The DENN protein module contains a longin domain, a DENN domain and a d-DENN domain. Nookala et al. (2012) identified a DENN module in folliculin (FLCN), the Birt-Hogg-Dube tumour suppressor. The DENN module is believed to be a GEF for Rab-GTPases, although FLCN is believed to act as a GAP for RagC (Tsun et al., 2013) as is its yeast homologue, LST7, in interaction with the yeast FNIP homologue Lst4 (Pacitto et al., 2015). A recent bioinformatic study identified DENN domains in several other proteins, including Folliculin Interacting Proteins (FNIP1/2), C9orf72 and SMCR8 (Zhang et al., 2012). SMCR8 was known to be involved in autophagy and C9orf72 in ALS and FTD (Behrends et al 2010; DeJesus-Hernandez et al., 2011; Renton et al., 2011; Smith et al., 2012). Now, Amick et al. (2016) show, interestingly, how they used genetic strategies to examine C9orf72 functions, interactions and subcellular localization.
The authors found that C9orf72 interacts with SMCR8 and that C9orf72 localizes to lysosomes in an amino acid-dependent way. In the absence of C9orf72, the authors observed impaired mTORC1 signalling responses to amino acid availability. Since FLCN and FNIP1 interact (Baba et al., 2006) it was hypothesised that C9orf72 would also interact with FLCN, FNIP1 or SMCR8. Immunoprecipitation assays showed strong interactions of C9orf72 with SMCR8 but not FLCN or FNIP1. Knockdown of SMCR8 by siRNA lead to reduction in C9orf72 protein levels leading the authors to suggest that interaction with SMCR8 is likely to be crucial for C9orf72 function. Clustering analyses revealed that SMCR8’s evolutionary history is most highly correlated with C9orf72, suggesting that their interaction is functionally significant. Other genes that shared correlated evolutionary history with C9orf72 and SMCR8 were proteins involved in regulating lysosomal protease activity and endosomal membrane traffic.
Knowing that FLCN recruitment to lysosomes is negatively regulated by amino acid availability (Petit et al., 2013), the authors investigated the effect of starvation on C9orf72 localization and observed that C9orf72 was recruited to lysosomes in starved cells. After amino acid re-feeding, C9orf72 was redistributed back into the cytoplasm. Starvation treatment, however, did not affect C9orf72-SMCR8 interaction. While C9orf72 localization to lysosomes was regulated by amino acid availability, direct inhibition of mTOR did not affect C9orf72 localization, suggesting that C9orf72’s recruitment to lysosomes is not a result of mTORC1 inactivation triggered by starvation but may be a more direct response to different amino acid levels.
To study the functions of C9orf72 and SMCR8, C9orf72 KO, SMCR8 KO and C9orf72+SMCR8 double KO cell lines were generated. The smcr8 knockout cell lines showed depleted levels of C9orf72 protein and vice-versa. This mutual dependence of C9orf72 and SMCR8 protein levels further supports the idea that their interaction is functionally important. Lysosomes were enlarged and more clustered in the perinuclear region in C9orf72 KO cells than in wild type or SMCR8 KO cells supporting the idea of the lysosome as site of action for C9orf72. While not showing the same lysosome phenotype, the SMCR8 KO cells were larger than the wild type or C9orf72 KO. Interestingly, C9orf72-SMCR8 double KO cells reversed the lysosome clustering and enlargement of C9orf72 single KO and the increased cell size of the SMCR8 KO. mTORC1, being a regulator of cell size (reviewed in Eltschinger and Loewith, 2015), could also contribute this increased cell size. The authors measured mTORC1 signalling by assessing phosphorylation of ribosomal protein S6. Basal S6 phosphorylation was similar to wild type in C9orf72 KO and C9orf72-SMCR8 double KO but increased in the SMCR8 KO cells. In addition, mTORC1 inhibition eliminated the size difference between control and SMCR8 siRNA transfected cells. mTORC1 recruitment to the lysosome is mediated by Rag GTPase (reviewed in Ferguson 2015). Responsiveness of mTORC1 to amino acid re-feeding was impaired in both the C9orf72 and SMCR8 single KO cells. Although C9orf72 and SMCR8 KO cells show defects in mTORC1 regulation by amino acids, mTOR recruitment to lysosomes still occurs in amino acid re-feeding suggesting that the C9orf72-SMCR8 heterodimer is not required for controlling the Rag GTPase-mediated lysosome recruitment of mTORC1. The authors examined the effects of RagC depletion on mTOR recruitment to lysosomes and found that this recruitment was highly RagC-dependent in all WT and KO cell lines indicating that C9orf72 and SMCR8 contribute to the regulation of mTORC1 signalling by amino acid availability in a Rag-independent manner.
It would be interesting to look at the expression pattern of C9orf72 and SMCR8 in the context of FLCN and FNIP expression: it is unclear if there is any functional redundancy between these DENN-domain containing proteins. In summary, the authors identified a C9orf72-SMCR8 protein complex that shares with FLCN-FNIP1 the ability to be recruited to lysosomes in an amino acid availability-dependent way. Since the FLCN-FNIP complex acts as a GAP, C9orf72-SMCR8 might also act as a GAP. The findings give insights into the functions of this sub-family of DENN domain containing proteins. In addition, the role of C9orf72 and SMCR8 in the mTORC1 signalling pathway suggests that studying their functions may be important for cancer biology research.
- Amick J, Roczniak-Ferguson A, & Ferguson SM (2016). C9orf72 binds SMCR8, localizes to lysosomes and regulates mTORC1 signaling. Molecular biology of the cell PMID: 27559131