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Ras homolog enriched in brain (Rheb) enhances apoptotic signaling.

ABSTRACT: Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNF?, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNF? but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNF?-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications.

SUBMITTER: Karassek S

PROVIDER: S-EPMC2962498 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Ras homolog enriched in brain (Rheb) enhances apoptotic signaling.

Karassek Sascha S Berghaus Carsten C Schwarten Melanie M Goemans Christoph G CG Ohse Nadine N Kock Gerd G Jockers Katharina K Neumann Sebastian S Gottfried Sebastian S Herrmann Christian C Heumann Rolf R Stoll Raphael R

The Journal of biological chemistry 20100804 44

Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applyin ...[more]

PMID: 20685651

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Project description:Postexercise protein ingestion can elevate rates of myofibrillar protein synthesis (MyoPS), mTORC1 activity, and mTOR translocation/protein-protein interactions. However, it is unclear if leucine-enriched essential amino acids (LEAA) can similarly facilitate intracellular mTOR trafficking in humans after exercise. The purpose of this study was to determine the effect of postexercise LEAA (4 g total EAAs, 1.6 g leucine) on acute MyoPS and mTORC1 translocation and signaling. Recreationally active men performed lower-body resistance exercise (5 × 8-10 leg press and leg extension) to volitional failure. Following exercise participants consumed LEAA (n = 8) or an isocaloric carbohydrate drink (PLA; n = 10). MyoPS was measured over 1.5-4 h of recovery by oral pulse of l-[ring-2H5]-phenylalanine. Phosphorylation of proteins in the mTORC1 pathway were analyzed via immunoblotting and mTORC1-LAMP2/WGA/Rheb colocalization via immunofluorescence microscopy. There was no difference in MyoPS between groups (LEAA = 0.098 ± 0.01%/h; PL = 0.090 ± 0.01%/h; P > 0.05). Exercise increased (P < 0.05) rpS6Ser240/244(LEAA = 35.3-fold; PLA = 20.6-fold), mTORSer2448(LEAA = 1.8-fold; PLA = 1.2-fold) and 4EBP1Thr37/46(LEAA = 1.5-fold; PLA = 1.4-fold) phosphorylation irrespective of nutrition (P > 0.05). LAT1 and SNAT2 protein expression were not affected by exercise or nutrient ingestion. mTOR-LAMP2 colocalization was greater in LEAA preexercise and decreased following exercise and supplement ingestion (P < 0.05), yet was unchanged in PLA. mTOR-WGA (cell periphery marker) and mTOR-Rheb colocalization was greater in LEAA compared with PLA irrespective of time-point (P < 0.05). In conclusion, the postexercise consumption of 4 g of LEAA maintains mTOR in peripheral regions of muscle fibers, in closer proximity to its direct activator Rheb, during prolonged recovery independent of differences in MyoPS or mTORC1 signaling compared with PLA ingestion. This intracellular localization of mTOR may serve to "prime" the kinase for future anabolic stimuli.NEW & NOTEWORTHY This is the first study to investigate whether postexercise leucine-enriched amino acid (LEAA) ingestion elevates mTORC1 translocation and protein-protein interactions in human skeletal muscle. Here, we observed that although LEAA ingestion did not further elevate postexercise MyoPS or mTORC1 signaling compared with placebo, mTORC1 peripheral location and interaction with Rheb were maintained. This may serve to "prime" mTORC1 for subsequent anabolic stimuli.

Dataset Information

Ras homolog enriched in brain (Rheb) enhances apoptotic signaling.

Publications

Ras homolog enriched in brain (Rheb) enhances apoptotic signaling.

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