Investigation Of The Interactions Between FLCN And PAT1 On The Regulation Of MTORC1 Signaling Pathway

BHD syndrome is a rare genetic disease, which is caused by the loss of FLCN gene. FLCN is widespread in nature, indicating it might be involve in certain rudimentary cellular functions. Previous works have shown a close interaction between FLCN and m TORC1, a conservative signal pathway enbales cells to sense the fluctuation of environmental amino acid levels. We have shown elsewhere that FLCN regulates the activity of m TORC1 by controlling the level of leucine in lysosome. This work aims to elucidate the underlying mechanisms. As an amino acid transporter, the lysosome-associated PAT1 functions to transport amino acid from the lysosome lumen into cytosol, which tends to down-regulate m TORC1. In this work, we found in the HEK293 cells that FLCN and PAT1 antagonize each other to control m TORC1. Firstly, overexpression of FLCN makes cells to maintain a relatively high level of m TORC1 upon amino acid starvation. Secondly, elevated FLCN or high leucine supply can counteract the suppressive effect of PAT1 on m TORC1. Conversely, knockdown of PAT1 inhibits the decrease of m TORC1 under amino acid starvation condition. Moreover, this result can be reversed by inhibiting FLCN simultaneously. The main results are as follows:1. Stable cell lines expressing FLCN-HA constitutively were generated(in pc DNA3.1).2. Upon amino acid starvation, overexpresison of FLCN activates mTORC1, which is manifested by a reduced sensitivity to the leucine level.3. Stable cell lines expressing EGFP-PAT1 constitutively were generated(in p EGFP-C1).4. Overexpression of PAT1 inhibits mTORC1.5. Elevated PAT1 suppresses the FLCN-overexpression-stimulated m TORC1.6. FLCN or high leucine supply counteractes the suppressive effect of PAT1 on m TORC1.7. Knockdown of PAT1 maintains mTORC1 acitivity upon starvation, which is in turn reversed by co-suppression of FLCN.Together, these results suggest that PAT1 and FLCN antagonize each other to modulate the leucine level in lysosome, which serves as the key signal to turn on m TORC1.