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There are 39 results for Rapamycin (displaying 11 to 20).

The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells

The regulation of autophagy in metazoans is only partly understood, and there is a need to identify the proteins that control this process. The diabetes‐ and obesity‐regulated gene (DOR), a recently reported nuclear cofactor of thyroid hormone receptors, is expressed abundantly in metabolically active tissues such as muscle. Here, we show that DOR shuttles between the nucleus and the cytoplasm, depending on cellular stress conditions, and re‐localizes to autophagosomes on autophagy activation. We demonstrate that DOR interacts physically with autophagic proteins Golgi‐associated ATPase enhancer of 16 kDa (GATE16) and microtubule‐associated protein 1A/1B‐light chain 3. Gain‐of‐function and loss‐of‐function studies indicate that DOR stimulates autophagosome formation and accelerates the degradation of stable proteins. CG11347, the DOR Drosophila homologue, has been predicted to interact with the Drosophila Atg8 homologues, which suggests functional conservation in autophagy. Flies lacking CG11347 show reduced autophagy in the fat body during pupal development. All together, our data indicate that DOR regulates autophagosome formation and protein degradation in mammalian and Drosophila cells.

DOR localizes to autophagosomes when autophagy is activated. (A) Confocal images of HeLa cells transiently transfected with DOR and GFP‐LC3, and incubated for 1h with DMEM, HBSS (starvation), 2 μM rapamycin or HBSS containing 50 μM chloroquine. Nuclei were labelled with DAPI. Scale bars, 10 μm. (B,C) Confocal images of HeLa cells transiently transfected with DOR or GFP‐LC3 and incubated with 50 nM Lysotracker in DMEM or HBSS. Scale bars, 10 μm. (D) Confocal images of HeLa cells transiently …

… is increased by inhibitors of proteasomes such as MG132 or lactacystin ( online). Incubation of DOR‐transfected HeLa cells with cellular stressors caused DOR to leave the nucleus ( online). Activation of autophagy by amino‐acid starvation (here referred to as starvation) or by rapamycin treatment of DOR‐transfected HeLa or C2C12 muscle cells—which show endogenous DOR expression—caused DOR to leave the nucleus and re‐localize to cytoplasmic punctate structures ( and ; online). We next analysed DOR …

Caroline Mauvezin et al. EMBO Reports December 2009

A lysosome‐to‐nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB

The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. Here, we show that the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB activity. Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. In addition, the transcriptional response of lysosomal and autophagic genes to either lysosomal dysfunction or pharmacological inhibition of mTORC1 is suppressed in TFEB−/− cells. Interestingly, the Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to regulate starvation‐ and stress‐induced nuclear translocation of TFEB. These data indicate that the lysosome senses its content and regulates its own biogenesis by a lysosome‐to‐nucleus signalling mechanism that involves TFEB and mTOR.

… and subsequently stimulated as indicated for at least 3 h. Correct subcellular fractionation was verified with H3 and tubulin antibodies. (C) Effects of ERK and mTOR inhibitors on TFEB nuclear translocation. TFEB–GFP HeLa cells were seeded in 96‐well plates, cultured for 12 h, and then treated with the indicated concentrations of the ERK inhibitor U0126, or the mTOR inhibitors Rapamycin, Torin 1, and Torin 2. After 3 h at 37°C, cells were processed and images were acquired using the OPERA automated …

… on TFEB nuclear translocation may be mediated by mTORC1. Consistent with this idea, chloroquine or SalA inhibited mTORC1 activity as measured by level of p‐P70S6K, a known mTORC1 substrate ( ; ). The involvement of mTOR appears in contrast with our previous observation that Rapamycin, a known mTOR inhibitor, did not affect TFEB activity. However, recent data indicate that Rapamycin is a partial inhibitor of mTOR, as some substrates are still efficiently phosphorylated in the presence of this drug …

Carmine Settembre et al. The EMBO Journal March 2012

Inhibition of autophagy by TAB2 and TAB3

Autophagic responses are coupled to the activation of the inhibitor of NF‐κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFβ‐activated kinase 1 (TAK1)‐binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1‐IKK signalling axis, constitutively interact with each other via their coiled‐coil domains (CCDs). Upon autophagy induction, TAB2 and TAB3 dissociate from Beclin 1 and bind TAK1. Moreover, overexpression of TAB2 and TAB3 suppresses, while their depletion triggers, autophagy. The expression of the C‐terminal domain of TAB2 or TAB3 or that of the CCD of Beclin 1 competitively disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3, hence stimulating autophagy through a pathway that requires endogenous Beclin 1, TAK1 and IKK to be optimally efficient. These results point to the existence of an autophagy‐stimulatory ‘switch’ whereby TAB2 and TAB3 abandon inhibitory interactions with Beclin 1 to engage in a stimulatory liaison with TAK1.

Reduced interaction between Beclin 1, TAB and TAB3 in conditions of autophagy induction. (A, B) Inhibition of autophagy by dominant‐negative (DN) TAK1. HeLa cells were co‐transfected with a GFP–LC3‐encoding construct plus pcDNA3.1 (empty vector), or plasmids for the expression of WT TAK1 (TAK1WT) or the DN TAK1K63W mutant. One day later, cells were either left untreated (control) or driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα …

… immunoprecipitated with antibodies specific for HA (D) or T7 (E) and the precipitate was separated by SDS–PAGE and revealed with an antibody specific for Flag. (F) Immunoprecipitation of endogenous BCN1 with endogenous TAB2 or TAB3. HeLa cells were subjected to autophagy induction with starvation conditions, 1 μM rapamycin or 30 μM pifithrin α (PFTα) for the indicated time and then processed for TAB2 or TAB3 immunoprecipitation followed by the immunodetection of BCN1, TAK1, TAB2 and TAB3. Results in (E) and (F) are representative for three independent experiments.

Inhibition of autophagy by full‐length TAB2 and TAB3 but induction by their C‐terminal fragments. (A) Effects of full‐length TAB2 and TAB3 or their deletion mutants (as in Figure 1C) on autophagy. HeLa cells stably expressing GFP–LC3 were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the indicated TAB2 and TAB3 variants for 24 h, then driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα) for 4 h. Finally, the frequency …

… , where it is usually retained by IκB, to the nucleus, where NF‐κB then becomes active as a cytoprotective and pro‐inflammatory transcription factor ( ). In both murine and human cells, the genetic inhibition of TAK1 or any of the IKK subunits (but not that of the NF‐κB subunit p65) prevents the induction of autophagy in response to a panoply of different stimuli including starvation, rapamycin, p53 inhibition and endoplasmic reticulum stress ( ; ; ). Conversely, constitutively active IKK subunits …

Alfredo Criollo et al. The EMBO Journal December 2011

Defective autophagy is a key feature of cerebral cavernous malformations

Cerebral cavernous malformation ( CCM ) is a major cerebrovascular disease affecting approximately 0.3–0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss‐of‐function mutations have been identified in three genes, KRIT 1 ( CCM 1 ), CCM 2 ( MGC 4607), and PDCD 10 ( CCM 3 ), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT 1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/ SQSTM 1, defective quality control systems, and increased intracellular stress. KRIT 1 loss‐of‐function activates the mTOR ‐ ULK 1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the mole‐cular and cellular phenotypes associated with CCM . Insufficient autophagy is also evident in CCM 2 ‐silenced human endothelial cells and in both cells and tissues from an endothelial‐specific CCM 3 ‐knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial‐to‐mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions.

Cd44, PAI1 (also known as Serpine1), and Id1 mRNA expression levels in KRIT1 wt and KRIT1‐KO endothelial cells were assessed by quantitative real‐time PCR. Where indicated, KRIT1 wt and KRIT1‐KO endothelial cells were treated with 100 nM Torin1 or 500 nM rapamycin for 16h. The data are expressed as the mean ± s.e.m. Cd44: *P = 0.02848 (KO ctrl vs. KO Rapa); *P = 0.02605 (KO ctrl vs. KO Tor1). PAI1: *P = 0.04446 (KO ctrl vs. KO Rapa); *P = 0.03996 (KO ctrl vs. KO Tor1). Id1: *P = 0.00266 (KO …

… cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments. Immunoblot analysis of p62, LC3 I/II, and actin in KRIT1 wt and KRIT1‐KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments. Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 …

… ‐of‐function impair autophagy through the up‐regulation of the mechanistic target of rapamycin (mTOR) pathway, leading to a defective quality control system and the accumulation of aberrant and aggregated proteins. Our data raise the possibility that therapeutic activation of autophagy might prevent or reverse adverse clinical outcomes, thus improving the long‐term prognosis of CCM patients. To study the contribution of autophagy to CCM pathogenesis, we investigated whether KRIT1 down‐regulation …

Saverio Marchi et al. EMBO molecular medicine November 2015

PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L‐associated PI3K activity

The Beclin1–VPS34 complex is recognized as a central node in regulating autophagy via interacting with diverse molecules such as ATG14L for autophagy initiation and UVRAG for autophagosome maturation. However, the underlying molecular mechanism that coordinates the timely activation of VPS34 complex is poorly understood. Here, we identify that PAQR3 governs the preferential formation and activation of ATG14L‐linked VPS34 complex for autophagy initiation via two levels of regulation. Firstly, PAQR3 functions as a scaffold protein that facilitates the formation of ATG14L‐ but not UVRAG‐linked VPS34 complex, leading to elevated capacity of PI(3) P generation ahead of starvation signals. Secondly, AMPK phosphorylates PAQR3 at threonine 32 and switches on PI(3) P production to initiate autophagosome formation swiftly after glucose starvation. Deletion of PAQR3 leads to reduction of exercise‐induced autophagy in mice, accompanied by a certain degree of disaggregation of ATG14L‐associated VPS34 complex. Together, this study uncovers that PAQR3 can not only enhance the capacity of pro‐autophagy class III PI3K due to its scaffold function, but also integrate AMPK signal to activation of ATG14L‐linked VPS34 complex upon glucose starvation.

… HBSS treatment, amino acid starvation (AS), or rapamycin (50 nM) treatment for different times as indicated. EWT and PAQR3‐deficient HeLa (PAQR3 Cas9‐1/2) cells were incubated with normal medium (NM), GS or treated with rapamycin (50 nM) for 4 h, followed by IB. FHeLa cells stably transfected with PAQR3 were incubated in GS for different times as indicated, followed by IB. GHeLa cells were infected with PAQR3 or PAQR11 overexpression of lentivirus, respectively. After GS for 2 or 4 h, the whole‐cell lysates were harvested for IB. HHeLa cells were infected by control or PAQR3‐expressed lentivirus. After GS for 4 h, the cells were fixed for immunofluorescence staining with LC3 antibody (red). Scale bar: 10 μm.

… , the cells were treated with normal medium (NM), amino acid starvation (AS) or glucose starvation (GS), HBSS solution, or rapamycin (Rapa, 50 nM) for 4 h, respectively. The cell lysates were then used in IP and IB. Four VPS34 complexes of MEFs were immunopurified using the indicated antibodies under NM, AS, or GS conditions. The relative abundance of VPS34‐binding partners was determined by IB. Each IP was normalized to the amount of VPS34. HEK293T cells were transfected with the plasmids …

… and degradation of p62 were drastically decreased in PAQR3‐deficient MEFs (Feng et al , ) (Fig  B). We next investigated whether PAQR3 could impact autophagy induced by other autophagy‐promoting conditions. Autophagy was significantly blunted by PAQR3 deficiency in MEFs treated with Hank's balanced salt solution (HBSS) or amino acid‐free medium (Fig  B and C). Besides, PAQR3‐deleted MEFs also showed attenuated autophagy activity after rapamycin treatment (Fig  D). Then, we confirmed the regulatory …

Da‐Qian Xu et al. The EMBO Journal February 2016

Nucleotide degradation and ribose salvage in yeast

Nucleotide degradation is a universal metabolic capability. Here we combine metabolomics, genetics and biochemistry to characterize the yeast pathway. Nutrient starvation, via PKA, AMPK/SNF1, and TOR, triggers autophagic breakdown of ribosomes into nucleotides. A protein not previously associated with nucleotide degradation, Phm8, converts nucleotide monophosphates into nucleosides. Downstream steps, which involve the purine nucleoside phosphorylase, Pnp1, and pyrimidine nucleoside hydrolase, Urh1, funnel ribose into the nonoxidative pentose phosphate pathway. During carbon starvation, the ribose‐derived carbon accumulates as sedoheptulose‐7‐phosphate, whose consumption by transaldolase is impaired due to depletion of transaldolase's other substrate, glyceraldehyde‐3‐phosphate. Oxidative stress increases glyceraldehyde‐3‐phosphate, resulting in rapid consumption of sedoheptulose‐7‐phosphate to make NADPH for antioxidant defense. Ablation of Phm8 or double deletion of Pnp1 and Urh1 prevent effective nucleotide salvage, resulting in metabolite depletion and impaired survival of starving yeast. Thus, ribose salvage provides means of surviving nutrient starvation and oxidative stress.

… nucleosides, nucleic bases and PPP intermediates as a function of starvation time, in wild‐type and autophagy deficient (atg7 deletion) yeast. The x axis represents minutes after carbon starvation, and the y axis represents fraction of unlabeled metabolites (mean±range of N=2 biological replicates). (C) Ratio of metabolite levels in atg7 strain versus wild‐type strain in carbon starvation. (D) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild‐type strain in carbon starvation. (E) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild‐type yeast. In (C) to (E), all reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.

… , proteins such as kinases sense metabolic conditions and activate signaling cascades to coordinate metabolism and overall cellular activity. Important examples of such signaling enzymes include protein kinase A (PKA, activated by glucose), AMP‐activated protein kinase (SNF1, repressed by glucose), and target of rapamycin (TOR, activated by abundant nitrogen) ( ; ; ; ). A particularly important function of these kinases is to control levels of ribosomes, which constitute about 10% of yeast dry weight …

Yi‐Fan Xu et al. Molecular Systems Biology May 2013

Listeria phospholipases subvert host autophagic defenses by stalling pre‐autophagosomal structures

Listeria can escape host autophagy defense pathways through mechanisms that remain poorly understood. We show here that in epithelial cells, Listeriolysin (LLO)‐dependent cytosolic escape of Listeria triggered a transient amino‐acid starvation host response characterized by GCN2 phosphorylation, ATF3 induction and mTOR inhibition, the latter favouring a pro‐autophagic cellular environment. Surprisingly, rapid recovery of mTOR signalling was neither sufficient nor necessary for Listeria avoidance of autophagic targeting. Instead, we observed that Listeria phospholipases PlcA and PlcB reduced autophagic flux and phosphatidylinositol 3‐phosphate (PI3P) levels, causing pre‐autophagosomal structure stalling and preventing efficient targeting of cytosolic bacteria. In co‐infection experiments, wild‐type Listeria protected PlcA/B‐deficient bacteria from autophagy‐mediated clearance. Thus, our results uncover a critical role for Listeria phospholipases C in the inhibition of autophagic flux, favouring bacterial escape from host autophagic defense.

… , analysed by IF using antibodies against mTOR and LAMP2. The arrow shows a Listeria vacuole positive for mTOR and LAMP2, and arrowheads indicate mTOR‐ and LAMP2‐positive late endosomes or lysosomes. (D, E) Percentage of cells infected with WT, PlcA/B− or LLO− Listeria strains displaying one or several LAMP2+ (D) or NDP52+ (E) Listeria vesicles. Values are means±s.e.m. n=3. **P<0.01. (F) Percentage of cells infected with WT Listeria, in the presence (black bars) or absence (white bars) of rapamycin, displaying one or several GFP‐LC3+ Listeria autophagosomes. Values are means±s.e.m. n=2. Scale bars: 5 μm.Source data for this figure is available on the online supplementary information page. …

… that are associated with the endoplasmic reticulum (ER), and the ER/mitochondria interface then provides the membranes necessary for the progressive extension of a double membrane that surrounds the cargo ( ), until full engulfment and formation of an autophagosome that is targeted to lysosomes for degradation of its content. While autophagy is inhibited by the checkpoint kinase mammalian target of rapamycin (mTOR) in metabolically replete cells, this process is strongly upregulated following mTOR inhibition …

Ivan Tattoli et al. The EMBO Journal November 2013

Functional and physical interaction between Bcl‐XL and a BH3‐like domain in Beclin‐1

The anti‐apoptotic proteins Bcl‐2 and Bcl‐X L bind and inhibit Beclin‐1, an essential mediator of autophagy. Here, we demonstrate that this interaction involves a BH3 domain within Beclin‐1 (residues 114–123). The physical interaction between Beclin‐1 and Bcl‐X L is lost when the BH3 domain of Beclin‐1 or the BH3 receptor domain of Bcl‐X L is mutated. Mutation of the BH3 domain of Beclin‐1 or of the BH3 receptor domain of Bcl‐X L abolishes the Bcl‐X L ‐mediated inhibition of autophagy triggered by Beclin‐1. The pharmacological BH3 mimetic ABT737 competitively inhibits the interaction between Beclin‐1 and Bcl‐2/Bcl‐X L , antagonizes autophagy inhibition by Bcl‐2/Bcl‐X L and hence stimulates autophagy. Knockout or knockdown of the BH3‐only protein Bad reduces starvation‐induced autophagy, whereas Bad overexpression induces autophagy in human cells. Gain‐of‐function mutation of the sole BH3‐only protein from Caenorhabditis elegans , EGL‐1, induces autophagy, while deletion of EGL‐1 compromises starvation‐induced autophagy. These results reveal a novel autophagy‐stimulatory function of BH3‐only proteins beyond their established role as apoptosis inducers. BH3‐only proteins and pharmacological BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin‐1 and Bcl‐2 or Bcl‐X L .

Impact of the BH3‐only protein Bad on autophagy. (A, B) Interactions between Bcl‐XL, Beclin‐1 and Bad in conditions of autophagy induction. Cells were either treated by nutrient depletion (A) or addition of 1 μM rapamycin, 1 mM lithium chloride, 100 μM L‐690,330 or 50 μM carbamazepine (B), followed by immunprecipitation of Bcl‐XL (as in Figure 2C) and revealing the immunoblots by antibodies specific for Bcl‐XL, Beclin‐1 or Bad. (C) Interaction between endogenous Bad and Bcl‐2. HeLa cells were …

… , whereas the amount of the BH3 protein Bad (whose activation is known to be triggered by serum withdrawal) ( ) that co‐immunoprecipitated with Bcl‐X L increased ( ). In contrast, addition of rapamycin (which induces autophagy by inhibition of mTOR) ( ) or other autophagy inducers that affect the level of phosphatidyl inositol‐3‐phoshate ( ) had less marked effects on the interactions between Beclin‐1, Bcl‐X L and Bad ( ). Upon starvation (but not upon ABT737 addition), the amount of endogenous Bad …

M Chiara Maiuri et al. The EMBO Journal May 2007

The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9 ALS / FTD ). C9orf72 encodes two C9orf72 protein isoforms of unclear function. Reduced levels of C9orf72 expression have been reported in C9 ALS / FTD patients, and although C9orf72 haploinsufficiency has been proposed to contribute to C9 ALS / FTD , its significance is not yet clear. Here, we report that C9orf72 interacts with Rab1a and the Unc‐51‐like kinase 1 ( ULK 1) autophagy initiation complex. As a Rab1a effector, C9orf72 controls initiation of autophagy by regulating the Rab1a‐dependent trafficking of the ULK 1 autophagy initiation complex to the phagophore. Accordingly, reduction of C9orf72 expression in cell lines and primary neurons attenuated autophagy and caused accumulation of p62‐positive puncta reminiscent of the p62 pathology observed in C9 ALS / FTD patients. Finally, basal levels of autophagy were markedly reduced in C9 ALS / FTD patient‐derived iN eurons. Thus, our data identify C9orf72 as a novel Rab1a effector in the regulation of autophagy and indicate that C9orf72 haploinsufficiency and associated reductions in autophagy might be the underlying cause of C9 ALS / FTD ‐associated p62 pathology.

… ‐targeting (Ctrl) or C9orf72 siRNA were incubated with vehicle (Ctrl), rapamycin, BafA1, or BafA1 + rapamycin for 6 h, and levels of LC3‐I and II were determined on immunoblots. Levels of LC3‐II were normalized against α‐tubulin and are shown relative to the BafA1‐treated sample (mean ± SEM; one‐way ANOVA with Fisher's LSD test: ns, not significant; **P ≤ 0.01, ***P ≤ 0.001; N = 3 experiments). These data are also shown in Fig D.

… /Ctrl: 120; Ctrl/Torin1: 101; C9orf72/Ctrl: 99; C9orf72/Torin1: 106; Ctrl/BafA1: 116; Ctrl/Torin1/BafA1: 118; C9orf72/BafA1: 109; C9orf72/Torin1/BafA1: 106). Scale bar = 20 μm. C9orf72 knockdown was confirmed by RT–qPCR (Appendix Fig S2). C, DHEK293 cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were incubated with BafA1, BafA1 + Torin1 (C), or BafA1 + rapamycin (D), and levels of LC3‐I and II were determined by immunoblots. Levels of LC3‐II were normalized against α‐tubulin …

HEK293 cells were transfected with non‐targeting (Ctrl) or C9orf72 siRNA. Cells were treated with rapamycin for 6 h to induce autophagy. Activation of ULK1 was determined on immunoblots using phospho‐ULK1 (Ser757), total ULK1, and GAPDH Abs (loading control). HeLa cells treated with non‐targeting (Ctrl) or C9orf72 siRNA were transfected with mCherry‐FIP200. Twenty‐four hours post‐transfection, cells were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 …

… remains membrane associated throughout autophagy, and in the process, it is degraded in autolysosomes. Thus, turnover of LC3‐II reflects the progression of autophagy. Due to questionable specificity of commercial C9orf72 antibodies, we used mRNA levels and RT–qPCR to confirm C9orf72 knockdown throughout this study ( ). In mammalian cells, ULK1 controls the initial stages of autophagosome formation and is itself regulated in response to nutrient starvation by mammalian target of rapamycin (mTOR …

Christopher P Webster et al. The EMBO Journal August 2016

PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2‐dependent actin remodeling

Altered abundance of phosphatidyl inositides ( PI s) is a feature of cancer. Various PI s mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5‐bisphosphate ( PI (4,5)P 2 ) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN 2 and PLC β1 enzymatically reduce plasma membrane PI (4,5)P 2 levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI (4,5)P 2 abundance by these enzymes releases the PI (4,5)P 2 ‐binding protein cofilin from its inactive membrane‐associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid‐dependent sequestration of an actin‐remodeling factor.

A, BLM2 cells transfected with siRNA targeting PTPRN2 (A), PLCβ1 (B) or a control siRNA were transfected with Lyn11‐FRB and INPP5E‐FKBP, treated with either DMSO or 100 nM rapamycin and subjected to the migration assay. N = 5 inserts/group. C, DMDA‐MB‐231 cells overexpressing PTPRN2 (C), PLCβ1 (D) or control vector were treated with carrier alone or carrier incubated with PI(4,5)P2 for 1 h and then immediately subjected to the migration assay. N = 5 inserts/group. EKaplan–Meier curve …

… membrane levels in metastatic migration, we manipulated the levels of this phosphoinositide in cancer cells using two methods. We first sought to determine whether the migration defect of PTPRN2‐ or PLCβ1‐depleted cells could be rescued by decreasing the plasma membrane PI(4,5)P 2 levels of these cells. To selectively deplete plasma membrane PI(4,5)P 2 , we used a rapamycin‐induced dimerization system previously developed for this purpose (Heo et al , ; Varnai et al , ). In this system, inositol …

Caitlin A Sengelaub et al. The EMBO Journal January 2016
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