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

点击翻译按钮获取中文摘要

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.