Mi Nam Lee

O‐GlcNAcase is essential for embryonic development and maintenance of genomic stability

Dysregulation of O‐GlcNAc modification catalyzed by O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, type 2 diabetes, and Alzheimer’s disease. Here we found that natural aging in wild‐type mice was marked by a decrease in OGA and OGT protein levels and an increase in O‐GlcNAcylation in various tissues. Genetic disruption of OGA resulted in constitutively elevated O‐GlcNAcylation in embryos and led to neonatal lethality with developmental delay. Importantly, we observed that serum‐stimulated cell cycle entry induced increased O‐GlcNAcylation and decreased its level after release from G2/M arrest, indicating that O‐GlcNAc cycling by OGT and OGA is required for precise cell cycle control. Constitutively, elevated O‐GlcNAcylation by OGA disruption impaired cell proliferation and resulted in mitotic defects with downregulation of mitotic regulators. OGA loss led to mitotic defects including cytokinesis failure and binucleation, increased lagging chromosomes, and micronuclei formation. These findings suggest an important role for O‐GlcNAc cycling by OGA in embryonic development and the regulation of the maintenance of genomic stability linked to the aging process.

Glycolytic Flux Signals to mTOR through Glyceraldehyde-3-Phosphate Dehydrogenase-Mediated Regulation of Rheb

ABSTRACT The mammalian target of rapamycin (mTOR) interacts with raptor to form the protein complex mTORC1 (mTOR complex 1), which plays a central role in the regulation of cell growth in response to environmental cues. Given that glucose is a primary fuel source and a biosynthetic precursor, how mTORC1 signaling is coordinated with glucose metabolism has been an important question. Here, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds Rheb and inhibits mTORC1 signaling. Under low-glucose conditions, GAPDH prevents Rheb from binding to mTOR and thereby inhibits mTORC1 signaling. High glycolytic flux suppresses the interaction between GAPDH and Rheb and thus allows Rheb to activate mTORC1. Silencing of GAPDH or blocking of the Rheb-GAPDH interaction desensitizes mTORC1 signaling to changes in the level of glucose. The GAPDH-dependent regulation of mTORC1 in response to glucose availability occurred even in TSC1-deficient cells and AMPK-silenced cells, supporting the idea that the GAPDH-Rheb pathway functions independently of the AMPK axis. Furthermore, we show that glyceraldehyde-3-phosphate, a glycolytic intermediate that binds GAPDH, destabilizes the Rheb-GAPDH interaction even under low-glucose conditions, explaining how high-glucose flux suppresses the interaction and activates mTORC1 signaling. Taken together, our results suggest that the glycolytic flux regulates mTORs access to Rheb by regulating the Rheb-GAPDH interaction, thereby allowing mTORC1 to coordinate cell growth with glucose availability.

Resveratrol induces autophagy by directly inhibiting mTOR through ATP competition

Resveratrol (RSV) is a natural polyphenol that has a beneficial effect on health, and resveratrol-induced autophagy has been suggested to be a key process in mediating many beneficial effects of resveratrol, such as reduction of inflammation and induction of cancer cell death. Although various resveratrol targets have been suggested, the molecule that mediates resveratrol-induced autophagy remains unknown. Here, we demonstrate that resveratrol induces autophagy by directly inhibiting the mTOR-ULK1 pathway. We found that inhibition of mTOR activity and presence of ULK1 are required for autophagy induction by resveratrol. In line with this mTOR dependency, we found that resveratrol suppresses the viability of MCF7 cells but not of SW620 cells, which are mTOR inhibitor sensitive and insensitive cancer cells, respectively. We also found that resveratrol-induced cancer cell suppression occurred ULK1 dependently. For the mechanism of action of resveratrol on mTOR inhibition, we demonstrate that resveratrol directly inhibits mTOR. We found that resveratrol inhibits mTOR by docking onto the ATP-binding pocket of mTOR (i.e., it competes with ATP). We propose mTOR as a novel direct target of resveratrol, and inhibition of mTOR is necessary for autophagy induction.

Cyclic AMP Controls mTOR through Regulation of the Dynamic Interaction between Rheb and Phosphodiesterase 4D

ABSTRACT The mammalian target of rapamycin complex 1 (mTORC1) is a molecular hub that regulates protein synthesis in response to a number of extracellular stimuli. Cyclic AMP (cAMP) is considered to be an important second messenger that controls mTOR; however, the signaling components of this pathway have not yet been elucidated. Here, we identify cAMP phosphodiesterase 4D (PDE4D) as a binding partner of Rheb that acts as a cAMP-specific negative regulator of mTORC1. Under basal conditions, PDE4D binds Rheb in a noncatalytic manner that does not require its cAMP-hydrolyzing activity and thereby inhibits the ability of Rheb to activate mTORC1. However, elevated cAMP levels disrupt the interaction of PDE4D with Rheb and increase the interaction between Rheb and mTOR. This enhanced Rheb-mTOR interaction induces the activation of mTORC1 and cap-dependent translation, a cellular function of mTORC1. Taken together, our results suggest a novel regulatory mechanism for mTORC1 in which the cAMP-determined dynamic interaction between Rheb and PDE4D provides a key, unique regulatory event. We also propose a new role for PDE4 as a molecular transducer for cAMP signaling.

Phospholipase D2 induces stress fiber formation through mediating nucleotide exchange for RhoA

Phospholipase D (PLD) is involved in diverse cellular processes including cell movement, adhesion, and vesicle trafficking through cytoskeletal rearrangements. However, the mechanism by which PLD induces cytoskeletal reorganization is still not fully understood. Here, we describe a new link to cytoskeletal changes that is mediated by PLD2 through direct nucleotide exchange on RhoA. We found that PLD2 induces RhoA activation independent of its lipase activity. PLD2 directly interacted with RhoA, and the PX domain of PLD2 specifically recognized nucleotide-free RhoA. Finally, we found that the PX domain of PLD2 has guanine nucleotide-exchange factor (GEF) activity for RhoA in vitro. In addition, we verified that overexpression of the PLD2-PX domain induces RhoA activation, thereby provoking stress fiber formation. Together, our findings suggest that PLD2 functions as an upstream regulator of RhoA, which enables us to understand how PLD2 regulates cytoskeletal reorganization in a lipase activity-independent manner.

A molecular chaperone glucose‐regulated protein 94 blocks apoptosis induced by virus infection

The hepatitis C virus (HCV) E2 protein has been shown to block apoptosis and has been suggested to facilitate persistent infection of the virus. Here, we report that the anti‐apoptotic activity of E2 is mediated by activation of nuclear factor kappa B (NF‐κB) that directs expression of survival gene products such as tumor necrosis factor (TNF‐α) receptor‐associated factor 2 (TRAF2), X‐chromosome–linked inhibitor of apoptosis protein (XIAP), FLICE‐like inhibitory protein (FLIP), and survivin. Increased levels of these proteins were observed in HCV‐infected cells and a cell line producing HCV E2 protein. The activation of NF‐κB was mediated by HCV‐E2–induced expression of the molecular chaperone glucose‐regulated protein 94 (GRP94). Overexpression of GRP94 alone resulted in expression of anti‐apoptotic proteins and blocked apoptosis induced by tumor‐necrosis–related apoptosis‐inducing ligand (TRAIL). Interestingly, increased levels of GRP94 were observed in cells supporting HCV proliferation that originated from liver tissues from HCV patients. Moreover, small interfering RNA (siRNA) knock‐down of GRP94 nullified the anti‐apoptotic activity of HCV E2. Conclusion: These data indicate that HCV E2 blocks apoptosis induced by HCV infection and the host immune system through overproduction of GRP94, and that HCV E2 plays an important role in persistent HCV infection. (HEPATOLOGY 2008.)

Osmotic Stress Regulates Mammalian Target of Rapamycin (mTOR) Complex 1 via c-Jun N-terminal Kinase (JNK)-mediated Raptor Protein Phosphorylation

Background: mTORC1 integrates diverse signals including stress to control cell growth. Results: JNK phosphorylates Raptor, a component of mTORC1, and activates mTORC1 kinase upon osmotic stress. Conclusion: mTORC1 is regulated by JNK during osmotic stress. Significance: Our findings provide the JNK-Raptor relationship as a potential mechanism by which stress activates mTORC1 signaling pathway. mTOR complex 1 (mTORC1) is a multiprotein complex that integrates diverse signals including growth factors, nutrients, and stress to control cell growth. Raptor is an essential component of mTORC1 that functions to recruit specific substrates. Recently, Raptor was suggested to be a key target of regulation of mTORC1. Here, we show that Raptor is phosphorylated by JNK upon osmotic stress. We identified that osmotic stress induces the phosphorylation of Raptor at Ser-696, Thr-706, and Ser-863 using liquid chromatography-tandem mass spectrometry. We found that JNK is responsible for the phosphorylation. The inhibition of JNK abolishes the phosphorylation of Raptor induced by osmotic stress in cells. Furthermore, JNK physically associates with Raptor and phosphorylates Raptor in vitro, implying that JNK is responsible for the phosphorylation of Raptor. Finally, we found that osmotic stress activates mTORC1 kinase activity in a JNK-dependent manner. Our findings suggest that the molecular link between JNK and Raptor is a potential mechanism by which stress regulates the mTORC1 signaling pathway.

C1-Ten Is a Protein Tyrosine Phosphatase of Insulin Receptor Substrate 1 (IRS-1), Regulating IRS-1 Stability and Muscle Atrophy

ABSTRACT Muscle atrophy occurs under various catabolic conditions, including insulin deficiency, insulin resistance, or increased levels of glucocorticoids. This results from reduced levels of insulin receptor substrate 1 (IRS-1), leading to decreased phosphatidylinositol 3-kinase activity and thereby activation of FoxO transcription factors. However, the precise mechanism of reduced IRS-1 under a catabolic condition is unknown. Here, we report that C1-Ten is a novel protein tyrosine phosphatase (PTPase) of IRS-1 that acts as a mediator to reduce IRS-1 under a catabolic condition, resulting in muscle atrophy. C1-Ten preferentially dephosphorylated Y612 of IRS-1, which accelerated IRS-1 degradation. These findings suggest a novel type of IRS-1 degradation mechanism which is dependent on C1-Ten and extends our understanding of the molecular mechanism of muscle atrophy under catabolic conditions. C1-Ten expression is increased by catabolic glucocorticoid and decreased by anabolic insulin. Reflecting these hormonal regulations, the muscle C1-Ten is upregulated in atrophy but downregulated in hypertrophy. This reveals a previously unidentified role of C1-Ten as a relevant PTPase contributing to skeletal muscle atrophy.

Deacetylated αβ-tubulin acts as a positive regulator of Rheb GTPase through increasing its GTP-loading.

Ras homolog enriched in brain (Rheb) regulates diverse cellular functions by modulating its nucleotide-bound status. Although Rheb contains a high basal GTP level, the regulatory mechanism of Rheb is not well understood. In this study, we propose soluble αβ-tubulin acts as a constitutively active Rheb activator, which may explain the reason why Rheb has a high basal GTP levels. We found that soluble αβ-tubulin is a direct Rheb-binding protein and that its deacetylated form has a high binding affinity for Rheb. Modulation of both soluble and acetylated αβ-tubulin levels affects the level of GTP-bound Rheb. This occurs in the mitotic phase in which the level of acetylated αβ-tubulin is increased but that of GTP-bound Rheb is decreased. Constitutively active Rheb-overexpressing cells showed an abnormal mitotic progression, suggesting the deacetylated αβ-tubulin-mediated regulation of Rheb status may be important for proper mitotic progression. Taken together, we propose that deacetylated soluble αβ-tubulin is a novel type of positive regulator of Rheb and may play a role as a temporal regulator for Rheb during the cell cycle.

Parkin ubiquitinates mTOR to regulate mTORC1 activity under mitochondrial stress

mTORC1, a kinase complex that is considered a master regulator of cellular growth and proliferation, is regulated by many extra- and intracellular signals. Among these signals, mitochondrial status is known to have an impact on the effects of mTORC1 on cell growth and survival. However, how mitochondrial status affects mTORC1 activity, notably the molecular link, is not fully elucidated. Here, we found that Parkin can interact with and ubiquitinate mTOR. We also identified K2066 and K2306 as Parkin-dependent and mitochondrial stress-induced mTOR ubiquitination residues. This ubiquitination by Parkin is required for maintenance of mTORC1 activity under mitochondrial stress. With regard to the physiological meaning of mTORC1 activity under mitochondrial stress, we suggest that mTORC1 plays a pro-survival role.