Last summer, Nookala et al., published the structure of the C-terminus of the FLCN protein, and showed that this portion of FLCN formed a divergent DENN domain. DENN domain proteins have Guanine nucleotide exchange factor (GEF) activity towards Rab GTPases, which are important for membrane trafficking, suggesting FLCN is a membrane trafficking protein. To read more about Rab, GEF and DENN proteins, please read our previous blog post on this subject.
The DENN domain of FLCN is non-canonical, and sequence homology was not sufficient to identify it as a DENN domain, suggesting that there could be further unidentified DENN domain proteins. In order to address this question, Zhang et al., used the protein sequence of FLCN’s DENN domain to search for such proteins.
The authors show that although this divergent DENN domain probably appeared later during evolutionary history than the classic DENN domain, elements of this divergent domain may have been present in the last eukaryotic common ancestor. Furthermore, the authors found six proteins with DENN domains similar to FLCN – C9ORF72, SMCR8, NPR2, NPR3, FNIP1 and FNIP2
C9orf72 and SMCR8 are both associated with human disease: expansion of a hexanucleotide repeat in C9orf72 causes Amyotrophic Lateral Sclerosis and Frontotemporal Dementia, while SMCR8 is within a region of chromosome 17 deleted in Smith-Magenis Syndrome. Npr2 and Npr3 form a heterodimer and, in S.cerevisiae, inhibit TORC1 signalling during nutrient starvation to prevent cell division (Neklesa and Davis, 2009). Of further interest, LST7, the S.cerevisiae homologue of FLCN, shows synthetic lethality with Sec13, which forms a trafficking complex with Npr2 and Npr3 (Dokudovskaya et al., 2011; Roberg et al., 1997).
FNIP1 and FNIP2, interact with FLCN and AMPK to regulate mTOR signalling (Baba et al., 2006; Hasumi et al., 2008) and were also found to have a DENN domain similar to FLCN’s. FNIP1 is required for correct B-cell growth and function in mice, while FNIP2 is required for MNU-induced apoptosis (Baba et al., 2012; Park et al., 2012; Sano et al., 2012).
Despite having a non-canonical DENN domain, FLCN has been shown to have GEF activity towards Rab35 in vitro (Nookala et al., 2012) and Rab27b expression has been found to be regulated by FLCN in three separate studies, suggesting that the DENN domain proteins identified in this study may also have GEF activity towards Rab GTPases. Determining which Rabs these proteins target in vivo, will shed light on these proteins’ functions, and how they contribute to human diseases, including Birt-Hogg-Dubé Syndrome. Indeed, Rab35 and Rab27b have roles in cadherin signalling and keratinocyte adhesion of the epidermis respectively, potentially implicating FLCN in these processes.
Interestingly, SMCR8, Npr2, Npr3 and FNIP1 have all been implicated in autophagy (Behrends et al., 2010; Wu and Tu, 2011), suggesting that FLCN may also function in autophagy. There are a number of autophagy Rabs (Hirota et al., 2013) and it would be interesting to find out whether any of these interact with FLCN or any of the DENN domain proteins identified in this study. Furthermore, given that FLCN, FNIP1, FNIP2, Npr2 and Npr3 are all known to function in mTOR signalling (Baba et al., 2006; Hasumi et al., 2008; Neklesa and Davis, 2009), it would be interesting to determine whether the effects of these proteins on mTOR signalling is mediated via membrane trafficking, and whether it is these proteins that link the coupled processes of mTOR signalling and autophagy.
- 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
- Baba M, Keller JR, Sun HW, Resch W, Kuchen S, Suh HC, Hasumi H, Hasumi Y, Kieffer-Kwon KR, Gonzalez CG, Hughes RM, Klein ME, Oh HF, Bible P, Southon E, Tessarollo L, Schmidt LS, Linehan WM, Casellas R (2012). The folliculin-FNIP1 pathway deleted in human Birt-Hogg-Dubé syndrome is required for murine B-cell development. Blood, 120 (6), 1254-61 PMID: 22709692
- Behrends C, Sowa ME, Gygi SP, Harper JW (2011). Network organization of the human autophagy system. Nature, 466 (7302), 68-76 PMID: 20562859
- Dokudovskaya S, Waharte F, Schlessinger A, Pieper U, Devos DP, Cristea IM, Williams R, Salamero J, Chait BT, Sali A, Field MC, Rout MP, Dargemont C (2011). A conserved coatomer-related complex containing Sec13 and Seh1 dynamically associates with the vacuole in Saccharomyces cerevisiae. Molecular & cellular proteomics, 10 (6), PMID: 21454883
- 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
- Yuko Hirota, Keiko Fujimoto and Yoshitaka Tanaka (2013). Rab GTPases in Autophagy, Autophagy – A Double-Edged Sword – Cell Survival or Death?, Dr. Yannick Bailly (Ed.), ISBN: 978-953-51-1062-0, InTech, DOI: 10.5772/54578
- Neklesa TK & Davis RW (2009). A genome-wide screen for regulators of TORC1 in response to amino acid starvation reveals a conserved Npr2/3 complex. PLoS Genetics, 5 (6), e1000515 PMID: 19521502
- Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montaño B, Donaldson JC, Blaszczyk BK, Chirgadze DY, Barr FA, Bazan JF, Blundell TL (2012). Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer. Open biology, 2 (8), 120071 PMID: 22977732
- Park H, Staehling K, Tsang M, Appleby MW, Brunkow ME, Margineantu D, Hockenbery DM, Habib T, Liggitt HD, Carlson G, Iritani BM (2012). Disruption of Fnip1 reveals a metabolic checkpoint controlling B lymphocyte development. Immunity, 36 (5), 769-81 PMID: 22608497
- Roberg KJ, Bickel S, Rowley N, Kaiser CA (1997). Control of amino acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7 and LST8. Genetics, 147 (4), 1569-84 PMID: 9409822
- Sano S, Sakagami R, Sekiguchi M, Hidaka M (2013). Stabilization of MAPO1 by specific binding with folliculin and AMP-activated protein kinase in O⁶-methylguanine-induced apoptosis. Biochemical and biophysical research communications, 430 (2), 810-5 PMID: 23201403
- Wu X & Tu BP (2011). Selective regulation of autophagy by the Iml1-Npr2-Npr3 complex in the absence of nitrogen starvation. Molecular biology of the cell, 22 (21), 4124-33 PMID: 21900499
- Zhang D, Iyer LM, He F, & Aravind L (2012). Discovery of Novel DENN Proteins: Implications for the Evolution of Eukaryotic Intracellular Membrane Structures and Human Disease. Frontiers in genetics, 3 PMID: 23248642
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.