Lysosomal localization of mammalian target of rapamycin complex 1 (mTORC1) is a critical step for activation of the molecule. Rag GTPases are essential for this translocation. Here, we demonstrate ...that Nudix-type motif 2 (NUDT2) is a novel positive regulator of mTORC1 activation. Activation of mTORC1 is impaired in NUDT2-silenced cells. Mechanistically, NUDT2 binds to Rag GTPase and controls mTORC1 translocation to the lysosomal membrane. Furthermore, NUDT2-dependent mTORC1 regulation is critical for proliferation of breast cancer cells, as NUDT2-silenced cells arrest in G0/G1 phases. Taken together, these results show that NUDT2 is a novel complex formation enhancing factor regulating mTORC1-Rag GTPase signaling that is crucial for cell growth control.
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•NUDT2 is identified as a novel regulator of Rag GTPase-mTORC1 signaling.•NUDT2 was associated with recruitment of mTORC1 to the lysosomal membrane.•The physical interaction between NUDT2 and Rag GTPase heterodimers is required for the lysosomal localization of mTORC1.•NUDT2-dependent mTORC1 regulation is critical for proliferation of breast cancer cells.
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.
mTORC1 integrates diverse signals including stress to control cell growth.
JNK phosphorylates Raptor, a component of mTORC1, and activates mTORC1 kinase upon osmotic stress.
mTORC1 is regulated by JNK during osmotic stress.
Our findings provide the JNK-Raptor relationship as a potential mechanism by which stress activates mTORC1 signaling pathway.
Interleukin 6 (IL6) is a pleiotropic cytokine that not only affects the immune system, but also plays an active role in many physiological events in various organs. Notably, 35% of systemic IL6 ...originates from adipose tissues under noninflammatory conditions. Here, we describe a previously unknown function of melanocortins in regulating Il6 gene expression and production in 3T3-L1 adipocytes through membrane receptors which are called melanocortin receptors (MCRs). Of the five MCRs that have been cloned, MC2R and MC5R are expressed during adipocyte differentiation. alpha-Melanocyte-stimulating hormone (alpha-MSH) or ACTH treatment of 3T3-L1 adipocytes induces Il6 gene expression and production in a time- and concentration-dependent manner via various signaling pathways including the protein kinase A, p38 mitogen-activated protein kinase, cJun N-terminal kinase, and IkappaB kinase pathways. Specific inhibition of MC2R and MC5R expression with short interfering Mc2r and Mc5r RNAs significantly attenuated the alpha-MSH-induced increase of intracellular cAMP and both the level of Il6 mRNA and secretion of IL6 in 3T3-L1 adipocytes. Finally, when injected into mouse tail vein, alpha-MSH dramatically increased the Il6 transcript levels in epididymal fat pads. These results suggest that alpha-MSH in addition to ACTH may function as a regulator of inflammation by regulating cytokine production.
Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, and available experimental and routine therapies result in limited survival benefits. A vulnerability of GBM cells to ...catastrophic vacuolization and cell death, a process termed methuosis, induced by Vacquinol-1 (VQ-1) has been described earlier. In the present study, we investigate the efficacy of VQ-1 treatment in two syngeneic rat GBM models, RG2 and NS1. VQ-1 treatment affected growth of both RG2 and NS1 cells
. Intracranially, significant reduction in RG2 tumor size was observed, although no effect was seen on overall survival. No survival advantage or effect on tumor size was seen in animals carrying the NS1 models compared to untreated controls. Furthermore, immunological staining of FOXP3, CD4 and CD8 showed no marked difference in immune cell infiltrate in tumor environment following treatment. Taken together, a survival advantage of VQ-1 treatment alone could not be demonstrated here, even though some effect upon tumor size was seen. Staining for immune cell markers did not indicate that VQ-1 either reduced or increased host anti-tumor immune response.
Increased membrane trafficking is observed in numerous cancer types, including glioblastoma. Targeting the oncogenic driven acquired alterations in membrane trafficking by synthetic cationic ...amphiphilic small molecules has recently been shown to induce death of glioblastoma cells, although the molecular targets are unknown.
The mechanism of action of the cationic amphiphilic drug Vacquinol-1 (Vacq1)-induced cytotoxicity was investigated using cell biology, biochemistry, functional experiments, chemical biology, unbiased antibody-based post-translation modification profiling, and mass spectrometry-based chemical proteomic analysis on patient-derived glioblastoma cells.
Vacq1 induced two types of abnormal endolysosomal vesicles, enlarged vacuoles and acidic vesicle organelles (AVOs). Mechanistically, enlarged vacuoles were formed by the impairment of lysosome reformation through the direct interaction and inhibition of calmodulin (CaM) by Vacq1, while AVO formation was induced by Vacq1 accumulation and acidification in the endosomal compartments through its activation of the v-ATPase. As a consequence of v-ATPase activation, cellular ATP consumption markedly increased, causing cellular energy shortage and cytotoxicity. This effect of Vacq1 was exacerbated by its inhibitory effects on calmodulin, causing lysosomal depletion and a failure of acidic vesicle organelle clearance.
Our study identifies the targets of Vacq1 and the mechanisms underlying its selective cytotoxicity in glioblastoma cells. The dual function of Vacq1 sets in motion a glioblastoma-specific vicious cycle of ATP consumption resulting in cellular energy crisis and cell death.
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.
There is a strong possibility that skeletal muscle can respond to irregular metabolic states by secreting specific cytokines. Obesity-related chronic inflammation, mediated by pro-inflammatory ...cytokines, is believed to be one of the causes of insulin resistance that results in type 2 diabetes. Here, we attempted to identify and characterize the members of the skeletal muscle secretome in response to tumor necrosis factor-alpha (TNF-α)-induced insulin resistance. To conduct this study, we comparatively analyzed the media levels of proteins released from L6 skeletal muscle cells. We found 28 TNF-α modulated secretory proteins by using separate filtering methods: Gene Ontology, SignalP, and SecretomeP, as well as the normalized Spectral Index for label-free quantification. Ten of these secretory proteins were increased and 18 secretory proteins were decreased by TNF-α treatment. Using microarray analysis of Zuker diabetic rat skeletal muscle combined with bioinformatics and Q-PCR, we found a correlation between TNF-α-mediated insulin resistance and type 2 diabetes. This novel approach combining analysis of the conditioned secretome and transcriptome has identified several previously unknown, TNF-α-dependent secretory proteins, which establish a foothold for research on the different causes of insulin resistance and their relationships with each other.
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 alpha beta -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 alpha beta -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 alpha beta -tubulin levels affects the level of GTP-bound Rheb. This occurs in the mitotic phase in which the level of acetylated alpha beta -tubulin is increased but that of GTP-bound Rheb is decreased. Constitutively active Rheb-overexpressing cells showed an abnormal mitotic progression, suggesting the deacetylated alpha beta -tubulin-mediated regulation of Rheb status may be important for proper mitotic progression. Taken together, we propose that deacetylated soluble alpha beta -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.
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 I-I2-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 I-I2-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 I-I2-tubulin levels affects the level of GTP-bound Rheb. This occurs in the mitotic phase in which the level of acetylated I-I2-tubulin is increased but that of GTP-bound Rheb is decreased. Constitutively active Rheb-overexpressing cells showed an abnormal mitotic progression, suggesting the deacetylated I-I2-tubulin-mediated regulation of Rheb status may be important for proper mitotic progression. Taken together, we propose that deacetylated soluble I-I2-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.
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.
► The mechanism of how GTP-bound Rheb is generated remains unclear. ► Deacetylated tubulin activates Rheb function by increasing the GTP-bound Rheb level. ► Accumulation of acetylated tubulins reduces Rheb-tubulin bindings and GTP-Rheb levels. ► Deacetylated tubulin acts as a temporal Rheb regulator during the cell cycle. ► Our findings provide new insights into small GTPases cycling and tubulin function.
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