FNIP1
FLCN-binding protein 1 (FNIP1), was identified in 2006 (Baba et al.) 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 external influences, 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.
FNIP2
FNIP2, a second FLCN-binding protein, was first designated KIAA1450 by Nagase et al. (2000) by sequencing clones obtained from a size-fractionated human brain cDNA library. Hasumi et al. (2008) subsequently identified it as FNIP2, with Takagi et al. (2008) identifying it as FNIPL (for FNIP1-Like) soon after. Additionally, Komori et al. (2009) cloned mouse FNIP2 (which they called MAPO1), and saw that it plays a role in apoptosis triggered by O6-methylguanine mispairing in DNA.
The accepted nomenclature for this protein is FNIP2, and it is homologous to FNIP1 (49% identity, 74% similarity). As with FNIP1, it is conserved across species and binds AMPK (Hasumi et al., 2008; Takagi et al., 2008). In vitro kinase assays also suggest that FNIP2 is phosphorylated by AMPK (Takagi et al., 2008). FNIP1 and FNIP2 are able to form homo- and heterodimers, as well as multimers (Takagi et al., 2008), suggesting a functional association between these two proteins.
Interactions with FLCN
Binding of FLCN to FNIP1 and FNIP2 is mediated specifically through the C-terminal region of FLCN (Baba et al., 2006; Hasumi et al., 2008; Takagi et al., 2008). In BHD syndrome, the majority of mutations are predicted to introduce a premature stop codon into FLCN, and therefore result in a protein truncation (Schmidt et al., 2005). However, it is presently unclear whether this truncated FLCN is targeted for nonsense-mediated decay, or remains in the cell with an altered function. Whatever the outcome, these mutations remove the ability of FLCN to interact with FNIP1 and FNIP2, which suggests that this interaction is functionally important. Further research has shown that serine 62 (ser62) is a phosphorylation site in FLCN (Wang et al., 2010). This work also suggests that ser62 phosphorylation is indirectly up-regulated by AMPK (Wang et al., 2010). FLCN also appears to be phosphorylated at ser302 by unknown kinases downstream of mTORC1 (Piao et al., 2009). Since mTORC1 is known to be indirectly down-regulated by AMPK, this process could be associated with an unknown feedback mechanism that regulates mTOR signalling.
