Zebrafish raptor mutation inhibits the activity of mTORC1, inducing craniofacial defects due to autophagy-induced neural crest cell death.

The mechanistic target of rapamycin (mTOR) coordinates metabolism and cell growth with environmental inputs. mTOR forms two functional complexes: mTORC1 and mTORC2. Proper development requires both complexes but mTORC1 has unique roles in numerous cellular processes, including cell growth, survival and autophagy. Here, we investigate the function of mTORC1 in craniofacial development. We created a zebrafish raptor mutant via CRISPR/Cas9, to specifically disrupt mTORC1. The entire craniofacial skeleton and eyes were reduced in size in mutants; however, overall body length and developmental timing were not affected. The craniofacial phenotype associates with decreased chondrocyte size and increased neural crest cell death. We found that autophagy is elevated in raptor mutants. Chemical inhibition of autophagy reduced cell death and improved craniofacial phenotypes in raptor mutants. Genetic inhibition of autophagy, via mutation of the autophagy gene atg7, improved facial phenotypes in atg7;raptor double mutants, relative to raptor single mutants. We conclude that finely regulated levels of autophagy, via mTORC1, are crucial for craniofacial development.

Role of Raptor Gene Variants in Hypertension: Influence on Blood Pressure Independent of Salt Intake in White Population.

BACKGROUND: The mTOR (mechanistic target of rapamycin) is an essential regulator of fundamental biological processes. mTOR forms 2 distinct complexes, mTORC1 (mTOR complex 1) when it binds with RAPTOR (Regulatory-associated Protein of mTOR) and mTORC2 (mTOR complex 2) when it associates with RICTOR (Rapamycin-insesitive companion of mTOR). Due to the previous link between the mTOR pathway, aldosterone, and blood pressure (BP), we anticipated that variants in the mTOR complex might be associated with salt-sensitive BP. METHODS: BP and other parameters were assessed after a one-week liberal Na+ (200 mmol/d) and a one-week restricted Na+ (10 mmol/d) diet in 608 White subjects from the Hypertensive Pathotype cohort, single-nucleotide variants in MTOR, RPTOR, and RICTOR genes were obtained for candidate genes analyses. RESULTS: The analysis revealed a significant association between a single nucleotide variants within the RPTOR gene and BP. Individuals carrying the RPTOR rs9901846 homozygous risk allele (AA) and heterozygous risk allele (GA) exhibited a 5 mm Hg increase in systolic BP on a liberal diet compared with nonrisk allele individuals (GG), but only in women. This single nucleotide variants effect was more pronounced on the restricted diet and present in both sexes, with AA carriers having a 9 mm Hg increase and GA carriers having a 5 mm Hg increase in systolic BP compared with GG. Interestingly, there were no significant associations between MTOR or RICTOR gene variants and BP. CONCLUSIONS: The RPTOR gene variation is associated with elevated BP in White participants, regardless of salt intake, specifically in females.

Selective disruption of mTORC1 and mTORC2 in VTA astrocytes induces depression and anxiety-like behaviors in mice.

Dysfunction of the mechanistic target of rapamycin (mTOR) signaling pathway is implicated in neuropsychiatric disorders including depression and anxiety. Most studies have been focusing on neurons, and the function of mTOR signaling pathway in astrocytes is less investigated. mTOR forms two distinct complexes, mTORC1 and mTORC2, with key scaffolding protein Raptor and Rictor, respectively. The ventral tegmental area (VTA), a vital component of the brain reward system, is enrolled in regulating both depression and anxiety. In the present study, we aimed to examine the regulation effect of VTA astrocytic mTOR signaling pathway on depression and anxiety. We specifically deleted Raptor or Rictor in VTA astrocytes in mice and performed a series of behavioral tests for depression and anxiety. Deletion of Raptor and Rictor both decreased the immobility time in the tail suspension test and the latency to eat in the novelty suppressed feeding test, and increased the horizontal activity and the movement time in locomotor activity. Deletion of Rictor decreased the number of total arm entries in the elevated plus-maze test and the vertical activity in locomotor activity. These data suggest that VTA astrocytic mTORC1 plays a role in regulating depression-related behaviors and mTORC2 is involved in both depression and anxiety-related behaviors. Our results indicate that VTA astrocytic mTOR signaling pathway might be new targets for the treatment of psychiatric disorders.

mTOR Regulates Mineralocorticoid Receptor Transcriptional Activity by ULK1-Dependent and -Independent Mechanisms.

The mineralocorticoid receptor (MR) is a transcription factor for genes mediating diverse, cell-specific functions, including trophic effects as well as promoting fluid/electrolyte homeostasis. It was reported that in intercalated cells, phosphorylation of the MR at serine 843 (S843) by Unc-51-like kinase (ULK1) inhibits MR activation and that phosphorylation of ULK1 by mechanistic target of rapamycin (mTOR) inactivates ULK1, and thereby prevents MR inactivation. We extended these findings with studies in M1 mouse cortical collecting duct cells stably expressing the rat MR and a reporter gene. Pharmacological inhibition of ULK1 dose-dependently increased ligand-induced MR transactivation, while ULK1 activation had no effect. Pharmacological inhibition of mTOR and CRISPR/gRNA gene knockdown of rapamycin-sensitive adapter protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) decreased phosphorylated ULK1 and ligand-induced activation of the MR reporter gene, as well as transcription of endogenous MR-target genes. As predicted, ULK1 inhibition had no effect on aldosterone-mediated transcription in M1 cells with the mutated MR-S843A (alanine cannot be phosphorylated). In contrast, mTOR inhibition dose-dependently decreased transcription in the MR-S843A cells, though not as completely as in cells with the wild-type MR-S843. mTOR, Raptor, and Rictor coprecipitated with the MR and addition of aldosterone increased their phosphorylated, active state. These results suggest that mTOR significantly regulates MR activity in at least 2 ways: by suppressing MR inactivation by ULK1, and by a yet ill-defined mechanism that involves direct association with MR. They also provide new insights into the diverse functions of ULK1 and mTOR, 2 key enzymes that monitor the cell's energy status.

RPTOR blockade suppresses brain metastases of NSCLC by interfering the ceramide metabolism via hijacking YY1 binding.

BACKGROUND: Ceramide metabolism is crucial in the progress of brain metastasis (BM). However, it remains unexplored whether targeting ceramide metabolism may arrest BM. METHODS: RNA sequencing was applied to screen different genes in primary and metastatic foci and whole-exome sequencing (WES) to seek crucial abnormal pathway in BM + and BM-patients. Cellular arrays were applied to analyze the permeability of blood-brain barrier (BBB) and the activation or inhibition of pathway. Database and Co-Immunoprecipitation (Co-IP) assay were adopted to verify the protein-protein interaction. Xenograft and zebrafish model were further employed to verify the cellular results. RESULTS: RNA sequencing and WES reported the involvement of RPTOR and ceramide metabolism in BM progress. RPTOR was significantly upregulated in BM foci and increased the permeability of BBB, while RPTOR deficiency attenuated the cell invasiveness and protected extracellular matrix. Exogenous RPTOR boosted the SPHK2/S1P/STAT3 cascades by binding YY1, in which YY1 bound to the regions of SPHK2 promoter (at -353 ~ -365 nt), further promoting the expression of SPHK2. The latter was rescued by YY1 RNAi. Xenograft and zebrafish model showed that RPTOR blockade suppressed BM of non-small cell lung cancer (NSCLC) and impaired the SPHK2/S1P/STAT3 pathway. CONCLUSION: RPTOR is a key driver gene in the brain metastasis of lung cancer, which signifies that RPTOR blockade may serve as a promising therapeutic candidate for clinical application.

p300 nucleocytoplasmic shuttling underlies mTORC1 hyperactivation in Hutchinson-Gilford progeria syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism and autophagy. Multiple pathways modulate mTORC1 in response to nutrients. Here we describe that nucleus-cytoplasmic shuttling of p300/EP300 regulates mTORC1 activity in response to amino acid or glucose levels. Depletion of these nutrients causes cytoplasm-to-nucleus relocalization of p300 that decreases acetylation of the mTORC1 component raptor, thereby reducing mTORC1 activity and activating autophagy. This is mediated by AMP-activated protein kinase-dependent phosphorylation of p300 at serine 89. Nutrient addition to starved cells results in protein phosphatase 2A-dependent dephosphorylation of nuclear p300, enabling its CRM1-dependent export to the cytoplasm to mediate mTORC1 reactivation. p300 shuttling regulates mTORC1 in most cell types and occurs in response to altered nutrients in diverse mouse tissues. Interestingly, p300 cytoplasm-nucleus shuttling is altered in cells from patients with Hutchinson-Gilford progeria syndrome. p300 mislocalization by the disease-causing protein, progerin, activates mTORC1 and inhibits autophagy, phenotypes that are normalized by modulating p300 shuttling. These results reveal how nutrients regulate mTORC1, a cytoplasmic complex, by shuttling its positive regulator p300 in and out of the nucleus, and how this pathway is misregulated in Hutchinson-Gilford progeria syndrome, causing mTORC1 hyperactivation and defective autophagy.

RPTOR mutation: a novel predictor of efficacious immunotherapy in melanoma.

Identifying biomarkers to evaluate the therapeutic effect of immune checkpoint inhibitors (ICIs) is crucial. Regulatory Associated Protein of MTOR Complex 1 (RPTOR), one of the genes in the mTOR pathway, plays a role in regulating tumor progression. However, the connection between RPTOR mutation and the efficacy of ICIs in melanoma remains unclear. The data of ICIs-treated melanoma patients in discovery (n = 384) and validation (n = 320) cohorts were obtained from cBioPortal databases. The genomic data in the two cohorts was used to investigate the connection between RPTOR mutation and immunotherapy efficacy. The underlying mechanisms were explored based on data from the The Cancer Genome Atlas (TCGA)-skin cutaneous melanoma (SKCM) cohort. Compared to melanoma patients with RPTOR wildtype (RPTOR-WT), RPTOR-mutation (RPTOR-Mut) patients achieved prolonged overall survival (OS) in both discovery cohort (median OS of 49.3 months vs. 21.7 months; HR = 0.41, 95% CI: 0.18-0.92; P = 0.026) and validation cohorts (not reached vs. 42.0 months; HR = 0.34, 95% CI: 0.11-1.06; P = 0.049). RPTOR-Mut melanoma patients exhibited a higher objective response rate (ORR) than RPTOR-WT patients in the discovery cohort (55.0% vs. 29.0%, P = 0.022). RPTOR-Mut patients exhibited higher TMB than RPTOR-WT patients in both discovery and validation cohorts (P < 0.001). RPTOR-Mut melanoma patients had an increased number of DNA damage response (DDR) mutations in TCGA-SKCM cohort. Immune cell infiltration analysis suggested that activated CD4 memory T cells were more enriched in RPTOR-Mut tumors. RPTOR-Mut melanoma patients had higher expression levels of immune-related genes than the RPTOR-WT patients. Our results suggest that RPTOR mutation could serve as a predictor of effective immunotherapy for melanoma.

Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide.

The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser791 following ß-adrenergic stimulation. The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.

Raptor mediates the selective inhibitory effect of cardamonin on RRAGC-mutant B cell lymphoma.

BACKGROUND: mTORC1 (mechanistic target of rapamycin complex 1) is associated with lymphoma progression. Oncogenic RRAGC (Rag guanosine triphosphatase C) mutations identified in patients with follicular lymphoma facilitate the interaction between Raptor (regulatory protein associated with mTOR) and Rag GTPase. It promotes the activation of mTORC1 and accelerates lymphomagenesis. Cardamonin inhibits mTORC1 by decreasing the protein level of Raptor. In the present study, we investigated the inhibitory effect and possible mechanism of action of cardamonin in RRAGC-mutant lymphoma. This could provide a precise targeted therapy for lymphoma with RRAGC mutations. METHODS: Cell viability was measured using a cell counting kit-8 (CCK-8) assay. Protein expression and phosphorylation levels were determined using western blotting. The interactions of mTOR and Raptor with RagC were determined by co-immunoprecipitation. Cells overexpressing RagC wild-type (RagCWT) and RagC Thr90Asn (RagCT90N) were generated by lentiviral infection. Raptor knockdown was performed by lentivirus-mediated shRNA transduction. The in vivo anti-tumour effect of cardamonin was assessed in a xenograft model. RESULTS: Cardamonin disrupted mTOR complex interactions by decreasing Raptor protein levels. RagCT90N overexpression via lentiviral infection increased cell proliferation and mTORC1 activation. The viability and tumour growth rate of RagCT90N-mutant cells were more sensitive to cardamonin treatment than those of normal and RagCWT cells. Cardamonin also exhibited a stronger inhibitory effect on the phosphorylation of mTOR and p70 S6 kinase 1 in RagCT90N-mutant cells. Raptor knockdown abolishes the inhibitory effects of cardamonin on mTOR. An in vivo xenograft model demonstrated that the RagCT90N-mutant showed significantly higher sensitivity to cardamonin treatment. CONCLUSIONS: Cardamonin exerts selective therapeutic effects on RagCT90N-mutant cells. Cardamonin can serve as a drug for individualised therapy for follicular lymphoma with RRAGC mutations.

Iron drives anabolic metabolism through active histone demethylation and mTORC1.

All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me2 at enhancers of a high-affinity leucine transporter, LAT3, and RPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1.

Comparative analysis of the fecal microbiota of healthy and injured common kestrel (Falco tinnunculus) from the Beijing Raptor Rescue Center.

The gut microbiota is a complex ecosystem that interacts with many other factors to affect the health and disease states of the host. The common kestrel (Falco tinnunculus) is protected at the national level in China. However, the available sequencing data of the gut microbiota from the feces of wild common kestrels, especially for being rescued individuals by professional organization, remains limited. In the present study, we characterized the fecal bacterial communities of healthy and injured common kestrels, and compared the structure of their fecal microbiota by analyzing the V3-V4 region of the 16S rRNA gene using high-throughput sequencing technology with the Illumina MiSeq platform. We found that Firmicutes, Proteobacteria and Actinobacteria were the most predominant phyla in common kestrels. Further, the beta diversity analysis showed that changes in gut microbes were associated with injuries to the common kestrel. The Bacteroides/Firmicutes ratio was significantly lower in the injured group. At the genus level, Glutamicibacter showed significant difference in the two groups. The aim of our current study was to characterize the basic bacterial composition and community structure in the feces of healthy common kestrels, and then compare the differences in the fecal microbiota between healthy and injured individuals. Patescibacteria, Spirochaetes, and Glutamicibacter may be studied as potential biomarkers for certain diseases in raptors. The results could provide the basic data for additional research on the fecal microbiota of common kestrels and contribute to the rescue of wild raptors in the future.

O-GlcNAcylation of Raptor transduces glucose signals to mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates metabolism and cell growth in response to nutrient levels. Dysregulation of mTORC1 results in a broad spectrum of diseases. Glucose is the primary energy supply of cells, and therefore, glucose levels must be accurately conveyed to mTORC1 through highly responsive signaling mechanisms to control mTORC1 activity. Here, we report that glucose-induced mTORC1 activation is regulated by O-GlcNAcylation of Raptor, a core component of mTORC1, in HEK293T cells. Mechanistically, O-GlcNAcylation of Raptor at threonine 700 facilitates the interactions between Raptor and Rag GTPases and promotes the translocation of mTOR to the lysosomal surface, consequently activating mTORC1. In addition, we show that AMPK-mediated phosphorylation of Raptor suppresses Raptor O-GlcNAcylation and inhibits Raptor-Rags interactions. Our findings reveal an exquisitely controlled mechanism, which suggests how glucose coordinately regulates cellular anabolism and catabolism.

mTORC2 acts as a gatekeeper for mTORC1 deficiency-mediated impairments in ILC3 development.

Group 3 innate lymphoid cells (ILC3s) are mediators of intestinal immunity and barrier function. Recent studies have investigated the role of the mammalian target of rapamycin complex (mTOR) in ILC3s, whereas the mTORC1-related mechanisms and crosstalk between mTORC1 and mTORC2 involved in regulating ILC3 homeostasis remain unknown. In this study, we found that mTORC1 but not mTORC2 was critical in ILC3 development, IL-22 production, and ILC3-mediated intestinal homeostasis. Single-cell RNA sequencing revealed that mTORC1 deficiency led to disruption of ILC3 heterogeneity, showing an increase in differentiation into ILC1-like phenotypes. Mechanistically, mTORC1 deficiency decreased the expression of NFIL3, which is a critical transcription factor responsible for ILC3 development. The activities of both mTORC1 and mTORC2 were increased in wild-type ILC3s after activation by IL-23, whereas inhibition of mTORC1 by Raptor deletion or rapamycin treatment resulted in increased mTORC2 activity. Previous studies have demonstrated that S6K, the main downstream target of mTORC1, can directly phosphorylate Rictor to dampen mTORC2 activity. Our data found that inhibition of mTORC1 activity by rapamycin reduced Rictor phosphorylation in ILC3s. Reversing the increased mTORC2 activity via heterozygous or homozygous knockout of Rictor in Raptor-deleted ILC3s resulted in severe ILC3 loss and complete susceptibility to intestinal infection in mice with mTORC1 deficiency (100% mortality). Thus, mTORC1 acts as a rheostat of ILC3 heterogeneity, and mTORC2 protects ILC3s from severe loss of cells and immune activity against intestinal infection when mTORC1 activity is diminished.

The role of Raptor in lymphocytes differentiation and function.

Raptor, a key component of mTORC1, is required for recruiting substrates to mTORC1 and contributing to its subcellular localization. Raptor has a highly conserved N-terminus domain and seven WD40 repeats, which interact with mTOR and other mTORC1-related proteins. mTORC1 participates in various cellular events and mediates differentiation and metabolism. Directly or indirectly, many factors mediate the differentiation and function of lymphocytes that is essential for immunity. In this review, we summarize the role of Raptor in lymphocytes differentiation and function, whereby Raptor mediates the secretion of cytokines to induce early lymphocyte metabolism, development, proliferation and migration. Additionally, Raptor regulates the function of lymphocytes by regulating their steady-state maintenance and activation.

Raptor couples mTORC1 and ERK1/2 inhibition by cardamonin with oxidative stress induction in ovarian cancer cells.

Background: A balance on nutrient supply and redox homeostasis is required for cell survival, and increased antioxidant capacity of cancer cells may lead to chemotherapy failure. Objective: To investigate the mechanism of anti-proliferation of cardamonin by inducing oxidative stress in ovarian cancer cells. Methods: After 24 h of drug treatment, CCK8 kit and wound healing test were used to detect cell viability and migration ability, respectively, and the ROS levels were detected by flow cytometry. The differential protein expression after cardamonin administration was analyzed by proteomics, and the protein level was detected by Western blotting. Results: Cardamonin inhibited the cell growth, which was related to ROS accumulation. Proteomic analysis suggested that MAPK pathway might be involved in cardamonin-induced oxidative stress. Western blotting showed that cardamonin decreased Raptor expression and the activity of mTORC1 and ERK1/2. Same results were observed in Raptor KO cells. Notably, in Raptor KO cells, the effect of cardamonin was weakened. Conclusion: Raptor mediated the function of cardamonin on cellular redox homeostasis and cell proliferation through mTORC1 and ERK1/2 pathways.

Discovery of the First Raptor (Regulatory-Associated Protein of mTOR) Inhibitor as a New Type of Antiadipogenic Agent.

Raptor, a regulatory-associated protein of mTOR, has been genetically proved to be an important regulator in lipogenesis. However, its druggable potential is rarely investigated, largely due to the lack of an inhibitor. In this study, the antiadipogenic screening of a daphnane diterpenoid library followed by target fishing led to the identification of a Raptor inhibitor, 1c (5/7/6 carbon ring with orthoester and chlorine functionalities). Pharmacodynamic studies verified that 1c is a potent and tolerable antiadipogenic agent in vitro and in vivo. Mechanistic studies revealed that the targeting of Raptor by 1c could block the formation of mTORC1 and then downregulate the downstream S6K1- and 4E-BP1-mediated C/EBPs/PPARγ signaling, eventually retarding adipocyte cell differentiation at the early stage. These findings suggest that Raptor can be explored as a novel therapeutic target for obesity and its related complications, and 1c as the first Raptor inhibitor may provide a new therapeutic option for these conditions.

Structure of the lysosomal mTORC1-TFEB-Rag-Ragulator megacomplex.

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy1. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1)2-5 is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN6,7. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the 'megacomplex'. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.

Phosphorylation of OTUB1 at Tyr 26 stabilizes the mTORC1 component, Raptor.

Raptor plays a critical role in mTORC1 signaling. High expression of Raptor is associated with resistance of cancer cells to PI3K/mTOR inhibitors. Here, we found that OTUB1-stabilized Raptor in a non-canonical manner. Using biochemical assays, we found that the tyrosine 26 residue (Y26) of OTUB1 played a critical role in the interaction between OTUB1 and Raptor. Furthermore, non-receptor tyrosine kinases (Src and SRMS kinases) induced phosphorylation of OTUB1 at Y26, which stabilized Raptor. Interestingly, phosphorylation of OTUB1 at Y26 did not affect the stability of other OTUB1-targeted substrates. However, dephosphorylation of OTUB1 destabilized Raptor and sensitized cancer cells to anti-cancer drugs via mitochondrial reactive oxygen species-mediated mitochondrial dysfunction. Furthermore, we detected high levels of phospho-OTUB1 and Raptor in samples of patients with renal clear carcinoma. Our results suggested that regulation of OTUB1 phosphorylation may be an effective and selective therapeutic target for treating cancers via down-regulation of Raptor.

Silencing RIPK1/mTORC1 signalling attenuated the inflammation and oxidative stress in diabetic cardiomyopathy.

BACKGROUND: Diabetes cardiomyopathy (DCM) is one of the major risk factors for the heart failure of the diabetic patients. RIPK1 maybe participate in the regulation of the oxidative stress and inflammation during DCM. METHODS: H&E and Masson staining were utilized to assess the inflammation and fibrosis in myocardial tissues. CCK-8 and TUNEL staining were utilized to analyze cell viability and apoptosis, respectively. SOD activity and MDA content were detected utilizing the kits. The formation of autophagosomes was measured by immunofluorescence assay. RESULTS: RIPK1 and RPTOR (a component of mTORC1) expression and oxidative stress level were upregulated, but autophagy was decreased in the myocardial tissues of DCM rat characterized by the high body weight and blood glucose, abnormal cardiac function, myocardial inflammation and fibrosis. High glucose (HG) treatment resulted in cell viability and autophagy level decreasing, inflammatory cytokines expression increasing and oxidative stress increasing in cardiac fibroblasts (CFs). Meanwhile, RIPK1 and RPTOR expression also was increased in HG-treated cells. HG-induced CFs apoptosis, inflammation, oxidative stress and the inhibition of HG to cell viability and autophagy was partly reversed by the inhibitor of RIPK1 and mTORC1. CONCLUSION: Overall, RIPK1/mTORC1 signalling suppression improved HG-induced apoptosis, inflammation and oxidative stress through activation autophagy. These data provided a reliable evidence that RIPK1 may be a potential target for DCM therapeutic.

Phosphorylation of the novel mTOR substrate Unkempt regulates cellular morphogenesis.

Mechanistic target of rapamycin (mTOR) is a protein kinase that integrates multiple inputs to regulate anabolic cellular processes. For example, mTOR complex 1 (mTORC1) has key functions in growth control, autophagy, and metabolism. However, much less is known about the signaling components that act downstream of mTORC1 to regulate cellular morphogenesis. Here, we show that the RNA-binding protein Unkempt, a key regulator of cellular morphogenesis, is a novel substrate of mTORC1. We show that Unkempt phosphorylation is regulated by nutrient levels and growth factors via mTORC1. To analyze Unkempt phosphorylation, we immunoprecipitated Unkempt from cells in the presence or the absence of the mTORC1 inhibitor rapamycin and used mass spectrometry to identify mTORC1-dependent phosphorylated residues. This analysis showed that mTORC1-dependent phosphorylation is concentrated in a serine-rich intrinsically disordered region in the C-terminal half of Unkempt. We also found that Unkempt physically interacts with and is directly phosphorylated by mTORC1 through binding to the regulatory-associated protein of mTOR, Raptor. Furthermore, analysis in the developing brain of mice lacking TSC1 expression showed that phosphorylation of Unkempt is mTORC1 dependent in vivo. Finally, mutation analysis of key serine/threonine residues in the serine-rich region indicates that phosphorylation inhibits the ability of Unkempt to induce a bipolar morphology. Phosphorylation within this serine-rich region thus profoundly affects the ability of Unkempt to regulate cellular morphogenesis. Taken together, our findings reveal a novel molecular link between mTORC1 signaling and cellular morphogenesis.