Requirement of FLCN tumor suppressor gene for mTORC1-mediated inhibition of TFE3 transcriptional activity

TFE3 is an oncogenic transcription factor whose subcellular localization and activity are regulated by post-translational modifications. TFE3 is hyper-phosphorylated in the cytoplasm of mammalian cells under normal growth conditions, but becomes hypo-phosphorylated and translocates into the nucleus of cells with genetic inactivation of the FLCN tumor suppressor gene, which is responsible for the development of renal cancer, lung cysts and cutaneous hamartomas in patients with Birt-Hogg-Dube syndrome (BHD). Since FLCN is suggested to play a role in metabolic signaling through its interaction with energy and nutrient sensing kinases AMPK and mTOR, we investigated whether metabolic signaling regulates TFE3 activity through post-translational modifications and subcellular localization. We found that TFE3 was activated by metabolic stresses, such as glucose or amino acid deprivation, and induced gene expression of lysosomal genes, cathepsins, V-ATPase and mucolipin-1. AMPK activation and/or mTORC1 inhibition by nutrient deprivation, AMPK activator, mTOR or lysosomal inhibitors induced hypo-phosphorylation and subsequent nuclear localization of TFE3. mTORC1 directly phosphorylated TFE3 in vitro and the mutation of a putative mTORC1-dependent TFE3 phosphorylation site, Ser 321, was critical for its interaction with 14-3-3 protein, and retention in the cytoplasm. Direct phosphorylation of TFE3 by AMPK was demonstrated by an in vitro kinase assay and the AMPK-mediated phosphorylation sites were determined by mass spectrometry; however, the physiological significance of AMPK-mediated TFE3 phosphorylation requires further investigation. mTORC1-mediated TFE3 phosphorylation under nutrient and growth factor replete conditions was dependent on FLCN expression. In summary, we have shown that TFE3 induces lysosomal gene expression upon activation of AMPK and inhibition_of mTORC1 activity by metabolic stresses or loss of FLCN, which would play important roles not only in catabolic processes but also in the pathogenicity of human disease including cancer.