{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T20:06:17Z","timestamp":1788465977250,"version":"build-2803163510"},"reference-count":48,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2021,6,27]],"date-time":"2021-06-27T00:00:00Z","timestamp":1624752000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["17K07752"],"award-info":[{"award-number":["17K07752"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["20K05837"],"award-info":[{"award-number":["20K05837"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["19K22275"],"award-info":[{"award-number":["19K22275"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator by controlling protein synthesis and autophagy in response to environmental cues. Amino acids, especially leucine and arginine, are known to be important activators of mTORC1 and to promote lysosomal translocation of mTORC1, where mTORC1 is thought to make contact with its activator Rheb GTPase. Although amino acids are believed to exclusively regulate lysosomal translocation of mTORC1 by Rag GTPases, how amino acids increase mTORC1 activity besides regulation of mTORC1 subcellular localization remains largely unclear. Here we report that amino acids also converge on regulation of the TSC2-Rheb GTPase axis via Ca2+\/calmodulin (CaM). We showed that the amino acid-mediated increase of intracellular Ca2+ is important for mTORC1 activation and thereby contributes to the promotion of nascent protein synthesis. We found that Ca2+\/CaM interacted with TSC2 at its GTPase activating protein (GAP) domain and that a CaM inhibitor reduced binding of CaM with TSC2. The inhibitory effect of a CaM inhibitor on mTORC1 activity was prevented by loss of TSC2 or by an active mutant of Rheb GTPase, suggesting that a CaM inhibitor acts through the TSC2-Rheb axis to inhibit mTORC1 activity. Taken together, in response to amino acids, Ca2+\/CaM-mediated regulation of the TSC2-Rheb axis contributes to proper mTORC1 activation, in addition to the well-known lysosomal translocation of mTORC1 by Rag GTPases.<\/jats:p>","DOI":"10.3390\/ijms22136897","type":"journal-article","created":{"date-parts":[[2021,6,27]],"date-time":"2021-06-27T22:24:57Z","timestamp":1624832697000},"page":"6897","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":25,"title":["Amino Acid-Mediated Intracellular Ca2+ Rise Modulates mTORC1 by Regulating the TSC2-Rheb Axis through Ca2+\/Calmodulin"],"prefix":"10.3390","volume":"22","author":[{"given":"Yuna","family":"Amemiya","sequence":"first","affiliation":[{"name":"Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nao","family":"Nakamura","sequence":"additional","affiliation":[{"name":"Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nao","family":"Ikeda","sequence":"additional","affiliation":[{"name":"Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Risa","family":"Sugiyama","sequence":"additional","affiliation":[{"name":"Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chiaki","family":"Ishii","sequence":"additional","affiliation":[{"name":"Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masatoshi","family":"Maki","sequence":"additional","affiliation":[{"name":"Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hideki","family":"Shibata","sequence":"additional","affiliation":[{"name":"Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Terunao","family":"Takahara","sequence":"additional","affiliation":[{"name":"Department of Applied Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,6,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1016\/j.cell.2006.01.016","article-title":"TOR signaling in growth and metabolism","volume":"124","author":"Wullschleger","year":"2006","journal-title":"Cell"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"400","DOI":"10.1016\/j.tcb.2014.03.003","article-title":"Regulation of mTORC1 by amino acids","volume":"24","author":"Sabatini","year":"2014","journal-title":"Trends Cell Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12929-020-00679-2","article-title":"Amino acid-dependent control of mTORC1 signaling: A variety of regulatory modes","volume":"27","author":"Takahara","year":"2020","journal-title":"J. Biomed. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1038\/s41556-018-0205-1","article-title":"mTOR as a central hub of nutrient signalling and cell growth","volume":"21","author":"Kim","year":"2019","journal-title":"Nat. Cell Biol."},{"key":"ref_5","first-page":"103","article-title":"Spatial regulation of mTORC1 signalling: Beyond the Rag GTPases. Semin","volume":"107","author":"Carroll","year":"2020","journal-title":"Cell Dev. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1016\/j.cell.2013.11.049","article-title":"Spatial Control of the TSC Complex Integrates Insulin and Nutrient Regulation of mTORC1 at the Lysosome","volume":"156","author":"Menon","year":"2014","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/j.devcel.2015.02.010","article-title":"MCRS1 binds and couples rheb to amino acid-dependent mTORC1 activation","volume":"33","author":"Fawal","year":"2015","journal-title":"Dev. Cell"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"jcs.208017","DOI":"10.1242\/jcs.208017","article-title":"Rheb localized on the Golgi membrane activates lysosome-localized mTORC1 at the Golgi-lysosome contact site","volume":"131","author":"Hao","year":"2017","journal-title":"J. Cell Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2750","DOI":"10.1091\/mbc.E19-03-0146","article-title":"Weak membrane interactions allow Rheb to activate mTORC1 signaling without major lysosome enrichment","volume":"30","author":"Angarola","year":"2019","journal-title":"Mol. Biol. Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"648","DOI":"10.1038\/ncb839","article-title":"TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling","volume":"4","author":"Inoki","year":"2002","journal-title":"Nat. Cell Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/S1097-2765(02)00568-3","article-title":"Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase\/akt pathway","volume":"10","author":"Manning","year":"2002","journal-title":"Mol. Cell"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"10662","DOI":"10.1038\/ncomms10662","article-title":"Lysosomal recruitment of TSC2 is a universal response to cellular stress","volume":"7","author":"Demetriades","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4776","DOI":"10.1074\/jbc.273.8.4776","article-title":"Differential regulation by calcium reveals distinct signaling requirements for the activation of Akt and p70S6k","volume":"273","author":"Conus","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1920","DOI":"10.1074\/jbc.272.3.1920","article-title":"An intracellular calcium signal activates p70 but not p90 ribosomal S6 kinase in liver epithelial cells","volume":"272","author":"Graves","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1016\/j.cmet.2008.03.002","article-title":"Amino acids activate mTOR complex 1 via Ca2+\/CaM signaling to hVps34","volume":"7","author":"Gulati","year":"2008","journal-title":"Cell Metab."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"33076","DOI":"10.1074\/jbc.M507201200","article-title":"hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase","volume":"280","author":"Byfield","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"747","DOI":"10.1042\/BJ20081865","article-title":"hVps15, but not Ca2+\/CaM, is required for the activity and regulation of hVps34 in mammalian cells","volume":"417","author":"Yan","year":"2009","journal-title":"Biochem. J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1496","DOI":"10.1126\/science.1157535","article-title":"The rag GTPases bind raptor and mediate amino acid signaling to mTORC1","volume":"320","author":"Sancak","year":"2008","journal-title":"Science"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"786","DOI":"10.1016\/j.cell.2014.01.024","article-title":"Regulation of TORC1 in Response to Amino Acid Starvation via Lysosomal Recruitment of TSC2","volume":"156","author":"Demetriades","year":"2014","journal-title":"Cell"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1128\/MCB.01285-12","article-title":"Novel Role for SHP-2 in Nutrient-Responsive Control of S6 Kinase 1 Signaling","volume":"33","author":"Mercan","year":"2012","journal-title":"Mol. Cell. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"17057","DOI":"10.1074\/jbc.M117.800201","article-title":"The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins","volume":"292","author":"Takahara","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1888","DOI":"10.1126\/science.1208592","article-title":"An expanded palette of genetically encoded Ca2+ indicators","volume":"333","author":"Zhao","year":"2011","journal-title":"Science"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"275","DOI":"10.1038\/nmeth.1314","article-title":"SUnSET, a nonradioactive method to monitor protein synthesis","volume":"6","author":"Schmidt","year":"2009","journal-title":"Nat. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1097\/JES.0b013e3182798a95","article-title":"Measuring protein synthesis with SUnSET: A valid alternative to traditional techniques?","volume":"41","author":"Goodman","year":"2013","journal-title":"Exerc. Sport Sci. Rev."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Zhang, W., Muramatsu, A., Matsuo, R., Teranishi, N., Kahara, Y., Takahara, T., Shibata, H., and Maki, M. (2020). The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21176315"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"404","DOI":"10.1038\/ncb3324","article-title":"A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation","volume":"18","author":"Li","year":"2016","journal-title":"Nat. Cell Biol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Li, R.J., Xu, J., Fu, C., Zhang, J., Zheng, Y.G., Jia, H., and Liu, J.O. (2016). Regulation of mTORC1 by lysosomal calcium and calmodulin. eLife, 5.","DOI":"10.7554\/eLife.19360"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1080\/15548627.2017.1389822","article-title":"A negative feedback regulation of MTORC1 activity by the lysosomal Ca2+ channel MCOLN1 (mucolipin 1) using a CALM (calmodulin)-dependent mechanism","volume":"14","author":"Sun","year":"2018","journal-title":"Autophagy"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1006\/abbi.2001.2682","article-title":"A calmodulin binding site in the tuberous sclerosis 2 gene product is essential for regulation of transcription events and is altered by mutations linked to tuberous sclerosis and lymphangioleiomyomatosis","volume":"398","author":"Noonan","year":"2002","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"944","DOI":"10.1021\/acs.biochem.6b01296","article-title":"Structural Consequences of Calmodulin EF Hand Mutations","volume":"56","author":"Piazza","year":"2017","journal-title":"Biochemistry"},{"key":"ref_31","first-page":"CR230","article-title":"Clinical symptoms of tuberous sclerosis complex in patients with an identical TSC2 mutation","volume":"11","author":"Rok","year":"2005","journal-title":"Med. Sci. Monit."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"171","DOI":"10.7600\/jpfsm.4.171","article-title":"Role of Ca2+ signaling in skeletal muscle hypertrophy and atrophy","volume":"4","author":"Ito","year":"2015","journal-title":"J. Phys. Fit. Sport. Med."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3009","DOI":"10.1128\/MCB.06599-11","article-title":"STIM1- Ca2+ Signaling Is Required for the Hypertrophic Growth of Skeletal Muscle in Mice","volume":"32","author":"Li","year":"2012","journal-title":"Mol. Cell. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Ito, N., Ruegg, U.T., and Takeda, S. (2018). ATP-Induced Increase in Intracellular Calcium Levels and Subsequent Activation of mTOR as Regulators of Skeletal Muscle Hypertrophy. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19092804"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.yjmcc.2020.03.004","article-title":"Saraf-dependent activation of mTORC1 regulates cardiac growth","volume":"141","author":"Sanlialp","year":"2020","journal-title":"J. Mol. Cell. Cardiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1136\/jitc-2020-002137","article-title":"Amino acids and RagD potentiate mTORC1 activation in CD8+ T cells to confer antitumor immunity","volume":"9","author":"Zhang","year":"2021","journal-title":"J. Immunother. Cancer"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1311","DOI":"10.1002\/emmm.201302989","article-title":"STIM1 and STIM2-mediated Ca2+ influx regulates antitumour immunity by CD8+ T cells","volume":"5","author":"Weidinger","year":"2013","journal-title":"EMBO Mol. Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"374","DOI":"10.1007\/s001250050690","article-title":"Mechanisms of arginine-induced increase in cytosolic calcium concentration in the beta-cell line NIT-1","volume":"40","author":"Weinhaus","year":"1997","journal-title":"Diabetologia"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1083\/jcb.201107033","article-title":"Class III PI-3-kinase activates phospholipase D in an amino acid-sensing mTORC1 pathway","volume":"195","author":"Yoon","year":"2011","journal-title":"J. Cell Biol."},{"key":"ref_40","first-page":"1","article-title":"Structural insights into TSC complex assembly and GAP activity on Rheb","volume":"12","author":"Yang","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1457","DOI":"10.1016\/S1097-2765(03)00220-X","article-title":"Insulin activation of Rheb, a mediator of mTOR\/S6K\/4E-BP signaling, is inhibited by TSC1 and 2","volume":"11","author":"Garami","year":"2003","journal-title":"Mol. Cell"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"13571","DOI":"10.1073\/pnas.202476899","article-title":"Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling","volume":"99","author":"Tee","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1016\/S0092-8674(03)00929-2","article-title":"TSC2 mediates cellular energy response to control cell growth and survival","volume":"115","author":"Inoki","year":"2003","journal-title":"Cell"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"18717","DOI":"10.1074\/jbc.M414499200","article-title":"The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses","volume":"280","author":"Smith","year":"2005","journal-title":"J. Biol. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1038\/sj.onc.1209106","article-title":"Regulation of the small GTPase Rheb by amino acids","volume":"25","author":"Roccio","year":"2006","journal-title":"Oncogene"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"31861","DOI":"10.1074\/jbc.M801546200","article-title":"Isolation of hyperactive mutants of mammalian target of rapamycin","volume":"283","author":"Ohne","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"3775","DOI":"10.1021\/bi0622874","article-title":"Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells","volume":"46","author":"Kremers","year":"2007","journal-title":"Biochemistry"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"28605","DOI":"10.1074\/jbc.M606087200","article-title":"Nutrient-dependent multimerization of the mammalian target of rapamycin through the N-terminal HEAT repeat region","volume":"281","author":"Takahara","year":"2006","journal-title":"J. Biol. Chem."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/13\/6897\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:25:06Z","timestamp":1760163906000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/13\/6897"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,6,27]]},"references-count":48,"journal-issue":{"issue":"13","published-online":{"date-parts":[[2021,7]]}},"alternative-id":["ijms22136897"],"URL":"https:\/\/doi.org\/10.3390\/ijms22136897","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,6,27]]}}}