FLCN phosphorylation was first identified by Baba et al. (2006) when multiple FLCN bands were seen on Western blots. Subsequent studies identified and characterised Serine 62 (S62) and Serine 302 (S302) as phosphorylated residues (Wang et al., 2010; Piao et al., 2009). A third FLCN phosphorylation site, Serine 73 (S73) was identified in a large study by Dephoure et al. (2008). Interestingly, this study found that all three FLCN phosphorylation sites were regulated by the cell cycle.
The cell division cycle involves the replication and separation of cellular material into two daughter cells. The cycle is tightly regulated by a series of kinases, which affect downstream proteins through phosphorylation. During the mitosis phase of the cell cycle, cellular processes undergo several changes, such as the termination of transcription and translation, the condensation of chromosomes and the breakdown of the nuclear envelope. Phosphorylation is instrumental in regulating these processes.
Dephoure et al. used stable isotope labelling, phosphopeptide enrichment and mass spectrometry to identify proteins whose phosphorylation was regulated by the cell cycle. The experiments were performed in HeLa cells arrested in either the G1 or mitotic phase of the cell cycle. The authors identified 14,265 unique phosphorylation sites on 3,682 different proteins. Overall, phosphorylation was upregulated during mitosis – 5,845 sites were phosphorylated only during mitosis, compared to 2,848 sites only phosphorylated during G1 phase. The remaining sites were phosphorylated during both phases of the cell cycle.
Three phosphorylation sites were identified on FLCN: S62, S73 and S302. S62 and S73 were found to be phosphorylated only during mitosis, whereas S302 was phosphorylated only during G1 phase. These results suggest that aspects of FLCN’s function or cellular localisation change during the cell cycle. Indeed, Nahorski et al. (2012) observed FLCN to be localised at cell junctions during interphase (which includes the G1 phase), but at the midbody during cytokinesis (part of the mitotic phase) (discussed in this previous blog post). Perhaps phosphorylation regulates this change in cellular localisation. Phosphorylation of FNIP1 and FNIP2 were not identified in this study, suggesting that these proteins are not phosphorylated during mitosis or the G1 phase.
The authors identified phosphorylation motifs for several kinases and the motif for Cyclin-dependent kinases, [pS/pT]-P, was found in FLCN at sites S62 and S73. However, previous studies have suggested that FLCN is phosphorylated by the AMPK and mTOR signalling pathways, either directly by these kinases or via downstream targets (Baba et al., 2006; Piao et al., 2009; Wang et al., 2010).
The effect of phosphorylation on FLCN’s function is unknown. Phosphorylation can affect many aspects of a protein, including its cellular localisation, conformation, stability, activity and its interactions with other molecules. It would be interesting to look at the phosphorylation status of FLCN during different cellular processes and when bound to an interacting partner, to decipher the function of this post-translational modification.
- Dephoure N, Zhou C, Villén J, Beausoleil SA, Bakalarski CE, Elledge SJ, Gygi SP. A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10762-7. PMID: 18669648
- Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, 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. 2006 Oct 17;103(42):15552-7. PMID: 17028174
- 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. 2010 Jan 4;584(1):39-43. PMID: 19914239
- Piao X, Kobayashi T, Wang L, Shiono M, Takagi Y, Sun G, Abe M, Hagiwara Y, Zhang D, Okimoto K, Kouchi M, Matsumoto I, Hino O. Regulation of folliculin (the BHD gene product) phosphorylation by Tsc2-mTOR pathway. Biochem Biophys Res Commun. 2009 Nov 6;389(1):16-21. PMID: 19695222
- Nahorski MS, Seabra L, Straatman-Iwanowska A, Wingenfeld A, Reiman A, Lu X, Klomp JA, Teh BT, Hatzfeld M, Gissen P, Maher ER. Folliculin interacts with p0071 (Plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis. Hum Mol Genet. 2012 Sep 10. [Epub ahead of print] PMID: 22965878
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.