Ser62 – Another piece of the puzzle…

The molecular functions of Folliculin (FLCN) are poorly understood, but indirect interactions between FLCN and AMPK (of the mTOR signaling network) mediated by FNIP1 and FNIP2 have been firmly established (Baba M et al, 2006; Hasumi H et al, 2008; Takagi Y et al, 2008), suggesting a role for FLCN in nutrient/energy-sensing through the AMPK/mTORC1 signaling pathway.

The functional role of FLCN in mTOR signaling is controversial since several recent publications have reported opposite impacts on phosphorylated ribosomal protein S6 (p-S6) signaling 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 p-S6 (Hartman TR et al, 2009; Takagi Y et al, 2008). Reduction of p-S6 was also observed in renal cysts developing in mice heterozygous for Flcn (Hartman TR et al, 2009). In contrast, kidney-specific homozygous knockout of Flcn results in an increase of p-S6, which contributed to the development of polycystic kidneys (Baba M et al, 2008; Chen J et al, 2008). To confuse the matter further, knockout of the FLCN homolog in S. pombe showed that FLCN, which is thought to act at the level of AMPK, has an opposite biochemical role of the AMPK target TSC2 in amino acid transport (van Slegtenhorst M et al, 2007). Such opposite roles are surprising given the overlapping clinical characteristics (skin harmatomas, lung and kidney cysts as well as renal carcinomas) associated with both BHD syndrome and tuberous sclerosis complex (TSC) disease and further indicate that the function of FLCN in AMPK/mTORC1 signaling is complex.

To better understand the molecular mechanism of tumorigenesis underlying BHD syndrome, it is clearly a priority to elucidate the function of FLCN. One of the ways in which this is achievable is by characterising the phosphorylation sites of a protein i.e. FLCN and identifying which kinases are responsible and in what order this occurs if multiple phosphorylation sites are present.

Classically a range of antibodies are utilised to detect these sites since specific antibodies can detect whether a protein is phosphorylated at a particular site: anti-phospho-tyrosine monoclonal antibodies have been widely used because they react with plethora of proteins containing phosphorylated tyrosine residues. In contrast, the monoclonal antibodies against phospho-serine or threonine residues are unpopular, since their affinity and specificity are less than optimal. To achieve precise characterization of signaling events, it is desirable to raise a good anti-phospho-site-specific antibody to clearly detect phosphorylated species.

Baba et al (2006) previously observed that rat FLCN migrated as multiple bands in immunoblot analysis. Through a series of experiments they identified that the migration of FLCN was affected by phosphorylation in the amino-terminal region and that rat FLCN has multiple phosphorylation sites. Wang et al (2009) generated a rabbit polyclonal antibody (BHD-P1) specific for a phosphorylated residue identified using site directed mutagenesis assays – Ser62, and successfully tested it’s reactivity to endogenous FLCN from human, mouse and rat cell lines, concluding that FLCN is phosphorylated at Ser62 in vivo. Subsequent studies showed that:

* Ser62 phosphorylation is indirectly up-regulated by AMPK and that another residue is directly phosphorylated by AMPK and;
* Ser62 phosphorylation is increased by binding with FLCN-interacting proteins (FNIP1 and FNIP2/).

These results highlight an important aspectof FLCN biochemistry, which is that the FLCN-AMPK-FNIP complex is regulated by Ser62 phosphorylation, and gets us one step further to understanding the role of FLCN in the mTOR signalling pathway. Still, many questions remain unanswered and the fact that FLCN has multiple phosphorylation sites will no doubt make it harder to elucidate its role but it is clear that progress is being made and that clues are being found by researchers. The generation of the BHD-P1 antibody is also remarkable since this can now be used by other labs to shed light on similar aspects of FLCN interactions.

www.BHDSyndrome.org – the online reference for anyone interested in Birt-Hogg-Dube Syndrome

References

Baba M., Hong S.B., Sharma N., Warren M.B., Nickerson M.L., Iwamatsu A., Esposito D., Gillette W.K., Hopkins 3rd R.F., Hartley J.L., Furihata M., Oishi S., Zhen W., Burke Jr. T.R., Linehan W.M., Schmidt L.S. and Zbar B., Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling, Proc. Natl. Acad. Sci. USA 103 (2006), pp. 15552–15557

Baba M, Furihata M, Hong SB, et al. Kidney-targeted Birt-Hogg-Dube gene inactivation in a mouse model: Erk1/2 and Akt-mTOR activation, cell hyperproliferation, and polycystic kidneys. J Natl Cancer Inst 2008;100(2):140- 54.

Chen J, Futami K, Petillo D, et al. Deficiency of FLCN in mouse kidney led to development of polycystic kidneys and renal neoplasia. PLoS ONE 2008;3(10):e3581.

Hartman TR, Nicolas E, Klein-Szanto A, et al. The role of the Birt-Hogg-Dube protein in mTOR activation and renal tumorigenesis. Oncogene 2009.

Hasumi H., Baba M.,. Hong S.B, Hasumi Y., Huang Y., Yao M., Valera V.A., Linehan W.M. and Schmidt L.S., Identification and characterization of a novel folliculin-interacting protein FNIP2, Gene 415 (2008), pp. 60–67

Takagi Y., Kobayashi T., Shiono M., Wang L., Piao X., Sun G., Zhang D., Abe M., Hagiwara Y., Takahashi K.and Hino O., Interaction of folliculin (Birt–Hogg–Dubé gene product) with a novel Fnip1-like (FnipL/Fnip2) protein, Oncogene 27 (2008), pp. 5339–5347.

van Slegtenhorst M, Khabibullin D, Hartman TR, et al. The Birt-Hogg-Dube and tuberous sclerosis complex homologs have opposing roles in amino acid homeostasis in Schizosaccharomyces pombe. J Biol Chem 2007;282(34):24583-90.

Wang L, Kobayashi T, Piao X, Shiono M, Takagi Y, Mineki R, Taka H, Zhang D, Abe M, Sun G, Hagiwara Y, Okimoto K, Matsumoto I, Kouchi M, Hino O. Serine 62 is a phosphorylation site in folliculin, the Birt-Hogg-Dubé gene product. FEBS Lett. 2009 Nov 13.

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