The identification of the FLCN interacting proteins FNIP1 and FNIP2 led to the discovery that FLCN functions in the AMPK signalling pathway (Baba et al., 2006; Hasumi et al., 2008; Takagi et al., 2008). Nahorski et al. (2012) have now identified Plakophilin-4 (PKP4, also known as p0071) as a novel FLCN interactor, and this has implicated FLCN in the regulation of RhoA signalling.
The FLCN-PKP4 interaction was identified by yeast-2-hybrid analysis and confirmed in vitro using co-immunoprecipitation studies in both Hek293 and ACHN cells. PKP4 is a member of the armadillo superfamily of proteins, which interact with cadherins and have a role in cell-cell contacts. PKP4 is also known to bind the desmosomal protein desmocollin 3a, plakoglobin and desmoplakin, and it has been linked to the regulation of RhoA signalling, cytokinesis and intercellular junction formation. Through its interaction with PKP4, Nahorski et al. suggest that FLCN may also regulate these processes and proceeded to investigate the function of the FLCN-PKP4 complex.
Immunofluorescence microscopic analysis, using MCF-7 cells, and bimolecular fluorescence complementation (BiFC) analysis, using HeLa cells, was used to investigate the intracellular localisation of FLCN and PKP4. As the localisation of PKP4 changes during mitosis, different stages of the cell cycle were studied. During interphase, FLCN and PKP4 were shown to co-localise most strongly at cell junctions, with a more dispersed co-localisation throughout the cytoplasm. The authors note that the FLCN signal at cell junctions is more consistent with a transient role, rather than that of a structural component. During cytokinesis, FLCN and PKP4 co-localise at the midbody.
PKP4 regulates RhoA signalling through a direct interaction with RhoA and Ect2, a RhoA GEF. Ect2 has been associated with renal cell carcinoma, as described in this previous blog post. To investigate FLCN’s effect on RhoA signalling, paired isogenic cell lines were used which demonstrated that FLCN deficiency is associated with increased expression of RhoA. Moreover, FLCN deficiency also resulted in increased activity of RhoA, as measured by the levels of GTP-bound RhoA present in the cells.
PKP4 loss is known to cause cytokinesis defects, multinucleation and a dysregulation of RhoA signalling. The authors demonstrate that FLCN loss also leads to an increase in multinucleated cells, suggesting that FLCN is required for cytokinesis. Furthermore, a wound healing assay showed that FLCN deficient cells migrated faster than cells which contained FLCN. Interestingly, inhibiting the downstream RhoA signalling pathway, using the ROCK inhibitor Y-27632, ameliorated this migratory phenotype. Finally, the effect of FLCN on cell junction formation was investigated and it was seen that knockdown of FLCN delayed tight junction formation and reduced the amount of Claudin-1 and E-Cadherin, components of tight junctions and adherin junctions respectively.
The exact function of the FLCN-PKP4 complex remains unknown, however this study has suggested a new role for FLCN in cytokinesis, cell junction formation and the regulation of RhoA signalling. RhoA is often overexpressed in cancers and it has been linked to metastasis. Perhaps the inhibition of this signalling pathway could be a potential therapy to treat metastatic renal tumours associated with BHD syndrome.
- Nahorski MS, Seabra L, Straatman-Iwanowska A, Wingenfeld A, Reiman A, Lu X, Klomp JA, Teh BT, Hatzfeld M, Gissen P, & Maher ER (2012). Folliculin interacts with p0071 (Plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis. Human molecular genetics PMID: 22965878
- 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 (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proceedings of the National Academy of Sciences of the United States of America, 103 (42), 15552-7 PMID: 17028174
- Hasumi H, Baba M, Hong SB, Hasumi Y, Huang Y, Yao M, Valera VA, Linehan WM, & Schmidt LS (2008). Identification and characterization of a novel folliculin-interacting protein FNIP2. Gene, 415 (1-2), 60-7 PMID: 18403135
- Takagi Y, Kobayashi T, Shiono M, Wang L, Piao X, Sun G, Zhang D, Abe M, Hagiwara Y, Takahashi K, & Hino O (2008). Interaction of folliculin (Birt-Hogg-Dubé gene product) with a novel Fnip1-like (FnipL/Fnip2) protein. Oncogene, 27 (40), 5339-47 PMID: 18663353