Elucidation of tumor suppressor function of Birt-Hogg-Dube syndrome gene(BHD)

在本研究中，通过同源搜索鉴定了一个新的Bhd产物(Flcn)结合蛋白(Fnipl-like Protein=FnipL)，并研究了它与Flcn和AMPK的结合。FnipL与Fnipl之间的相互作用可能主要通过各蛋白的C-末端结构域以及Fnipl与Fnipl之间的相互作用来实现。异位表达的Flcn主要定位于细胞核。共转染分析表明，FnipL或Fnipl可将HEN定位于细胞质。FnipL中羧基末端的Flcn结合域的缺失取消了Flcn的细胞质定位，表明Fnip蛋白通过形成复合体来调节Flcn的定位。通过使用siRNA，可以观察到BHD抑制的细胞中S6K1的磷酸化水平降低。在FNIPL或FN/P/抑制的细胞中，S6K1的磷酸化水平也降低。这些结果表明，Flcn-FnipL和Flcn-Fnipl复合体正向调节S6K1的磷酸化。我们还分析了Flcn的磷酸化。在TSC2基因缺失的肾癌细胞中，雷帕霉素处理或TSC2的表达可抑制Flcn的磷酸化。293细胞中强制表达rheb可诱导Flcn的磷酸化。相反，RNAi介导的对Raptor的抑制降低了Flcn的磷酸化。这些结果表明，TSC2-mTOR途径调节Flcn的磷酸化。通过定点突变和质谱分析发现，氨基末端的丝氨酸残基是主要的磷酸化位点。然而，该位点的磷酸化被认为不受TSC2-mTOR的调节。还鉴定了其他几个候选的磷酸化位点。综上所述，本研究揭示了Flcn和mTOR的累加功能关系，为阐明BHD缺乏致癌的分子机制提供了线索。

In this study, a novel Bhd product (Flcn)-binding protein (Fnipl-like protein = FnipL) was identified by homology search and its binding with Flcn and AMPK was characterized. The interaction between FnipL and Flcn may be mediated mainly by the C-terminal domains of each proteins as well as Flcn-Fnipl interaction. Ectopically expressed Flcn was localized mainly in the nucleus. Co-transfection analysis revealed that Hen is localized to the cytoplasm by FnipL or Fnipl. A deletion of carboxy-terminal Flcn-binding domain in FnipL cancelled cytoplasmic localization of Flcn, suggesting that the Fnip proteins regulate localization of Flcn through the complex formation. By the employment of siRNA, a decrease in S6K1 phosphorylation in the BHD-suppressed cell was observed. A decrease in S6K1 phosphorylation in FNIPL- or FN/P/ -suppressed cells was also observed. These results suggest that Flcn-FnipL and Flcn-Fnipl complexes positively regulate S6K1 phosphorylation. We also analyzed phosphorylation of Flcn. Phosphorylation of Flcn was suppressed by rapamycin-treatment or expression of Tsc2 in the Tsc2-deficient renal carcinoma cells. Forced expression of Rheb in 293 cells induced Flcn phosphorylation. Conversely, RNAi-mediated inhibition of raptor reduced Flcn phosphorylation. These results suggest that Tsc2-mTOR pathway regulates Flcn phosphorylation. By site-directed mutagenesis and mass spectrometry revealed that a serine residue in the amino-terminal region is a major phosphorylation site. However phosphorylation of this site was thought to be not regulated by Tsc2-mTOR. Several other candidate phosphorylation sites were identified. Together, in this study, reciplocal functional relationship between Flcn and mTOR has been revelead as a clue to elucidate the molecular mechanism of carcinogenesis associated with BHD-deficiency.