Investigation Of The Involvement Of The BHD Syndrome Gene FLCN In The Leucine Stimulated MTORC1 Signaling Pathway

The Birt–Hogg–Dubé(BHD) syndrome is characterized by benign skin tumours, lung cysts and a high risk for developing kidney cancers. The molecular basis of BHD was associated with mutations of the folliculin(FLCN) gene. However the FLCN functions are not fully understood. In a previos work, we generated a Drosophila BHD model and found that the FLCN(DBHD) mutant flies showed similar phenotypes with those of protein starvation or impaired mTOR signaling pathway. Interestingly, supplementation with high level of leucine in the medium markedly rescued the growth phenotypes. These results suggest that loss of DBHD sensitizes flies to the leucine signal that activates the mTOR signaling. However, the underlying mechanisms and if the resuces by leucine are conserved in mammals remain to be elucidated.In this work, I used the human embryonic kidney 293(HEK293) cells as a model to investigate the above questions and obtained the following results:1. High level of leucine reversed the decrease of m TORC1 casued by FLCN knockdown.Suppression of FLCN by RNAi decreased mTORC1 activity; this consequence could be reversed by high leucine supplementation in the cultural medium.2. Suppression of FLCN sensitizes cells to the available leucine for mTORC1 induction.Overexpressing FLCN did not increase the mTORC1 activity in complete culture medium.However in the absence of amino acids, overexpressing FLCN inhibited the decreased of m TORC1.3. FLCN was localized on the lysosome, which was affected by amino acid supplies.Immunostaining and western blot analysis showed that some FLCN proteins bond with the lysosome. This binding was significantly stimulated by starvation of amino acids.4. FLCN maintain leucine level within the lysosomes. Suppression of FLCN lead to reduced lysosomal leucine levels. On the contrary, overexpressing FLCN inhibited the reduction of lysosomal leucine under amino acid starvation conditions.5. FLCN and the transmembrane amino acid transporter protein PAT1 antagonize each other to control mTORC1 activity. Suppression of FLCN decreased the mTORC1 activity, thisconsequence was inhibited by co-suppression of PAT1.6. FLCN inhibits the accumulation of PAT1 on lysosome. Based on the results of both immunostaining and western blot, we found that suppression of FLCN significantly increased the lysosome-achored PAT1. We also found that starvation of amino acids recruited PAT1 to the lysosome. However, this effect was inhibited by overexpressing FLCN.7. FLCN and PAT1 show close interaction with each other. We found that PAT1co-immunoprecipitated with FLCN in a reciprocal manner..In summary, our results identify that FLCN controls m TORC1 by modulating the leucine signal in lysosome. Inaddition, we provided evidence that FLCN exerted this role by inhibiting the accumulation of the amino acid transporter PAT1 on the lysosome surface,thereby maintaining the signal strength.