@article {Paquette2021.01.27.428292, author = {Mathieu Paquette and Leeanna El-Houjeiri and Linda C. Zirden and Pietri Puustinen and Paola Blanchette and Hyeonju Jeong and Kurt Dejgaard and Peter M. Siegel and Arnim Pause}, title = {AMPK-dependent phosphorylation is required for transcriptional activation of TFEB/TFE3}, elocation-id = {2021.01.27.428292}, year = {2021}, doi = {10.1101/2021.01.27.428292}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Increased autophagy and lysosomal activity promote tumor growth, survival and chemo-resistance. During acute starvation, autophagy is rapidly engaged by AMPK activation and mTORC1 inhibition to maintain energy homeostasis and cell survival. TFEB and TFE3 are master transcriptional regulators of autophagy and lysosomal activity and their cytoplasm/nuclear shuttling is controlled by mTORC1-dependent multisite phosphorylation. However, it is not known whether and how the transcriptional activity of TFEB or TFE3 is regulated. We show that AMPK mediates phosphorylation of TFEB and TFE3 on three serine residues, leading to TFEB/TFE3 transcriptional activity upon nutrient starvation, FLCN depletion and pharmacological manipulation of mTORC1 or AMPK. AMPK loss does not affect TFEB/TFE3 nuclear localization nor protein levels but reduces their transcriptional activity. Collectively, we show that mTORC1 specifically controls TFEB/TFE3 cytosolic retention whereas AMPK is essential for TFEB/TFE3 transcriptional activity. This dual and opposing regulation of TFEB/TFE3 by mTORC1 and AMPK is reminiscent of the regulation of another critical regulator of autophagy, ULK1. Surprisingly, we show that chemoresistance is mediated by AMPK-dependent activation of TFEB, which is abolished by pharmacological inhibition of AMPK or mutation of serine 466/467/469 to alanine residues within TFEB. Altogether, we show that AMPK is a key regulator of TFEB/TFE3 transcriptional activity, and we validate AMPK as a promising target in cancer therapy to evade chemotherapeutic resistance.Competing Interest StatementThe authors have declared no competing interest.ACC1/2acetyl-CoA carboxylase 1 or 2ACTBactin betaAICAR5-aminoimidazole-4-carboxamide ribonucleotideAMPKAMP-activated protein kinaseAMPKiAMPK inhibitor, SBI-0206965CAconstitutively activeCARM1coactivator-associated arginine methyltransferaseCFPcyan fluorescent proteinCLEARcoordinated lysosomal expression and regulationDKOdouble knockoutDMEMDulbecco{\textquoteright}s modified Eagle{\textquoteright}s mediumDMSOdimethyl sulfoxideDQ-BSAselfquenched BODIPY{\textregistered}dye conjugates of bovine serum albuminEBSSEarle{\textquoteright}s balanced salt solutionFLCNfolliculinGFPgreen fluorescent proteinGSTglutathione S-transferasesHDHuntington diseaseHTThuntingtinKOknock-outLAMP1lysosomal-associated membrane protein 1MEFmouse embryonic fibroblastsMITFmelanocyte inducing transcription factormTORC1mechanistic target of rapamycin complex 1PolyQpolyglutamineRT-qPCRreverse transcription quantitative polymerase chain reactionS6ribosomal protein S6TCLtotal cell lysatesTFE3transcription factor E3TFEBtranscription factor EBTKOtriple knock-outULK1unc-51-like kinase 1}, URL = {https://www.biorxiv.org/content/early/2021/01/27/2021.01.27.428292}, eprint = {https://www.biorxiv.org/content/early/2021/01/27/2021.01.27.428292.full.pdf}, journal = {bioRxiv} }