FLCN-interacting protein 1 (FNIP1), was identified in 2006 (Baba et al., 2006.) as an evolutionarily conserved protein that interacts with and phosphorylates FLCN. FNIP1 also binds AMPK, which is a negative regulator of mTOR and a key protein for energy sensing in cells (Inoki et al., 2003; Gwinn et al., 2008). Baba et al. (2006) demonstrated that both FLCN and FNIP1 are phosphorylated by AMPK. This interaction between FNIP1 and FLCN was also shown to be modified by additional factors, since treatment with an AMPK inhibitor (compound C), rapamycin or amino acid starvation affected the phosphorylation status of FLCN, further indicating a role for FLCN in energy sensing and the mTOR pathway. FNIP1 has also been shown to be phosphorylated by mTORC1 (Yu et al., 2011) and is ubiquitinated at lysine 161 (Wagner et al., 2011).
FNIP1 and FNIP2 are required for FLCN’s localisation to lysosomes during amino acid starvation, where FLCN interacts with the Rag proteins in order to activate mTORC1 signalling once amino acid levels are restored (Petit et al., 2013; Tsun et al., 2013). FLCN’s interaction with and activation of the Rag proteins is also facilitated by FNIP1 and FNIP2 (Petit et al., 2013; Tsun et al., 2013).
Zhang et al., (2012) identified a divergent DENN domain in FNIP1 and FNIP2, similar to that of FLCN. Using X-ray crystallography Pacitto et al. (2015) confirmed that the yeast orthologue Lst4 does contain a structural DENN-family protein. Lst4 forms a 1:1 heterodimer with the yeast orthologue of FLCN, Lst7, and like the mammalian proteins this heterodimer localises to the vacuolar membrane to act on TOR signalling (Péli-Gulli et al. 2015).
Behrends et al. (2010) have suggested that FNIP1 is also involved in autophagy, the process by which cellular components are degraded. FNIP1 was found to interact with GABARAP, a member of the ATG8-family of proteins which are required for autophagosomal development. Moreover, knockdown of FNIP1 led to an increase in autophagosome production. However, this perceived increase could reflect an accumulation of autophagosomes due to a block in a later step of the pathway.
Two independent studies published in 2012 suggest that FNIP1 is required for B cell development in mice (Park et al. 2012; Baba et al. 2012). Using flow cytometry, Park et al. (2012) observed an increase in p-S6R in FNIP1-null pre-B cells, suggesting an increase in mTOR-mediated metabolism. In addition, AMPK activation failed to inhibit this phosphorylation in FNIP1-null B cells, which indicates that FNIP1 may be important for AMPK to inhibit mTOR (Park et al. 2012). In contrast Baba et al. (2012) found that their FNIP1-null mice did not show a change in the levels of mTOR and p-S6R in pro-B cells, but did exhibit a marked increase in pre-B cell apoptosis (Baba et al. 2012).
Park et al. (2012) suggest FNIP1 loss leads to energy stress, causing the B cell arrest, whereas Baba et al. (2012) suggest FNIP1 loss leads to increased apoptosis. The differences observed between these studies may be due to the different methods used to generate the FNIP1-null mice (described in 6.3.2). However, Baba et al. (2012) also showed that FLCN knockout mice have the same B cell phenotype as FNIP1 knockout mice, indicating the FLCN gene may also function in B cell development.
Park et al. (2014) show that invariant Natural Killer T (iNKT) cells failed to develop normally in FNIP1-null mice. Although stage 0,1 and 2 cells were found, very few mature stage 3 iNKT cells were found in these mice, suggesting the block happened at some point between stage 2 and stage 3. Mitochondrial mass was reduced, ATP levels were low, cell size was increased and mTOR signalling was dysregulated in FNIP1-null iNKT cells. BrdU pulse experiments showed that FNIP1-null cells over-proliferated in stage 3 of iNKT cell development, making cells vulnerable to apoptosis, as shown by an increase in Caspase 3-positive cells. Together, this suggests that dysregulated mTOR signalling leads to higher energy consumption, meaning that cells do not have the required energy reserves for proliferation and maturation to stage 3, and die somewhere between stage 2 and 3. However, mTOR dysregulation is not fully responsible for this phenotype, as in vivo treatment of pups, beginning in utero, did not rescue the iNKT cell phenotype.
FNIP1 has been shown to be alternatively spliced during the later stages of mesenchyme differentiation (Venables et al., 2013), further suggesting that FNIP1 is important for B cell differentiation and development. In primary fibroblasts, a shorter isoform of FNIP1 (Uniprot: Q8TF40-3) – lacking the final 84 bps of exon 6, corresponding to amino acids 208-235 – predominates. Reprogramming of these fibroblasts to induced pluripotent stem cells (iPSCs) caused the longer, canonical, isoform of FNIP1 to predominate. This splicing pattern was fully reversed upon in vitro differentiation of iPSCs into fibroblasts, where the shorter isoform was again observed.
Reyes et al. (2014) reported that Fnip1 also has a role in specification of mammalian skeletal muscle fibres. Loss of Fnip1 resulted in increased numbers of Type 1 muscle fibres, increased AMPK activation and increased expression of the AMPK-target and coactivator PGC1α. Further work is required to fully understand the role of Fnip1 in muscle development and specification.
Sequence analysis across species shows that two thirds of the alternatively spliced exons found in this study (including FNIP1 exon 6) arose during the emergence of jawed vertebrates, indicating that alternative splicing of these genes may play an important role in the evolution and physiology of jawed vertebrates (Venables et al., 2013).