines the functional domain for the down-regulation of Rheb (20). Mutations in the Tsc1 or Tsc2 gene lead to tuberous sclerosis whose symptoms include the appearance of benign tumors called hamartomas at different parts of the body as well as neurological symptoms (21, 22). Overexpression of Rheb results in constitutive activation of mTOR even in the absence of nutrients (15, 16). Two mTOR complexes, mTORC1 and mTORC2, have been identified (23, 24). Whereas mTORC1 is involved in protein synthesis activation mediated by S6K and 4EBP1, mTORC2 is involved in the phosphorylation of Akt in response to insulin. It has been suggested that Rheb is involved in the activation of mTORC1 but not mTORC2 (25).Although Rheb is clearly involved in the activation of mTOR, the mechanism of activation has not been established. We as well as others have suggested a model that involves the interaction of Rheb with the TOR complex (26–28). Rheb activation of mTOR kinase activity using immunoprecipitated mTORC1 was reported (29). Rheb has been shown to interact with mTOR (27, 30), and this may involve direct interaction of Rheb with the kinase domain of mTOR (27). However, this Rheb/mTOR interaction is a weak interaction and is not dependent on the presence of GTP bound to Rheb (27, 28). Recently, a different model proposing that FKBP38 (FK506-binding protein 38) mediates the activation of mTORC1 by Rheb was proposed (31, 32). In this model, FKBP38 binds mTOR and negatively regulates mTOR activity, and this negative regulation is blocked by the binding of Rheb to FKBP38. However, recent reports dispute this idea (33).To further characterize Rheb activation of mTOR, we have utilized an in vitro system that reproduces activation of mTORC1 by the addition of recombinant Rheb. We used mTORC1 immunoprecipitated from nutrient-starved cells using anti-raptor antibody and have shown that its kinase activity against 4E-BP1 is dramatically increased by the addition of recombinant Rheb. Importantly, the activation of mTORC1 is specific to Rheb and is dependent on the presence of bound GTP as well as an intact effector domain. FKBP38 is not detected in our preparation and further investigation suggests that FKBP38 is not an essential component for the activation of mTORC1 by Rheb. Our study revealed that Rheb enhances the binding of a substrate 4E-BP1 with mTORC1 rather than increasing the kinase activity of mTOR.     

RAV-Like1 Maintains Brassinosteroid Homeostasis via the Coordinated Activation of BRI1 and Biosynthetic Genes in Rice

Temporal and spatial variation in the levels of and sensitivity to hormones are essential for the development of higher organisms. Traditionally, end-product feedback regulation has been considered as the key mechanism for the achievement of cellular homeostasis. Brassinosteroids (BRs) are plant steroid hormones that are perceived by the cell surface receptor kinase Brassinosteroid Insensitive1. Binding of these hormones to the receptor activates BR signaling and eventually suppresses BR synthesis. This report shows that RAVL1 regulates the expression of the BR receptor. Furthermore, RAVL1 is also required for the expression of the BR biosynthetic genes D2, D11, and BRD1 that are subject to BR negative feedback. Activation by RAVL1 was coordinated via E-box cis-elements in the promoters of the receptor and biosynthetic genes. Also, RAVL1 is necessary for the response of these genes to changes in cellular BR homeostasis. Genetic evidence is presented to strengthen the observation that the primary action of RAVL1 mediates the expression of genes involved in BR signaling and biosynthesis. This study thus describes a regulatory circuit modulating the homeostasis of BR in which RAVL1 ensures the basal activity of both the signaling and the biosynthetic pathways.     

Ceramide inhibits insulin-stimulated Akt phosphorylation through activation of Rheb/mTORC1/S6K signaling in skeletal muscle

Ceramide is a negative regulator of insulin activity. At the molecular level, it causes a decrease in insulin-stimulated Akt Ser473 phosphorylation in C2C12 myotubes. Interestingly, we found that the phosphorylation of S6K at Thr389 was increased under the same conditions. Utilizing both rapamycin to inhibit mTORC1 activity and shRNA to knock down Rheb, we demonstrated that the decrease in Akt Ser473 phosphorylation stimulated by insulin after C2-ceramide incubation can be prevented. The mechanism by which C2-ceramide impairs signaling would seem to involve a negative feedback of activated S6K via phosphorylation of insulin receptor substrate-1 at Ser636/639, since S6K inhibitor can block this phenomenon. Finally, rapamycin treatment was found not to affect C2-ceramide-induced PKCζ activation, suggesting that the pathway revealed in this study is parallel to the one involving PKCζ activation. We proposed a novel pathway/mechanism involving Rheb/mTORC1/S6K signaling to explain how C2-ceramide impairs insulin signaling via Akt phosphorylation. The existence of multiple pathways involved in insulin signaling impairment by C2-ceramide treatment implies that different strategies might be needed to ameliorate insulin resistance caused by C2-ceramide.     

GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37°C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-free YPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.     

An Autoinhibited State in the Structure of Thermotoga maritima NusG

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Highlights? Thermotoga maritima NusG shows a dynamic intramolecular NTD-CTD interaction ? The NTD-CTD interaction hides the binding surfaces for RNA polymerase, S10, and Rho ? Domain interaction contributes to thermostability ? Thermotoga maritima NusG does not complement a NusG-deficient E. coli strain     

Regulation of mTORC1 signaling by pH

Background

Acidification of the cytoplasm and the extracellular environment is associated with many physiological and pathological conditions, such as intense exercise, hypoxia and tumourigenesis. Acidification affects important cellular functions including protein synthesis, growth, and proliferation. Many of these vital functions are controlled by mTORC1, a master regulator protein kinase that is activated by various growth-stimulating signals and inactivated by starvation conditions. Whether mTORC1 can also respond to changes in extracellular or cytoplasmic pH and play a role in limiting anabolic processes in acidic conditions is not known.

Methodology/Findings

We examined the effects of acidifying the extracellular medium from pH 7.4 to 6.4 on human breast carcinoma MCF-7 cells and immortalized mouse embryo fibroblasts. Decreasing the extracellular pH caused intracellular acidification and rapid, graded and reversible inhibition of mTORC1, assessed by measuring the phosphorylation of the mTORC1 substrate S6K. Fibroblasts deleted of the tuberous sclerosis complex TSC2 gene, a major negative regulator of mTORC1, were unable to inhibit mTORC1 in acidic extracellular conditions, showing that the TSC1–TSC2 complex is required for this response. Examination of the major upstream pathways converging on the TSC1–TSC2 complex showed that Akt signaling was unaffected by pH but that the Raf/MEK/ERK pathway was inhibited. Inhibition of MEK with drugs caused only modest mTORC1 inhibition, implying that other unidentified pathways also play major roles.

Conclusions

This study reveals a novel role for the TSC1/TSC2 complex and mTORC1 in sensing variations in ambient pH. As a common feature of low tissue perfusion, low glucose availability and high energy expenditure, acidic pH may serve as a signal for mTORC1 to downregulate energy-consuming anabolic processes such as protein synthesis as an adaptive response to metabolically stressful conditions.     

Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgi- derived COP-coated vesicles

The cycle of nucleotide exchange and hydrolysis by a small GTP-binding protein, ADP-ribosylation factor (ARF), helps to provide vectoriality to vesicle transport. Coat assembly is triggered when ARF binds GTP, initiating transport vesicle budding, and coat disassembly is triggered when ARF hydrolyzes GTP, allowing the uncoated vesicle to fuse.