PP2A-dependent TFEB activation is blocked by PIKfyve-induced mTORC1 activity

Transcriptional factor EB (TFEB) is a master regulator of genes required for autophagy and lysosomal function. The nuclear localization of TFEB is blocked by the mechanistic target of rapamycin complex 1 (mTORC1)-dependent phosphorylation of TFEB at multiple sites including Ser-211. Here we show that inhibition of PIKfyve, which produces phosphatidylinositol 3,5-bisphosphate on endosomes and lysosomes, causes a loss of Ser-211 phosphorylation and concomitant nuclear localization of TFEB. We found that while mTORC1 activity toward S6K1, as well as other major mTORC1 substrates, is not impaired, PIKfyve inhibition specifically impedes the interaction of TFEB with mTORC1. This suggests that mTORC1 activity on TFEB is selectively inhibited due to loss of mTORC1 access to TFEB. In addition, we found that TFEB activation during inhibition of PIKfyve relies on the ability of protein phosphatase 2A (PP2A) but not calcineurin/PPP3, to dephosphorylate TFEB Ser-211. Thus, when PIKfyve is inhibited, PP2A is dominant over mTORC1 for control of TFEB phosphorylation at Ser-S211. Together these findings suggest that mTORC1 and PP2A have opposing roles on TFEB via phosphorylation and dephosphorylation of Ser-211, respectively, and further, that PIKfyve inhibits TFEB activity by facilitating mTORC1-dependent phosphorylation of TFEB.