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

Birt-Hogg-Dube综合征基因（BHD）抑癌功能的阐明

• 批准号: 18590380
• 负责人: KOBAYASHI Toshiyuki
• 金额: $ 2.57万
• 依托单位: Juntendo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590380/
• 关键词: BHDsyndrome; FnipL; mTOR; AMPK; Tsc2; Fnip 1; Bhd症候群; Fnip1; 腎癌; Bhd; Nihonラット; Ekerラット; 動物モデル; リン酸化

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.

在本研究中，通过同源性搜索鉴定了一种新型 Bhd 产物 (Flcn) 结合蛋白（Fnipl 样蛋白 = FnipL），并对其与 Flcn 和 AMPK 的结合进行了表征。 FnipL 和 Flcn 之间的相互作用可能主要由每种蛋白质的 C 端结构域以及 Flcn-Fnipl 相互作用介导。异位表达的 Flcn 主要位于细胞核中。共转染分析表明，Hen 通过 FnipL 或 Fnipl 定位于细胞质。 FnipL 中羧基末端 Flcn 结合域的缺失取消了 Flcn 的细胞质定位，表明 Fnip 蛋白通过复合物形成调节 Flcn 的定位。通过使用 siRNA，观察到 BHD 抑制细胞中 S6K1 磷酸化的减少。还观察到 FNIPL 或 FN/P/ 抑制细胞中 S6K1 磷酸化减少。这些结果表明 Flcn-FnipL 和 Flcn-Fnipl 复合物正向调节 S6K1 磷酸化。我们还分析了 Flcn 的磷酸化。雷帕霉素处理或 Tsc2 缺陷型肾癌细胞中 Tsc2 的表达可抑制 Flcn 的磷酸化。在 293 细胞中强制表达 Rheb 会诱导 Flcn 磷酸化。相反，RNAi 介导的 raptor 抑制会降低 Flcn 磷酸化。这些结果表明 Tsc2-mTOR 途径调节 Flcn 磷酸化。通过定点诱变和质谱分析表明氨基末端区域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 调节。还鉴定了其他几个候选磷酸化位点。在这项研究中，Flcn 和 mTOR 之间的相互功能关系已被揭示，作为阐明与 BHD 缺乏相关的致癌分子机制的线索。

项目成果

Interaction of Fox01 and TSC2 induces insulin resistance through activation of the mammalian target of rapamycin/p70 S6K pathway.

Fox01 和 TSC2 的相互作用通过激活哺乳动物雷帕霉素/p70 S6K 通路靶标诱导胰岛素抵抗。

• 发表时间: 2006
• 期刊: J.Biol.Chem 281
• 作者: Cao Y;et al.
• 通讯作者: et al.

GADD34 inhibits mammalian target of rapamycin signaling via tuberous sclerosiscomplex and controls cell survival under bioenergetic stress.

GADD34 通过结节性硬化症抑制哺乳动物雷帕霉素信号传导靶标，并控制生物能应激下的细胞存活。

• 发表时间: 2007
• 期刊: Int J Mol Med 19(3)
• 作者: Watanabe R;Kobayashi T;Hino O;Okabe H;Chano T
• 通讯作者: Chano T

Tuberous sclerosis complex 2 loss-of-function mutation regulates reactive oxygen species production through Racl activation.

结节性硬化症复合体 2 功能丧失突变通过 Racl 激活调节活性氧的产生。

• 发表时间: 2008
• 期刊: Biochem. Biophys. Res. Commun 368
• 作者: Suzuki T.;Das S.K.;Inoue H.;Kazami M.;Hino O.;Kobayashi T.;Yeung R.S.;Kobayashi K.;Tadokoro T.;Yamamoto Y.
• 通讯作者: Yamamoto Y.

Identification of a novel binding protein Fnip 1-Like(FnipL) to Flcn encoded by the BHD(Birt-Hogg-Dube) gene

BHD(Birt-Hogg-Dube)基因编码的新型Fnip 1-Like(FnipL)与Flcn结合蛋白的鉴定

• 发表时间: 2007
• 作者: Takagi;Y.;et. al.
• 通讯作者: et. al.

Suppression of viral rephcation by stress-inducible GADD34 protein via the mammalian serine/threonine_protein kinase mTOR pathway

应激诱导的 GADD34 蛋白通过哺乳动物丝氨酸/苏氨酸_蛋白激酶 mTOR 途径抑制病毒复制

• 发表时间: 2007
• 期刊: Journal of Virology 81
• 作者: Minami;K;et. al.
• 通讯作者: et. al.