Birt-Hogg-Dube´(BHD) syndrome is characterized by the development of pneumothorax, hair folliculomas and renal tumors and the responsible BHD gene is thought to be a tumor suppressor. The function of folliculin (Flcn), encoded by BHD, is totally unknown, although its interaction with Fnip1 has been reported. In this study, we identified a novel protein binding to Flcn, which is highly homologous to Fnip1, and which we named FnipL (recently reported in an independent study as Fnip2). The interaction between FnipL/Fnip2 and Flcn may be mediated mainly by the C-terminal domains of each protein as is the case for the Flcn-Fnip1 interaction. FnipL/Fnip2 and Flcn were located together in the cytoplasm in a reticular pattern, although solely expressed Flcn was found mainly in the nucleus. Cytoplasmic retention of Flcn was canceled with C-terminal truncation of FnipL/Fnip2, suggesting that FnipL/Fnip2 regulates Flcn distribution through their complex formation. By the employment of siRNA, we observed a decrease in S6K1 phosphorylation in the BHDsuppressed cell. We also observed a decrease in S6K1 phosphorylation in FNIP1-and, to a lesser extent, in FNIPL/FNIP2-suppressed cells. These results suggest that Flcn-FnipL/Fnip2 and Flcn-Fnip1 complexes positively regulate S6K1 phosphorylation and that FnipL/Fnip2 provides an important clue to elucidating the function of Flcn and the pathogenesis of BHD.

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Edited by Berend WieringaKeywords: Birt-Hogg-Dubé syndrome Folliculin 5 0 -AMP-activated protein kinase Phosphorylation FLCN-interacting protein a b s t r a c t Recently, it was reported that the product of Birt-Hogg-Dubé syndrome gene (folliculin, FLCN) is directly phosphorylated by 5 0 -AMP-activated protein kinase (AMPK). In this study, we identified serine 62 (Ser62) as a phosphorylation site in FLCN and generated an anti-phospho-Ser62-FLCN antibody. Our analysis suggests that Ser62 phosphorylation is indirectly up-regulated by AMPK and that another residue is directly phosphorylated by AMPK. By binding with FLCN-interacting proteins (FNIP1 and FNIP2/FNIPL), Ser62 phosphorylation is increased. A phospho-mimic mutation at Ser62 enhanced the formation of the FLCN-AMPK complex. These results suggest that function(s) of FLCN-AMPK-FNIP complex is regulated by Ser62 phosphorylation.

Tuberin, a tumor-suppressor protein produced by the tuberous sclerosis gene TSC2, downregulates the RhebmTOR-S6K pathway (mTOR axis). Comparison of the effects of human tuberin mutations, such as G1556S, suggests that pathways other than the mTOR axis might also be involved in the pathogenesis of tuberous sclerosis. Here we test this possibility using the rat G1556S-type mutation (GSM) and a transgenic Eker (Tsc2 mutant) rat system. Cells expressing GSM-tuberin failed to downregulate the mTOR axis. GSM-tuberin had an altered localization, which underlie its reduced ability to form a complex with hamartin, and a site-specific alteration in phosphorylation status indicating diverse regulation by Akt. GSM-transgenic (GSM-Tg) rats exhibited suppression of macroscopic renal tumors following N-ethyl-Nnitrosourea treatment. Intriguingly, rats with weaker GSM-Tg expression showed microscopic cystic and pre-tumorous lesions that were restricted in size and expansion, although they had hyper-phosphorylation of ribosomal protein S6. These results highlight a novel pathway involving tuberin that regulates tumor suppression independently of the mTOR inhibitory function. Identification of such a novel pathway will provide clear implications for generation of new therapeutic targets in the treatment of these tumors.

The Tsc2 product, tuberin, negatively regulates the mTOR pathway. We have exploited the Eker (Tsc2-mutant) rat system to analyse various Tsc2 mutations. Here, we focus on the N525S-Tsc2 variant (NSM), which is known to cause distinct symptoms in patients even though normal suppression of mTOR is observed. Unexpectedly, we were repeatedly unable to generate viable rats carrying the NSM transgene. Genotypic analysis revealed that most of the embryos carrying the transgene died around embryonic day after 14.5—similar to the stage of lethality observed for Eker homozygotes. Thus, the NSM transgene appeared to have a dominant lethal effect in our rat model. Further, no significant differences were observed for various signal transduction molecules in transiently expressed NSM cells compared to WT. These results indicate that a non-mTOR pathway, critical for embryogenesis, is being regulated by tuberin, providing a link between tuberin expression and the severity of Tsc2 mutation-related pathogenesis.