It was recently reported that FLCN activates mTORC1 signalling in response to amino acid stimulation by interacting with RagA and RagB at the cytosolic lysosome surface, possibly by acting as a GEF towards these proteins (Petit et al., 2013). Another recent study from a group at MIT also reports that FLCN activates mTORC1 signalling in response to amino acid stimulation, but instead reports that this is due to FLCN acting as a G-activating protein towards RagC and RagD (Tsun et al., 2013).
Like the Rab proteins, which have been discussed previously, the Rag proteins (RagA-D) are GTPase proteins which cycle between an active GTP-bound state, and an inactive GDP-bound state. Guanine nucleotide exchange factors (GEFs) catalyse the release of the GDP molecule, allowing the protein to reload a GTP molecule. GTPase activating proteins (GAPs) stimulate the activity of active GTP-bound GTPases, which initiates a signalling event – in this case the mTOR signalling – and causes the hydrolysis of GTP to GDP, thereby inactivating the GTPase. Therefore GEFs and GAPs work antagonistically to regulate the activity of GTPases.
Tsun et al. found that GDP-bound RagC is required to recruit the Raptor subunit of the mTORC1 protein complex to lysosomes, where mTORC1 signalling is activated in response to amino acid stimulation. Furthermore, the authors show that during amino acid starvation, FNIP2 recruits FLCN to the lysosome, where it interacts with the Rag proteins. Upon restimulation with amino acids, FLCN and FNIP2 act as a GTPase activating protein (GAP) towards RagC and RagD, facilitating the hydrolysis of GTP-bound RagC to GDP, allowing it to recruit Raptor to the lysosome and subsequently activate mTORC1 signalling.
These results show broad similarities with the Petit et al. study. Both studies show that when amino acid availability is low, FLCN localises to lysosomes, and when amino acid levels are restored, FLCN activates mTORC1 signalling and disperses from lysosomes.
However, there are several differences between the two studies. Firstly, the Petit et al. study found that FLCN interacts with the GTPase domain of RagA and RagB, and possibly acts as a GEF towards these proteins. Conversely, Tsun et al. found that FLCN interacts with RagC and has GAP activity towards it. Secondly, Petit et al. found that FLCN’s recruitment to lysosomes and interactions with the Rag proteins was dependent on FNIP1, whilst Tsun et al. report FNIP2 to be required for these processes.
The reason for the differences between the two studies is unclear. For example, FNIP1 and FNIP2 may control whether FLCN interacts with RagA/B or RagC/D and also whether it acts as a GEF or a GAP. This may mean that FLCN activates mTORC1 through a different mechanism in different cell types, depending on whether FNIP1 or FNIP2 is predominantly expressed. Indeed, the two studies used different cell lines – Petit et al. used HeLa cells, while Tsun et al. used HEK293T cells – which may account for this difference. However, the C-terminal region of FLCN is a non-canonical DENN domain, and while DENN domains are know to have GEF activity (Wu et al., 2011), the Tsun et al. study is the first report of a DENN domain showing GAP activity, meaning that at present the implications of this finding are intriguing.
It is currently unknown how the results of both this study and the Petit et al. study relate to BHD syndrome. However, both studies do identify the Rag proteins as FLCN-interacting proteins, meaning that if Rag dysregulation is a contributing factor in the development of BHD symptoms, compounds that target these or any downstream proteins might prove to be viable treatments for BHD.
- Petit CS, Roczniak-Ferguson A, & Ferguson SM (2013). Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases. The Journal of cell biology, 202 (7), 1107-22 PMID: 24081491
- Tsun ZY, Bar-Peled L, Chantranupong L, Zoncu R, Wang T, Kim C, Spooner E, & Sabatini DM (2013). The Folliculin Tumor Suppressor Is a GAP for the RagC/D GTPases That Signal Amino Acid Levels to mTORC1. Molecular cell PMID: 24095279
- Wu X, Bradley MJ, Cai Y, Kümmel D, De La Cruz EM, Barr FA, & Reinisch KM. (2011) Insights regarding guanine nucleotide exchange from the structure of a DENN-domain protein complexed with its Rab GTPase substrate. Proceedings of the National Academy of Sciences of the United States of America, 108 (46), 18672-7 PMID: 22065758
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.