FLCN-binding protein 1 (FNIP1), was identified in 2006 (Baba et al) as a protein able to interact with and phosphorylate FLCN. FNIP1 was also determined to be able to bind 5′-AMP-activated protein kinase (AMPK), 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 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 in the mTOR pathway.

FNIP2, a second FLCN-binding protein, was first identified by Hasumi et al, (2008). Takagi et al, (2008) subsequently identified FNIP2 as FNIPL. The accepted nomenclature for this protein is FNIP2, it is homologous to FNIP1 (49% identity, 74% similarity), is conserved across species, and also binds to AMPK. Interestingly, FNIP1 and FNIP2 are able to form homo- and heterodimers, as well as multimers (Takagi et al, 2008), suggesting a coordinated functional relationship between these proteins.

Significantly, approximately 84% of reported BHD kindreds have FLCN mutations that are predicted to prematurely truncate FLCN (Nickerson et al, 2002; Khoo et al, 2002; Schmidt et al, 2005; Leter et al, 2008).  This removes FLCNs ability to interact with FNIP-1 and -2, which suggests that the interaction between FLCN and the FNIPs is functionally important. Recent research (Wang et al, 2009) has shown that serine 62 (Ser62) is the major phosphorylation site in FLCN. Their analysis suggests that Ser62 phosphorylation is indirectly up-regulated by AMPK.

The expression patterns of FLCN, FNIP-1 and -2 in human tissues were determined by Hasumi et al, (2008) using real-time PCR. Expression patterns of FLCN, FNIP1 and FNIP2 were generally similar, and consistently similar in specific tissues such as muscle, nasal mucosa, salivary gland and uvula, suggesting that FLCN, FNIP1 and FNIP2 may cooperate together in those organs. However, FNIP2 expression is higher relative to FNIP1 in fat, liver and pancreas, which suggests that FNIP2 may have a specific function in those metabolic tissues (Hasumi et al, 2008). FLCN/FNIP1 and FLCN/FNIP2 dimers have been shown to co-localize in the cytoplasm in a reticular pattern and binding of FLCN to FNIP1 and -2 is mediated through the C-terminal region of FLCN (Baba et al, 2006; Hasumi et al, 2008; Takagi et al, 2008). Co-expression studies of FLCN, FNIP1 and FNIP2 indicate that both FNIPs regulate the cytoplasmic distribution of FLCN since expression studies have shown that when expressed alone, FNIP2 constructs are distributed within the cytoplasm with condensed features around the nucleus. When FLCN constructs are expressed alone they are found mainly in the nucleus. However, when FNIP2 and FLCN are co-expressed they co-localise together in the cytoplasm in a reticular pattern, which is similar to the co-localisation of FNIP1 and FLCN (Baba et al, 2006; Hasumi et al, 2008; Takagi et al, 2008)

The molecular functions of FLCN are poorly understood, but indirect interactions between FLCN and AMPK within the mTOR signalling networks mediated by FNIP1 and -2 have been firmly established (Baba et al, 2006; Hasumi et al, 2008). However, the functional role of FLCN in mTOR signalling is undetermined since several recent publications have reported opposite impacts on phosphorylated ribosomal protein S6 (p-S6; an indicator of mTOR activation) signalling as a consequence of FLCN downregulation. Two studies recently reported that transient downregulation of FLCN by siRNA in human cell lines results in reduction of phosphorylation of p-S6 (Takagi et al, 2008; Hartman et al, 2009). Reduction of p-S6 was also observed in renal cysts developing in mice heterozygous for FLCN (Hartman et al, 2009). In contrast, kidney-specific homozygous knockout of FLCN results in an increase in phosphorylated p-S6, which contributed to the development of polycystic kidneys (Baba et al, 2008; Chen et al, 2008). This data suggests a role for FLCN in nutrient/energy-sensing mediated through the mTOR signalling pathway.

Further studies in Schizosaccharomyces pombe (S. Pombe) revealed that yeast FLCN homologue and yeast TSC1/2 regulate common downstream targets but have opposing roles, specifically TSC1/2 inhibit the activation of Tor2 and subsequent downstream elements but S.pombe FLCN upregulates the same elements (van Slegtenhorst, 2007). If this relationship between BHD and TSC1/TSC2 is recapitulated in mammalian cells and mTOR is inhibited in cells lacking BHD, there may be important clinical implications for BHD patients.

Recent work using a kidney-targeted FLCN gene inactivation in a mouse model has indicated that the Raf-MEK-Erk pathway, which is activated in many cancers and regulates cell proliferation (Roberts et al, 2007), was activated in FLCN-knockout kidneys (Baba et al, 2008). This suggests that a common upstream effector of these two pathways may be activated by loss of FLCN tumour suppressor function, resulting in cell growth and proliferation within the FLCN-null kidney cell.

Quick Links:

Back to: Contents

Backwards to: 5  Folliculin expression

Forwards to: 7.  Animal Models of BHD syndrome

References