D Lysates from FT cells transfected with the indicated plasmids were separated on Phos-tag gel. Arrowheads indicate multiple bands that may represent phosphorylated Girdin NT. E Lysates from FT cells stimulated with FBS in the presence or absence of U were subjected to IP to enrich for endogenous Girdin, followed by the enrichment of phosphopeptides using a Titansphere Phos-TiO kit.

The phosphopeptides were analyzed by mass spectrometry. Several reports have indicated that the components of mTORC1 signaling undergo ubiquitination in response to amino acids [ 18 , 19 ].

Therefore, we first tested endogenous 4F2hc ubiquitination by tandem-repeated ubiquitin-binding entities TUBEs also known as ubiquitin traps , which enabled us to measure 4F2hc ubiquitination under a non-denaturing physiological condition see Materials and methods. We found that 4F2hc was ubiquitinated in FT cells, and ubiquitination was significantly increased in response to amino acid stimulation Fig 3A.

Next, we transfected FT cells with Flag-tagged 4F2hc and His-tagged ubiquitin and then isolated ubiquitinated proteins using Ni-NTA beads under denaturing conditions. The result showed that amino acid stimulation dramatically promoted 4F2hc ubiquitination Fig 3B. A and B FT cells transfected with the indicated plasmids were starved for amino acids for 1 h in amino acid—free medium supplemented with dialyzed FBS, followed by stimulation with complete growth medium for 30 min.

Ubiquitinated proteins were isolated with glutathione-sepharose beads under a non-denaturing condition A or Ni-NTA agarose beads under a denaturing condition B to detect 4F2hc ubiquitination. C Primary structures of 4F2hc short and long isoforms and its mutants used in the study.

D Lysates from FT cells transfected with the indicated plasmids were pulled down under denaturing conditions using Ni-NTA agarose beads, showing the ubiquitination of the transfected 4F2hc and its mutants. E FT cells transfected with the indicated combinations of 4F2hc, GST, and GST-NT.

The lysates were pulled down with glutathione beads, followed by WB with the indicated antibodies. We identified eight potential ubiquitination sites in the cytoplasmic domain of 4F2hc from the PhosphoSite database, mutated them in various combinations by substituting lysine K residues to arginines Rs , and compared the ubiquitination of wild-type WT 4F2hc and the mutants Fig 3C and 3D.

Moreover, the ubiquitination of 4F2hc was almost undetectable in 8KR mutant, which means that all or most of these eight K residues are involved in 4F2hc ubiquitination.

Consistently, the 5KR mutant showed a weaker interaction with the Girdin NT domain than its WT counterpart, and the interaction between the Girdin NT domain and 6KR or 8KR mutant was almost undetectable Fig 3E.

These data suggested that the ubiquitination of 4F2hc plays an important role in its interaction with Girdin. Consistent with a previous study, small interfering RNA siRNA -mediated depletion of 4F2hc inhibited mTORC1 activity, as indicated by the decreased phosphorylation of the ribosomal protein S6 kinase beta1 S6K1 and S6, which were monitored as readouts for mTORC1 activation Fig 4A , S1A Fig , S1 Data.

In contrast, Girdin depletion by two independent interfering sequences significantly increased the basal level of mTORC1 activity Fig 4A and 4B , S1A and S1B Fig , S1 Data , which was reciprocally confirmed by the fact that overexpression of WT Girdin, but not its AA mutant that had lost the capacity to bind 4F2hc, significantly suppressed mTORC1 activity Fig 4C , S1C Fig , S1 Data.

Throughout the study, to achieve equal expression levels of exogenously introduced cDNAs and to exclude interclonal differences among cell lines, we utilized the Flp-In system, in which the cDNAs are inserted by homologous recombination into a single genomic recombination target site introduced into the host Flp-In cells see Materials and methods to generate cell lines.

In all of HeLa, FT, and Flp-In cells, Girdin knockout cells showed higher basal mTORC1 activity than WT cells. In addition, re-expression of WT Girdin, but not Girdin AA mutant, significantly decreased mTORC1 activity in Girdin knockout Flp-In cells S1F Fig.

Unexpectedly, the overexpression of 4F2hc also inhibited basal mTORC1 activation, which might be due to mislocalization of amino acid transporters or disruption of endogenous amino acid transporter complexes by overexpressed 4F2hc Fig 4C.

In addition, 4F2hc knockdown abrogated Girdin depletion-induced mTORC1 activation, indicating that Girdin regulates mTORC1 activity through 4F2hc Fig 4A. A and B FT cells were transfected or transduced with the indicated siRNA or shRNA, respectively, followed by WB to monitor mTORC1 activity.

In blot images for S6K1 and pS6K1, the lower bands represent S6K1 or pS6K1, as indicated by arrowhead or asterisk, respectively. Quantitative data are shown in S1A and S1B Fig. C Basal mTORC1 activity in control Flp-In cells and cells stably overexpressing Girdin WT, Girdin AA, and 4F2hc.

Quantitative data are shown in S1C Fig. D Brain sections from heterogeneous left and Girdin knockout right P14 mice were stained for pS6. The brown staining indicates pS6 signal.

The regions within the black boxes are shown below at a higher magnification. E, F, G FT cells E , primary mouse embryonic fibroblasts isolated from WT and Girdin-deficient mice F , or Flp-In cells stably transduced with the indicated constructs F were starved for amino acids for 1 h in medium supplemented with dialyzed FBS, followed by stimulation with complete medium for the indicated time.

mTORC1 activity and Girdin expression were monitored by WB. Quantitative data are shown in S1G—S1I Fig. FBS, fetal bovine serum; FH, Flag-HA epitope; Girdin, girders of actin filaments; HP, hippocampus; Mr , molecular marker; mTORC1, mechanistic target of rapamycin complex 1; OB, olfactory bulb; RMS, rostral migratory stream; shRNA, short hairpin RNA; siRNA, small interfering RNA; S6K1, S6 kinase beta1; WB, western blot; WT, wild-type; 4F2hc, 4F2 heavy chain.

The significance of Girdin in mTORC1 regulation was further supported by the observation that neuroblasts in the rostral migratory stream RMS and the dentate gyrus DG of the brain, the development of which have been shown to be specifically regulated by Girdin [ 23 , 34 ], exhibited significantly high S6 activity in Girdin-deficient as compared to control mice Fig 4D.

This result supported the notion that Girdin suppresses mTORC1 activity. Next, we examined the kinetics of mTORC1 activation in amino acid—stimulated cells. Girdin depletion significantly augmented mTORC1 activity over time upon amino acid stimulation in FT cells cultured in the presence of dialyzed serum Fig 4E , S1G Fig , S1 Data.

This was confirmed in primary mouse embryonic fibroblasts isolated from control and Girdin-deficient mice Fig 4F , S1H Fig , S1 Data. Overexpression of WT Girdin, but not its AA mutant, significantly inhibited amino acid—induced mTORC1 activation, supporting that Girdin is a negative regulator of mTORC1 in the presence of serum and amino acid signals Fig 4G , S1I Fig , S1 Data.

This notion was corroborated by the fact that Girdin depletion inhibited autophagy induced by amino acid withdrawal, as shown by the reduced lipidation of microtubule-associated protein light chain 3 LC3 S2A Fig , S1 Data. In this experiment, we also tested the effect of the lysosome inhibitor Bafilomycin A1, which inhibits the late phase of autophagy, including autophagosome—lysosome fusion and autolysosome acidification [ 35 ].

In cells treated with Bafilomycin A1, Girdin knockdown still inhibited amino acid starvation—induced autophagy, indicating that Girdin affects the early autophagy phase, as mTORC1 reportedly does S2A Fig [ 36 ].

Furthermore, the overexpression of WT Girdin, but not its AA mutant, accelerated autophagy, as indicated by the decreased appearance of green fluorescent protein GFP -LC3 puncta S2B—S2D Fig , S1 Data.

These findings suggested that Girdin does not directly regulate S6K1 and S6 activities but rather affects the entire mTORC1 pathway. Given that Girdin was previously reported as a critical regulator of intracellular membrane trafficking [ 24 ], we hypothesized that Girdin-mediated negative regulation of mTORC1 activity was attributed to 4F2hc internalization.

Immunofluorescence analysis, in which the specificity of the 4F2hc antibody used clone HBJ [ 37 ] was verified by the knockdown of endogenous 4F2hc Fig 5A and an IP test followed by western blot WB analysis using a different, commercially available anti-4F2hc antibody Fig 5B , showed that 4F2hc localized to the plasma membrane.

The expression of WT Girdin, but not the AA mutant, promoted 4F2hc translocation to the lysosomes after amino acid stimulation Fig 5C and 5D , S1 Data.

Consistently, cell fractionation showed that Girdin overexpression and depletion decreased and increased the cell surface level of 4F2hc, respectively Fig 5E and 5F , S1 Data.

A In-house-developed 4F2hc antibody was used for IF in HeLa cells and FT cells; staining intensity was markedly reduced in 4F2hc knockdown cells. B In-house-developed 4F2hc antibody was used for IP in HeLa cells, and the sample was detected by commercial 4F2hc antibody to verify the quality of the antibody.

C Flp-In cells stably transduced with the indicated plasmids were starved for amino acids for 1 h and stimulated with amino acids for 30 min, followed by immunofluorescence staining for 4F2hc green and Lamp1 red. Arrowheads indicate the localization of 4F2hc in the lysosome.

D Quantification of the cells shown in C with 4F2hc localized on the lysosome cells from three independent experiments. The data are presented as means ± SEs. The data underlying this figure can be found in S1 Data.

E Cell surface proteins of HeLa cells transfected with the indicated siRNAs or plasmids were biotinylated and isolated, followed by WB with the indicated antibodies. F Quantification of the bands intensity of cell surface 4F2hc from 3 independent experiments is shown.

The value of the surface 4F2hc in the cells transfected with empty vector or control siRNA were set as Girdin, girders of actin filaments; IgG, immunoglobulin G; Mr , molecular marker; N.

Previous studies have shown that Gln, Leu, and arginine Arg are three intracellular amino acids that activate mTORC1 [ 10 , 38 , 39 ]. Our comprehensive measurement of cytosolic amino acids showed that min stimulation with total amino acids, which is sufficient to activate mTORC1, led to increases in Gln and Leu Fig 6A , S3A and S3B Fig.

Interestingly, while most amino acids increased in concentration upon total amino acid stimulation, Arg did not change Fig 6A , S1 Data , which may be due to the activity of some amino acid exchangers that mediate Arg efflux [ 40 ].

These results confirmed the correlation between mTORC1 activation and intracellular amino acid contents—at least those of Gln and Leu—in our experimental setup. Amino acid concentrations were measured with an HPLC system. Quantification of each amino acid concentration from 3 independent experiments is shown; the data are presented as means ± SEs.

B, C FT cells transfected with indicated siRNA B or Flp-in cells stably expressing empty vector, Girdin WT, Girdin AA, and 4F2hc C were starved for amino acids for 1 h and stimulated with total amino acids for 10 min.

The values in control cells control siRNA or empty vector starved for amino acids were set as 1. D, E FT cells transduced with control or Girdin shRNA were starved for 1 h in amino acid—free medium and stimulated with Gln D or Leu E for the indicated times, followed by WB.

Note that the activation of mTORC1 was observed at 60 min in Gln stimulated cells D. AA, amino acids; Arg, arginine; A. Next, we examined the effects of Girdin and 4F2hc depletion and overexpression on intracellular amino acid contents Fig 6B and 6C , S3A and S3B Fig , S1 Data.

In cells stimulated with total amino acids, Girdin knockdown led to significantly increased Gln and Leu contents, in contrast to 4F2hc depletion, which led to decreases in the two amino acids Fig 6B , S3A Fig , S1 Data. Neither Girdin nor 4F2hc knockdown had an effect on intracellular Arg under the same condition.

These results were consistent with our finding that Girdin knockdown increased amino acid—stimulated mTORC1 activation, whereas 4F2hc knockdown decreased it Fig 4A , suggesting that Girdin and 4F2hc modulate mTORC1 activity through the regulation of intracellular Gln and Leu contents.

Overexpression of Girdin and 4F2hc led to significant decreases in Gln and Leu, but not Arg, in cells stimulated with total amino acids Fig 6C , S3B Fig , S1 Data.

Overexpression of Girdin AA mutant also resulted in decreased Gln and Leu contents, but less significantly than WT. These results are consistent with our findings that overexpression of Girdin WT and 4F2hc inhibited mTORC1 activation.

In these experiments, Gln and Leu were very low in starved cells Fig 6A , S3 Fig , which indicated the significance of uptake of these amino acids across the plasma membrane in determining cellular mTORC1 activation Fig 6B and 6C. In addition, although there were some differences in Gln and Arg concentration in starved Girdin or 4F2hc-overexpressing cells, the concentration was also very low Fig 6C , S3 Fig.

Supporting this notion, we found that Girdin and 4F2hc depletion significantly up-regulated and inhibited mTORC1 activation, respectively, in cells stimulated with either Gln or Leu Fig 6D and 6E. Notably, the kinetics of mTORC1 activation induced by Gln and Leu stimulation were different: Leu stimulation activated mTORC1 quickly, whereas Gln-stimulated activation of mTORC1 required about 1 h stimulation, as previously reported [ 10 ].

Altogether, the data implied that Girdin down-regulates the cell surface level of 4F2hc via endocytosis, which subsequently decreases intracellular Gln and Leu contents to negatively regulate mTORC1 activation Fig 7.

B In the presence of Girdin, it interacts with 4F2hc depending on MAPK activity and amino acid stimulation, which promotes endocytosis of 4F2hc into the lysosome, leading to changes in intracellular Gln, Leu, and mTORC1 activity. AA, amino acids; Girdin, girders of actin filaments; Gln, glutamine; Leu, leucine; MAPK, mitogen-activated protein kinase; mTORC1, mechanistic target of rapamycin complex 1; TSC, tuberous sclerosis; Ub, ubiquitin; 4F2hc, 4F2 heavy chain.

Our present study identified a novel negative regulatory mechanism for amino acid signaling. This process is mediated by Girdin—4F2hc interaction, which is regulated by Girdin phosphorylation and 4F2hc ubiquitination downstream of MAPK and amino acid stimulation, respectively.

The interaction between Girdin and 4F2hc modulates the cell surface level of 4F2hc through the regulation of 4F2hc endocytosis, which subsequently changes intracellular Gln and Leu contents to negatively regulate amino acid signaling.

The involvement of Arg in Girdin-mediated mTORC1 regulation remains unclear. In addition, because the changes of mTORC1 activation by Girdin knockdown were relatively modest Fig 4E—4G , additional mechanisms apart from Girdin—4F2hc interaction could also play a role in mTORC1 regulation.

We showed that MAPK-mediated Girdin phosphorylation is crucial for the endocytosis and lysosomal localization of 4F2hc. This finding is in accordance with a previous report that Girdin functions to activate dynamin GTPase, which is essential for the pinching off of clathrin-coated vesicles and their endocytosis [ 24 ].

In addition, another study reported that 4F2hc translocates to the lysosome through binding to the lysosomal protein, LAPTM4b [ 44 ].

We speculate that Girdin promotes amino acid—stimulated 4F2hc endocytosis, and the internalized 4F2hc may translocate to the lysosome via interaction with LAPTM4b. To clarify the mechanism of Girdin—4F2hc interaction in regulation of mTORC1 activity, we investigated the role of Girdin and 4F2hc on intracellular amino acids.

Although some studies have reported an essential role of lysosomal amino acids in mTORC1 activation [ 45 ], a very recent study showed that 1-h amino acid starvation led to a significant decrease in essential amino acids in whole cell lysate but not the lysosome fraction, which implicated that lysosomal amino acids may not be a major driving force for mTORC1 activity [ 46 ].

This was consistent with the fact that most amino acid sensors identified so far, such as Castor, sestrin2, and SAMTOR, are located in the cytosol [ 14 — 16 ].

Therefore, we focused on the effects of Girdin and 4F2hc on cytosolic amino acids, specifically, Leu, Gln, and Arg, all of which regulate mTORC1 activity. Girdin depletion—mediated mTORC1 activation was accompanied by an increase in intracellular Gln and Leu contents, whereas no obvious changes in Arg were detected among control and Girdin or 4F2hc knockdown cells under the given culture conditions.

Based on these findings, we speculated that Girdin negatively regulates amino acid signaling via modulating 4F2hc endocytosis and the cytosolic contents of Gln and Leu. In embryonic development and homeostasis in adult tissues, mTORC1 activity needs to be tightly controlled; deregulation of mTORC1 leads to stem cell aging, reduced tissue regeneration capacity, and the progression of diseases such as cancer [ 47 , 48 ].

One potential role of Girdin-mediated mTORC1 regulation was suggested by our finding that mTORC1 activity is significantly up-regulated in neuroblasts in the RMS and DG in the brain of Girdin-deficient mice Fig 4D. We and others have previously reported that Girdin is essential for the development of postnatal brain and adult neurogenesis, in which it controls the differentiation and migration of newly generated neuroblasts in the RMS and DG [ 23 , 34 ].

We therefore speculate that the negative regulatory effect of Girdin on mTORC1 may contribute to the proper development of those brain regions in the developmental and adult stages. Girdin is expressed in some types of human malignancies, in which it may have a role in maintaining appropriate activation of mTORC1.

Further experiments are required to verify the physiological functions of Girdin-mediated mTORC1 regulation. All animal protocols were approved by the Animal Care and Use Committee of Nagoya University Graduate School of Medicine Approval number The HeLa cell line was purchased from the American Tissue Type Culture Rockville, MD.

Primary mouse embryonic fibroblasts were isolated from WT and Girdin-deficient mouse E Amino acid—free DMEM Cell Science and Technology Institute, Inc. PolyFect Qiagen, Hilden, Germany was used to obtain high-level expression in HeLa cells.

A control shRNA vector provided by Clontech Laboratories was used as a negative control. The production of retroviral supernatants by GP packaging cells Clontech Laboratories is described below. Louis, MO , anti-Flag clone M2, Sigma , anti-β-actin clone AC, Sigma , normal mouse IgG Millipore, Milford, MA, 12— , and normal sheep IgG Millipore, 12— Phos-tag Acrylamide Wako, Saitama, Japan was used for the generation of the Phos-tag gel to analyze protein phosphorylation in cells.

The single guide RNAs sgRNAs in the pX vector 1 μg were transfected into targeting cells in a 3. Forty-eight hours after selection, the cells were trypsinized and seeded into a well plate 4 cells per well. Single clones were expanded and screened for Girdin expression by protein immunoblotting.

We isolated Girdin immunocomplex by co-IP. To prepare mouse brain lysate, adult mouse brains without cerebellum were lysed in IP lysis buffer 20 mM Tris-HCl, mM NaCl, 0. The brain lysate was cleared by centrifugation at , g for 1 h at 4°C, and the supernatant was collected as whole mouse brain lysate.

The lysate was precleared by incubation with Protein G beads for 1 h. One hundred milligrams of precleared brain lysate was incubated with 50 μL of Protein G beads cross-linked with the indicated antibody. After overnight rotation at 4°C, the beads were extensively washed with 10 mL of lysis buffer and 10 mL of PBS, followed by elution of the protein complex with μL of acidic buffer Thermo Scientific Pierce for 5 min at room temperature.

The eluate was neutralized by adding 10 μL of 1M Tris-HCl pH 9. For the identification of proteins included in the eluate, the whole eluates were digested with Trypsin Gold Promega, Madison, WI for 16 h at 37°C after reduction, alkylation, demineralization, and concentration, followed by analysis on the Orbitrap Fusion mass spectrometer Thermo Fisher Scientific, San Jose, CA.

All procedures were performed on ice or at 4°C with prechilled buffers. Samples for amino acid concentration measurement were prepared as previously described, with minor modifications [ 49 ]. After centrifugation at 15, rpm for 15 min, the supernatants were collected, and amino acids were measured using an Agilent HPLC System Wako, Osaka, Japan.

For each sample, replicate sets of cells that were prepared and treated identically were used for amino acid measurement and cell counting, respectively. cDNA encoding human 4F2hc28 was inserted into the pcDNA3. A cDNA fragment encoding the 4F2hc cytoplasmic domain — was inserted into the pGEX-5X-2 vector GE Healthcare, Waukesha, WI.

The construction of plasmids pGEX-5XGST-Girdin-NT 1— , pETa-Girdin-NT 1— -His6, pEF-BOS-GST, and pEF-BOS-GST-Girdin-NT was previously described [ 18 ].

GFP and Girdin-NT cDNAs were inserted into the pRetroQ-3xFlag vector. Ubiquitin cDNA Myc-Ub , MAPKK DA, and GFP-LC3 were gifts from Keiji Tanaka Tokyo Metropolitan Institute of Medical Science , Yukiko Gotoh The University of Tokyo , and Toyoshi Fujimoto Nagoya University , respectively.

Histidine-tagged Ub His-Ub was kindly provided by Dr. Hui-Kuan Lin University of Texas M. Anderson Cancer Center. Overall, Sestrin2 acts as a leucine sensor in the cytoplasm Overviews of multiple signals activating mTORC1. Amino acids, growth factors and energy signals can lead to mTORc1 activation.

Growth factors and energy signals activate mTORC1 primarily through the PI3K pathway and AMPK pathway, respectively. Amino acids signal to mTORC1 in Rag-dependent and Rag-independent pathway.

Sestrin2, CASTOR1 and SMATOR shown in red box are reported to be cytosolic amino acid sensors for leucine, arginine, and s-adenosyl-L-methionine, respectively.

Arrows and bars represent activation and inhibition, respectively, of downstream proteins. GATOR2 have been characterized as integrating multiple amino acid inputs to mTORC1, and thus specific amino acid sensors may interact with GATOR2, analogous to that of SESN2 Figure 1. Chantranupong and his colleagues searched a protein interaction database named BioPlex to identify potential GATOR2-interacting partners, and they found that CASTOR1 encoded by GATS protein-like 3 GATSL3 gene is one of putative GATOR2 interactors Subsequently, CASTOR1 has been characterized as a cytosolic arginine sensor.

The homodimeric CASTOR1 protein can interact with GATOR2 under the arginine deprivation condition to inhibit mTORC1 activation.

In vitro binding assays showed that CASTOR1 can directly and specifically bind to arginine at a highly affinity of ~30 μm Arginine-bound CASTOR1 can lead to dissociation of CASTOR1 from GATOR2, leading to mTORC1 activation.

Thus, CASTOR1 acts as an arginine sensor in the cytoplasm 75 , As the mentioned above, GATOR2 plays a role in integrating multiple amino acid inputs to mTORC1. Xin Gu et, al found that SAMTOR, previously named C7orf60, interacts with GATOR1 and KICSTOR through searching the BioPlex database and co-immunoprecipitation assays Methyl donor S-adenosylmethionine SAM can directly bind to GATOR1 and then disrupt the SAMTOR-GATOR1 at the constant of approximately 7 μM in media SAMTOR senses SAM to signal methionine sufficiency to mTORC1, while methionine may translate into SAM to activate mTORC1.

Thus, SAMTOR acts as a SAM sensor in the cytoplasm SLC38A9, an uncharacterized protein with sequence homology to amino acid transporters, functions as a positive regulator of mTORC1 in an amino acid-sensitive manner 79 , 80 Figure 1.

SLC38A9 has been characterized as a lysosomal transmembrane protein that interacts with GTPases and Ragulator which can regulate mTORC1 activation SLC38A9 binds to arginine with a high Michaelis constant Knockout of SLC38A9 inhibits mTORC1 activation by arginine, and overexpression of SLC38A9 makes mTORC1 activation more sensitive by arginine.

Thus, SLC38A9 is an excellent candidate for being an arginine sensor in the lysosome for activating mTORC1 Amino acids act as protein building blocks, precursor substrates for signaling molecule, and crucial nutrient signals to mTORC1 activation.

In the past few years, our understanding of amino acid sensing to mTORC1 has increased tremendously 8 , 28 , 29 , 33 , Of note, mTORC1 acts as a critical regulatory node that controls cell metabolism, including cell proliferation, cycle and death.

Dysfunction of mTORC1 is highly associated with many diseases such as insulin resistance 83 - 85 , diabetes 86 and various types of cancer 87 - the discovery of Rag proteins is a breakthrough in understanding of mTORC1 activation by nutrients, and it helps researchers identify other key components in the mTORC1 signaling pathway, especially amino acid sensors.

Recently, Sestrin2, CASTOR1, SMATOR and SLC39A1 have been identified as amino acid sensors. However, sensors for other amino acid such as serine and glutamine remain to be identified. Overall, the molecular mechanisms of amino acid sensing to mTORC1 is quite complex, and more regulatory components of mTORC1 require further investigation.

This article does not contain any studies with human participants or animals performed by any of the authors. Hence, no informed consent is required for any part of this review. Authors declare no conflict of interest. This study was supported by the China Postdoctoral Science Foundation M , the Key Research Project of Frontier Sciences of Chinese Academy of Sciences QYZDY-SSW-SMC , the Hunan Science and Technology Project XK , the Xiaoxiang Scholar Distinguished Professor Fund of Hunan Normal University, the National Thousand Young Talents Program and the Hunan Hundred Talents Program.

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CAS PubMed Google Scholar. Download references. The authors are grateful to their colleague R. Gong and the rest of the Guan laboratory for valuable discussions and insightful comments. In addition, the authors would like to thank V.

Tagliabracci for critical reading of this manuscript. The authors would like to apologize to their colleagues whose work could not be cited owing to space limitations.

The work in the Guan laboratory was supported by a National Institutes of Health NIH grant CA and a grant from the Department of Defense W81XWH to K.

J is supported by a grant from the National Cancer Institute T32CA , and R. R is supported by a grant from the Canadian Institute of Health Research CIHR. Ryan C. Russell and Kun-Liang Guan are at the Department of Pharmacology and Moores Cancer Center, Jenna L. Jewell, University of California, San Diego, La Jolla, California , USA.

Jenna L. Jewell, Ryan C. You can also search for this author in PubMed Google Scholar. Correspondence to Kun-Liang Guan. Kun-Liang Guan's homepage. Reprints and permissions. Jewell, J.

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Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Plants cope with energy shortage by activating respiratory pathways that use amino acids as alternative substrates.

The adaptation of amino acid metabolism to energy deprivation is mediated by the balance between TOR and SnRK signaling. Amino acids serve as signaling molecules to coordinate growth and stress responses. Journal Article. The role of amino acid metabolism in signaling and metabolic adaptation to stress-induced energy deficiency in plants.

Björn Heinemann , Björn Heinemann. Institute for Plant Genetics, Department of Plant Proteomics, Leibniz University Hannover. Oxford Academic. Tatjana M Hildebrandt. Correspondence: hildebrandt genetik. Editorial decision:. Corrected and typeset:. PDF Split View Views.

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Abstract The adaptation of plant metabolism to stress-induced energy deficiency involves profound changes in amino acid metabolism. Abiotic stress , alternative respiration , amino acid metabolism , branched-chain amino acid degradation , energy deficiency , lysine degradation , signaling , SnRK , TOR.

Open in new tab Download slide. Box 1. Amino acids as precursors for secondary metabolites during abiotic stress. Box 2. Key developments in understanding the role of amino acid metabolism in signaling and metabolic adaptation to stress-induced energy deficiency.

Box 3. TOR and SnRK1 signaling in plants. Google Scholar OpenURL Placeholder Text. Falcone Ferreyra. Google Scholar Google Preview OpenURL Placeholder Text. Jamsheer K. Van Leene. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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More from Oxford Academic. Biological Sciences. Plant Sciences and Forestry. Science and Mathematics. In addition, overexpression of Rab1A promotes mTORC1 signaling. Furthermore, Fan et al have revealed that SLC36A4, known as PAT4, is predominantly localized in the Golgi apparatus and engages mTORC1 interaction with Rab1A Sestrins have been initially identified as a family of stress-inducible proteins, which are capable of attenuating various stresses, stimulating autophagy, and regulating cell metabolism 64 , 65 Figure 1.

Apart from the function of redox regulation, Sestrin have been characterized as negative regulators of mTORC1 and positive regulators of AMPK through TSC2 66 - The Sestrins have been proposed to interact with and function as guanine nucleotide dissociation inhibitor for the Rag GTPases More recently, Sestrin1 and Sestrin2 showed a high affinity K d of 10~15 and ~20 μM to physically bind to leucine Moreover, Sestrin2 which purified from prokaryotic cell, has ability to directly bind to leucine but not arginine in vitro.

And the assay of mutation Sestrin2 and crystalized Sestrin2 strongly indicated residues of Sestrin2 directly bind leucine and importance for binding leucine capacity of the protein.

Sestrin2 interacts with GATOR2 to inhibit mTORC1 under leucine deprivation, not arginine deprivation 72 , When leucine added in media, it binds to Sestrin2 to dissociate the complex of Sestrin2-GATOR2.

Leucine must bind to Sestrin2 in order for leucine to activate mTORC1 in mammalian cells. Overall, Sestrin2 acts as a leucine sensor in the cytoplasm Overviews of multiple signals activating mTORC1.

Amino acids, growth factors and energy signals can lead to mTORc1 activation. Growth factors and energy signals activate mTORC1 primarily through the PI3K pathway and AMPK pathway, respectively. Amino acids signal to mTORC1 in Rag-dependent and Rag-independent pathway. Sestrin2, CASTOR1 and SMATOR shown in red box are reported to be cytosolic amino acid sensors for leucine, arginine, and s-adenosyl-L-methionine, respectively.

Arrows and bars represent activation and inhibition, respectively, of downstream proteins. GATOR2 have been characterized as integrating multiple amino acid inputs to mTORC1, and thus specific amino acid sensors may interact with GATOR2, analogous to that of SESN2 Figure 1. Chantranupong and his colleagues searched a protein interaction database named BioPlex to identify potential GATOR2-interacting partners, and they found that CASTOR1 encoded by GATS protein-like 3 GATSL3 gene is one of putative GATOR2 interactors Subsequently, CASTOR1 has been characterized as a cytosolic arginine sensor.

The homodimeric CASTOR1 protein can interact with GATOR2 under the arginine deprivation condition to inhibit mTORC1 activation. In vitro binding assays showed that CASTOR1 can directly and specifically bind to arginine at a highly affinity of ~30 μm Arginine-bound CASTOR1 can lead to dissociation of CASTOR1 from GATOR2, leading to mTORC1 activation.

Thus, CASTOR1 acts as an arginine sensor in the cytoplasm 75 , As the mentioned above, GATOR2 plays a role in integrating multiple amino acid inputs to mTORC1. Xin Gu et, al found that SAMTOR, previously named C7orf60, interacts with GATOR1 and KICSTOR through searching the BioPlex database and co-immunoprecipitation assays Methyl donor S-adenosylmethionine SAM can directly bind to GATOR1 and then disrupt the SAMTOR-GATOR1 at the constant of approximately 7 μM in media SAMTOR senses SAM to signal methionine sufficiency to mTORC1, while methionine may translate into SAM to activate mTORC1.

Thus, SAMTOR acts as a SAM sensor in the cytoplasm SLC38A9, an uncharacterized protein with sequence homology to amino acid transporters, functions as a positive regulator of mTORC1 in an amino acid-sensitive manner 79 , 80 Figure 1.

SLC38A9 has been characterized as a lysosomal transmembrane protein that interacts with GTPases and Ragulator which can regulate mTORC1 activation SLC38A9 binds to arginine with a high Michaelis constant Knockout of SLC38A9 inhibits mTORC1 activation by arginine, and overexpression of SLC38A9 makes mTORC1 activation more sensitive by arginine.

Thus, SLC38A9 is an excellent candidate for being an arginine sensor in the lysosome for activating mTORC1 Amino acids act as protein building blocks, precursor substrates for signaling molecule, and crucial nutrient signals to mTORC1 activation.

In the past few years, our understanding of amino acid sensing to mTORC1 has increased tremendously 8 , 28 , 29 , 33 , Of note, mTORC1 acts as a critical regulatory node that controls cell metabolism, including cell proliferation, cycle and death.

Dysfunction of mTORC1 is highly associated with many diseases such as insulin resistance 83 - 85 , diabetes 86 and various types of cancer 87 - the discovery of Rag proteins is a breakthrough in understanding of mTORC1 activation by nutrients, and it helps researchers identify other key components in the mTORC1 signaling pathway, especially amino acid sensors.

Recently, Sestrin2, CASTOR1, SMATOR and SLC39A1 have been identified as amino acid sensors. However, sensors for other amino acid such as serine and glutamine remain to be identified.

Overall, the molecular mechanisms of amino acid sensing to mTORC1 is quite complex, and more regulatory components of mTORC1 require further investigation. This article does not contain any studies with human participants or animals performed by any of the authors.

Hence, no informed consent is required for any part of this review. Authors declare no conflict of interest. This study was supported by the China Postdoctoral Science Foundation M , the Key Research Project of Frontier Sciences of Chinese Academy of Sciences QYZDY-SSW-SMC , the Hunan Science and Technology Project XK , the Xiaoxiang Scholar Distinguished Professor Fund of Hunan Normal University, the National Thousand Young Talents Program and the Hunan Hundred Talents Program.

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Other GTPases in amino acid signaling to mTORC1. Sestrin2: a leucine sensor in the cytoplasm. Figure 1. CASTOR1: an arginine sensor in the cytoplasm.

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Wu GY, Field CJ, Marliss EB d Glucose and glutamine metabolism in rat macrophages: enhanced glycolysis and unaltered glutaminolysis in spontaneously diabetic BB rats.

Wu G, Bazer FW, Cudd TA, Meininger CJ, Spencer TE Maternal nutrition and fetal development. Wu G, Bazer FW, Davis TA, Kim SW, Li P, Marc Rhoads J, Carey Satterfield M, Smith SB, Spencer TE, Yin Y Arginine metabolism and nutrition in growth, health and disease.

Wu G, Bazer FW, Johnson GA, Knabe DA, Burghardt RC, Spencer TE, Li XL, Wang JJ Triennial growth symposium: important roles for L-glutamine in swine nutrition and production.

J Anim Sci — Wu G, Bazer FW, Satterfield MC, Li X, Wang X, Johnson GA, Burghardt RC, Dai Z, Wang J, Wu Z Impacts of arginine nutrition on embryonic and fetal development in mammals.

Wu G, Bazer FW, Dai Z, Li D, Wang J, Wu Z Amino acid nutrition in animals: protein synthesis and beyond. Annu Rev Anim Biosci — Wu G, Bazer FW, Johnson GA, Herring C, Seo H, Dai Z, Wang J, Wu Z, Wang X Functional amino acids in the development of the pig placenta.

Mol Reprod Dev — Wu G, Bazer FW, Johnson GA, Hou Y Board-invited review: arginine nutrition and metabolism in growing, gestating, and lactating swine.

Article PubMed PubMed Central Google Scholar. Wullschleger S, Loewith R, Hall MN TOR signaling in growth and metabolism. Yang Q, Inoki K, Ikenoue T, Guan KL Identification of Sin1 as an essential TORC2 component required for complex formation and kinase activity.

Genes Dev — Yang J, Dolinger M, Ritaccio G, Mazurkiewicz J, Conti D, Zhu X, Huang Y Leucine stimulates insulin secretion via down-regulation of surface expression of adrenergic alpha2A receptor through the mTOR mammalian target of rapamycin pathway: implication in new-onset diabetes in renal transplantation.

Yeramian A, Martin L, Serrat N, Arpa L, Soler C, Bertran J, McLeod C, Palacin M, Modolell M, Lloberas J, Celada A Arginine transport via cationic amino acid transporter 2 plays a critical regulatory role in classical or alternative activation of macrophages.

J Immunol — Zajac A, Poprzecki S, Zebrowska A, Chalimoniuk M, Langfort J Arginine and ornithine supplementation increases growth hormone and insulin-like growth factor-1 serum levels after heavy-resistance exercise in strength-trained athletes. J Strength Conditioning Res — Article Google Scholar.

Zeng X, Wang F, Fan X, Yang W, Zhou B, Li P, Yin Y, Wu G, Wang J Dietary arginine supplementation during early pregnancy enhances embryonic survival in rats.

Zhang Y, Dong C Regulatory mechanisms of mitogen-activated kinase signaling. Cell Mol Life Sci — Zhang J, Xie Z, Dong Y, Wang S, Liu C, Zou MH Identification of nitric oxide as an endogenous activator of the AMP-activated protein kinase in vascular endothelial cells.

Zhao Y, Xiong X, Sun Y DEPTOR, an mTOR inhibitor, is a physiological substrate of SCF betaTrCP E3 ubiquitin ligase and regulates survival and autophagy. Zhuang Y, Wang XX, He J, He S, Yin Y Recent advances in understanding of amino acid signaling to mTORC1 activation.

Front Biosci landmark Ed — Download references. We apologize to those authors whose articles were not cited because of space limitations. Department of Animal Science, North Carolina State University, Raleigh, NC, , USA. The Comparative Medicine Institute, North Carolina State University, Raleigh, NC, , USA.

You can also search for this author in PubMed Google Scholar. Correspondence to Xiaoqiu Wang. Reprints and permissions. Paudel, S. Amino Acids in Cell Signaling: Regulation and Function. In: Wu, G. eds Amino Acids in Nutrition and Health.

Advances in Experimental Medicine and Biology, vol Springer, Cham. Published : 13 July Publisher Name : Springer, Cham.

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Provided by the Springer Nature SharedIt content-sharing initiative. Policies and ethics. Skip to main content. Abstract Amino acids are the main building blocks for life. Keywords Functional amino acids Transceptor Sensor mTORC1 AMPK MAPK. Buying options Chapter EUR eBook EUR Softcover Book EUR Hardcover Book EUR Tax calculation will be finalised at checkout Purchases are for personal use only Learn about institutional subscriptions.

Abbreviations AMPK: AMP-activated protein kinase Akt: Protein kinase B EAA: Nutritionally essential amino acids IGF: Insulin-like growth factor ERK: Extracellular signal-regulated kinases JNK: c-Jun NH 2 -terminal kinase LEL: Late endosome and lysosome MAPK: Mitogen-activated protein kinase mTOR: Mechanistic target of rapamycin NEAA: Nutritionally nonessential amino acids PI3K: Phosphatidylinositolkinase Tr: Trophectoderm.

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The lysate was precleared by incubation with Protein G beads for 1 h. One hundred milligrams of precleared brain lysate was incubated with 50 μL of Protein G beads cross-linked with the indicated antibody. After overnight rotation at 4°C, the beads were extensively washed with 10 mL of lysis buffer and 10 mL of PBS, followed by elution of the protein complex with μL of acidic buffer Thermo Scientific Pierce for 5 min at room temperature.

The eluate was neutralized by adding 10 μL of 1M Tris-HCl pH 9. For the identification of proteins included in the eluate, the whole eluates were digested with Trypsin Gold Promega, Madison, WI for 16 h at 37°C after reduction, alkylation, demineralization, and concentration, followed by analysis on the Orbitrap Fusion mass spectrometer Thermo Fisher Scientific, San Jose, CA.

All procedures were performed on ice or at 4°C with prechilled buffers. Samples for amino acid concentration measurement were prepared as previously described, with minor modifications [ 49 ]. After centrifugation at 15, rpm for 15 min, the supernatants were collected, and amino acids were measured using an Agilent HPLC System Wako, Osaka, Japan.

For each sample, replicate sets of cells that were prepared and treated identically were used for amino acid measurement and cell counting, respectively. cDNA encoding human 4F2hc28 was inserted into the pcDNA3. A cDNA fragment encoding the 4F2hc cytoplasmic domain — was inserted into the pGEX-5X-2 vector GE Healthcare, Waukesha, WI.

The construction of plasmids pGEX-5XGST-Girdin-NT 1— , pETa-Girdin-NT 1— -His6, pEF-BOS-GST, and pEF-BOS-GST-Girdin-NT was previously described [ 18 ]. GFP and Girdin-NT cDNAs were inserted into the pRetroQ-3xFlag vector. Ubiquitin cDNA Myc-Ub , MAPKK DA, and GFP-LC3 were gifts from Keiji Tanaka Tokyo Metropolitan Institute of Medical Science , Yukiko Gotoh The University of Tokyo , and Toyoshi Fujimoto Nagoya University , respectively.

Histidine-tagged Ub His-Ub was kindly provided by Dr. Hui-Kuan Lin University of Texas M. Anderson Cancer Center. To generate stable Girdin knockdown FT cells, 24 μg of either control or Girdin shRNA and 4 μg of vesicular stomatitis virus G protein pVSV-G vector Clontech were cotransfected into GP packaging cells Clontech Laboratories.

Cells were lysed in IP lysis buffer 20 mM Tris-HCl, mM NaCl, 0. The lysates were cleared via centrifugation at 12, × g for 10 min and the supernatants were incubated with 2 μg of the appropriate primary antibodies or normal IgG on a rotator at 4°C overnight, followed by the addition of 20 μL of protein A or G Sepharose beads Sigma at 4°C for 3 h.

Then, the beads were washed three times with IP lysis buffer, and the protein complex was eluted using μL of 1× SDS sample buffer.

For IP with anti-Flag antibody, the cell lysates were incubated with 20 μL of ANTI-FLAG M2 Affinity Gel Sigma for 3 h.

Then, the beads were washed three times with IP lysis buffer followed by elution of the protein complex using μL of 1× SDS sample buffer. For WB analysis, samples separated by SDS-PAGE were transferred onto polyvinylidene difluoride membranes.

Band intensities were quantified using ImageJ NIH, Bethesda, ML. The IP products, including the beads, were denatured with μL of 7 M guanidine hydrochloride. After centrifugation for 5 min at 10, × g at room temperature, the supernatant was transferred to a new 1.

Protein expression was induced by adding μM isopropyl beta- d -thiogalactoside IPTG , followed by continuous culture at 25°C for an additional 4 h. The cell pellets were suspended in homogenizing buffer 20 mM Tris-HCl, pH 8. The lysates were cleared via centrifugation at 37, rpm for 1 h and applied to a column of Glutathione Sepharose 4B beads with a 1-mL bed volume GE Healthcare equilibrated with 20 mL of TED buffer 20 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, pH 8.

The column was washed extensively with 10 mL of TED buffer and the GST fusion protein was eluted using elution buffer 10 mM glutathione in TED buffer , followed by dialysis with TED buffer. The phosphorylation assay was performed as previously described [ 14 ].

In brief, MAPK was reacted with the recombinant proteins of Girdin-NT WT and mutants SA, SA, and AA in 50 μL of reaction mixture 20 mM MOPS, pH 7.

Then, the reaction mixtures were boiled in SDS sample buffer and subjected to SDS-PAGE and silver staining. The radiolabeled proteins were visualized with an image analyzer Typhoon FLA ; GE Healthcare Life Sciences.

Two methods were used to test in vivo ubiquitination of 4F2hc. For the detection of endogenous 4F2hc ubiquitination, we exploited the recently developed TUBEs approach to capture ubiquitinated proteins [ 50 , 51 ].

The cells were then lysed with μL of IP lysis buffer supplemented with Complete Mini protease inhibitor and PhosSTOP phosphatase inhibitor cocktails Roche and 25 μg of GST-TUBE2 LifeSensors, UM Cleared cell lysates were incubated with 50 μL of Glutathione Sepharose 4B beads for 2 h.

The beads were washed three times with IP lysis buffer, followed by elution of the protein complex by μL 1× SDS sample buffer and WB analysis using anti-4F2hc antibody.

For the detection of exogenous 4F2hc ubiquitination, FT cells transfected with 4F2hc 1. Then, the cells were washed twice with ice-cold PBS, scraped off the plates in the PBS, and collected by centrifugation at × g for 5 min. The cell pellets were lysed in buffer C 6 M guanidine-HCl, 0.

The whole cell extracts were mixed with μL of Ni-NTA agarose beads Qiagen at 4°C overnight. The Ni-NTA beads were washed twice with buffer C, once with buffer D mixture of buffer C: buffer E , and once with buffer E 25 mM Tris-HCl, 20 mM imidazole, pH 6.

The bound proteins were eluted by boiling in 1× SDS loading buffer containing mM imidazole and resolved by SDS-PAGE, followed by WB analysis.

The cells were washed three times with ice-cold PBS and lysed in IP lysis buffer. The cell lysates were incubated with 20 μL of NeutrAvidin agarose Thermo Scientific for 1 h, followed by three washes with IP lysis buffer.

Bound proteins were eluted by boiling in 1× SDS loading buffer and were detected by WB analysis. Immunofluorescence studies were performed as previously described [ 18 ]. Cells were plated on glass base dishes Iwaki, Osaka, Japan , fixed, and stained with the indicated antibodies.

The cells were imaged using a confocal laser-scanning microscope LSM, Carl Zeiss, Oberkochen, Germany. Immunohistochemistry was performed as previously described [ 23 ].

Tissue sections were deparaffinized and rehydrated, and antigens were retrieved by boiling in target retrieval solution pH 9 Dako for 30 min. After washing with PBS containing 0. The data are presented as means ± standard errors SEs. Statistical significance was evaluated using Student t test.

All experiments were repeated at least 3 times. A—C Band intensities for pS6K1 and S6K1, and pS6 and S6 in Fig 4A—4C were quantified, and the ratios of pS6K1 to S6K1 and pS6 to S6 are presented as the mean ± SE in A related to Fig 4A , B related to Fig 4B , and C related to Fig 4C.

Values in control cells were set as 1. All experiments were repeated 3 times. Lysates from the WT parent cells and Girdin knockout cells were analysed by WB to detect the basal activation level of mTORC1.

F Girdin WT or AA mutant was re-expressed in Girdin knockout Flp-In cells, followed by detection of basal mTORC1 activity. G—I Band intensities for pS6K1 and S6K1, and pS6 and S6 in Fig 4E—4G were quantified, and the ratios of pS6K1 to S6K1 and pS6 to S6 are presented as the mean ± SE in G related to Fig 4E , H related to Fig 4F , I related to Fig 4G.

Values in control cells stimulated by amino acids for 1 h were set as 1. A FT cells transduced with the indicated shRNAs pretreated with or without nM Bafilomycin A1 for 3 h were starved for amino acids AA— for the indicated times, followed by WB with the indicated antibodies.

Red arrowheads indicate lipidated LC3. The ratio of lipidated to total LC3 is shown in the lower panel. Values in control cells starved for amino acids for 3 h were set as 1.

B Flp-In cells stably expressing the indicated constructs were starved for amino acids AA— for the indicated times followed by WB with the indicated antibodies.

Values in control cells starved for amino acids for 2 h were set as 1. C, D Flp-In cells stably expressing the indicated constructs were transfected with GFP-LC3, followed by starvation for amino acids for 2 h.

The cells were then fixed and visualized using confocal microscopy. GFP, green fluorescent protein; Girdin, girders of actin filaments; LC3, light chain 3; N. We thank Toyoshi Fujimoto Nagoya University , Yukiko Gotoh The University of Tokyo , Hui-Kuan Lin University of Texas M.

Anderson Cancer Center , and Keiji Tanaka Tokyo Metropolitan Institute of Medical Science for providing the GFP-LC3, MAPKK DA, His-Ub, and Myc-Ub plasmids, respectively. We also thank Kentaro Taki Nagoya University , Yasuhiro Funahashi Nagoya University , and Tomonari Hamaguchi Nagoya University for technical assistance with the proteomic analysis and in vitro kinase assay.

Article Authors Metrics Comments Media Coverage Reader Comments Figures. Abstract Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 mTORC1 is fundamental to cell growth and metabolism.

Author summary The mechanistic target of rapamycin complex 1 mTORC1 protein kinase is a master regulator of cell growth, which senses several extracellular signals, such as growth factors and nutrient levels, to coordinate cell metabolism.

Introduction Cells respond to extracellular stimuli through multiple signaling pathways, which govern and coordinate various cellular activities.

Results Identification of 4F2hc as a Girdin-interacting protein To investigate the role of Girdin in mTORC1 signaling, we employed large-scale co-immunoprecipitation co-IP to isolate Girdin-interacting proteins in mouse brain lysate.

Download: PPT. Fig 1. Fig 2. Phosphorylation of Girdin is required for its interaction with 4F2hc. Fig 3. Fig 4. Girdin regulates amino acid stimulation—induced mTORC1 activation through interaction with 4F2hc. Girdin induces internalization of 4F2hc by lysosomes Given that Girdin was previously reported as a critical regulator of intracellular membrane trafficking [ 24 ], we hypothesized that Girdin-mediated negative regulation of mTORC1 activity was attributed to 4F2hc internalization.

Fig 6. Girdin negatively regulates amino acid signaling through decreasing intracellular Gln and Leu contents. Fig 7. Discussion Our present study identified a novel negative regulatory mechanism for amino acid signaling.

Materials and methods Ethics statement All animal protocols were approved by the Animal Care and Use Committee of Nagoya University Graduate School of Medicine Approval number Cell culture, transfection, and RNA interference The HeLa cell line was purchased from the American Tissue Type Culture Rockville, MD.

Purification and identification of Girdin-interacting proteins We isolated Girdin immunocomplex by co-IP. Measurement of intracellular amino acids All procedures were performed on ice or at 4°C with prechilled buffers. Plasmids cDNA encoding human 4F2hc28 was inserted into the pcDNA3. Retrovirus infection To generate stable Girdin knockdown FT cells, 24 μg of either control or Girdin shRNA and 4 μg of vesicular stomatitis virus G protein pVSV-G vector Clontech were cotransfected into GP packaging cells Clontech Laboratories.

Immunoprecipitation and WB analysis Cells were lysed in IP lysis buffer 20 mM Tris-HCl, mM NaCl, 0. In vitro kinase assay The phosphorylation assay was performed as previously described [ 14 ]. Ubiquitination assay Two methods were used to test in vivo ubiquitination of 4F2hc.

Immunofluorescence staining Immunofluorescence studies were performed as previously described [ 18 ]. Immunohistochemistry Immunohistochemistry was performed as previously described [ 23 ].

Data analysis The data are presented as means ± standard errors SEs. Supporting information. S1 Table. Girdin-interacting proteins identified by IP and mass spectrometry. Girdin, girders of actin filaments. s XLSX. S1 Data. Raw data used for quantification in this work.

S1 Fig. Girdin negatively regulates basal mTORC1 activity. s TIF. S2 Fig. Girdin and 4F2hc regulate autophagy induced by amino acid depletion. S3 Fig. Comprehensive measurement of intracellular amino acids.

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