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Rheb regulates nuclear mTORC1 activity independent of farnesylation.

ABSTRACT: The small GTPase Ras homolog enriched in brain (Rheb) plays a critical role in activating the mechanistic target of rapamycin complex 1 (mTORC1), a signaling hub that regulates various cellular functions. We recently observed nuclear mTORC1 activity, raising an intriguing question as to how Rheb, which is known to be farnesylated and localized to intracellular membranes, regulates nuclear mTORC1. In this study, we found that active Rheb is present in the nucleus and required for nuclear mTORC1 activity. We showed that inhibition of farnesyltransferase reduced cytosolic, but not nuclear, mTORC1 activity. Furthermore, a farnesylation-deficient Rheb mutant, with preferential nuclear localization and specific lysosome tethering, enables nuclear and cytosolic mTORC1 activities, respectively. These data suggest that non-farnesylated Rheb is capable of interacting with and activating mTORC1, providing mechanistic insights into the molecular functioning of Rheb as well as regulation of the recently observed, active pool of nuclear mTORC1.

SUBMITTER: Zhong Y

PROVIDER: S-EPMC9667991 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Rheb regulates nuclear mTORC1 activity independent of farnesylation.

Zhong Yanghao Y Zhou Xin X Guan Kun-Liang KL Zhang Jin J

Cell chemical biology 20220315 6

The small GTPase Ras homolog enriched in brain (Rheb) plays a critical role in activating the mechanistic target of rapamycin complex 1 (mTORC1), a signaling hub that regulates various cellular functions. We recently observed nuclear mTORC1 activity, raising an intriguing question as to how Rheb, which is known to be farnesylated and localized to intracellular membranes, regulates nuclear mTORC1. In this study, we found that active Rheb is present in the nucleus and required for nuclear mTORC1 a ...[more]

PMID: 35294906

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Project description:RationaleEndoplasmic reticulum (ER) stress dysregulates ER proteostasis, which activates the transcription factor, ATF6 (activating transcription factor 6α), an inducer of genes that enhance protein folding and restore ER proteostasis. Because of increased protein synthesis, it is possible that protein folding and ER proteostasis are challenged during cardiac myocyte growth. However, it is not known whether ATF6 is activated, and if so, what its function is during hypertrophic growth of cardiac myocytes.ObjectiveTo examine the activity and function of ATF6 during cardiac hypertrophy.Methods and resultsWe found that ER stress and ATF6 were activated and ATF6 target genes were induced in mice subjected to an acute model of transverse aortic constriction, or to free-wheel exercise, both of which promote adaptive cardiac myocyte hypertrophy with preserved cardiac function. Cardiac myocyte-specific deletion of Atf6 (ATF6 cKO [conditional knockout]) blunted transverse aortic constriction and exercise-induced cardiac myocyte hypertrophy and impaired cardiac function, demonstrating a role for ATF6 in compensatory myocyte growth. Transcript profiling and chromatin immunoprecipitation identified RHEB (Ras homologue enriched in brain) as an ATF6 target gene in the heart. RHEB is an activator of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), a major inducer of protein synthesis and subsequent cell growth. Both transverse aortic constriction and exercise upregulated RHEB, activated mTORC1, and induced cardiac hypertrophy in wild type mouse hearts but not in ATF6 cKO hearts. Mechanistically, knockdown of ATF6 in neonatal rat ventricular myocytes blocked phenylephrine- and IGF1 (insulin-like growth factor 1)-mediated RHEB induction, mTORC1 activation, and myocyte growth, all of which were restored by ectopic RHEB expression. Moreover, adeno-associated virus 9- RHEB restored cardiac growth to ATF6 cKO mice subjected to transverse aortic constriction. Finally, ATF6 induced RHEB in response to growth factors, but not in response to other activators of ATF6 that do not induce growth, indicating that ATF6 target gene induction is stress specific.ConclusionsCompensatory cardiac hypertrophy activates ER stress and ATF6, which induces RHEB and activates mTORC1. Thus, ATF6 is a previously unrecognized link between growth stimuli and mTORC1-mediated cardiac growth.

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.

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Rheb regulates nuclear mTORC1 activity independent of farnesylation.

Publications

Rheb regulates nuclear mTORC1 activity independent of farnesylation.

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