A number of studies have suggested that FLCN regulates autophagy, although precisely how was unknown. A recent study by Dr Elaine Dunlop and Dr Andy Tee from Cardiff University report that FLCN regulates autophagy through interactions with GABARAP and ULK1.
Dunlop et al. (2014) first analysed whether autophagy was dysregulated in FLCN-deficient HK2 and MEF cells. They found that although acute starvation-induced autophagy was unperturbed, basal autophagic flux was reduced in these cells, as measured by increased SQSTM1 expression and impaired autophagosome maturation.
As FNIP1 has been previously shown to interact with the autophagy protein GABARAP (Behrends et al., 2010), Dunlop et al. investigated whether FLCN regulated autophagy through GABARAP. Mass spectrometry identified eight high-confidence interacting partners for GABARAP, including both FLCN and FNIP1. Further in vitro experiments confirmed this interaction, and showed that FLCN was only able to bind GABARAP in the presence of FNIP1 or FNIP2, and that FNIP2 in particular potentiated this interaction.
ULK1, a key activator of autophagy, was found to control the FLCN-FNIP-GABARAP interaction by phosphorylating three novel sites in the FLCN protein – S406, S537 and S542. However, ULK1 was still able to dissociate a FLCN-FNIP-GABARAP complex containing a triple serine-to-alanine unphosphorylateable FLCN mutant, suggesting that ULK1 targets additional sites in FLCN, the FNIPs or GABARAP.
Analysis of a BHD patient’s renal tumour showed elevated levels of SQSTM1, GABARAP and MAP1LC3B expression, suggesting autophagy was impaired in the tumour. Furthermore, mutant FLCN proteins derived from BHD-patient mutations interacted preferentially with ULK1 than GABARAP, and did not activate autophagy when the C-terminal domain was truncated. This suggests that the C-terminal end of the protein is important in order to dissociate from ULK1, interact with GABARAP and to ultimately activate autophagy.
These results suggest that FLCN binding to GABARAP is required for basal autophagy to proceed, and that ULK1 usually acts to inhibit the FLCN-FNIP-GABARAP complex. During nutrient sufficiency, ULK1 is inactivated by mTOR signalling, suggesting that the FLCN-FNIP-GABARAP complex is able stimulate basal autophagy, which is commonly activated during cell growth (Musiwaro et al., 2013). Conversely, during starvation conditions, AMPK activates ULK1 which, in turn, inhibits the FLCN-FNIP-GABARAP complex, thus concomitantly inhibiting basal autophagy and activating starvation-induced autophagy.
FLCN has been shown to regulate mTOR signalling and AMPK signalling, both of which function upstream of ULK1, and to affect MAP1LC3B and MAP1LC3C expression (Bastola et al., 2013), indicating that FLCN may control autophagy at multiple points in the pathway. Furthermore, FLCN has been shown to activate, inhibit, or have no effect on autophagy in different cell types, indicating that FLCN’s role in autophagy – like its role in other signalling pathways – is highly cell type specific. Indeed, as FNIP2 and FNIP1 were both required for the FLCN-GABARAP interaction in this study, it is likely that differential expression of FLCN’s interacting proteins modifies FLCN’s function in the cell.
However, dysregulated autophagy is a common feature of many kidney cancers, and the results of this study suggest that reduced autophagy may drive renal tumorigenesis in BHD. This lends further support to earlier reports that radiotherapy and Paclitaxel chemotherapy, both of which stimulate autophagy, may prove effective treatments for BHD-associated kidney cancer.
- Bastola P, Stratton Y, Kellner E, Mikhaylova O, Yi Y, Sartor MA, Medvedovic M, Biesiada J, Meller J, & Czyzyk-Krzeska MF (2013). Folliculin contributes to VHL tumor suppressing activity in renal cancer through regulation of autophagy. PloS one, 8 (7) PMID: 23922894
- Behrends C, Sowa ME, Gygi SP, & Harper JW (2010). Network organization of the human autophagy system. Nature, 466 (7302), 68-76 PMID: 20562859
- Dunlop EA, Seifan S, Claessens T, Behrends C, Kamps MA, Rozycka E, Kemp AJ, Nookala RK, Blenis J, Coull BJ, Murray JT, van Steensel MA, Wilkinson S, & Tee AR (2014). FLCN, a novel autophagy component, interacts with GABARAP and is regulated by ULK1 phosphorylation. Autophagy, 10 (10) PMID: 25126726
- Musiwaro P, Smith M, Manifava M, Walker SA, & Ktistakis NT (2013). Characteristics and requirements of basal autophagy in HEK 293 cells. Autophagy, 9 (9), 1407-17 PMID: 23800949
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.