Li Yong, Inoki Ken, Vikis Haris, Guan Kun-Liang
Life Sciences Institute, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
Methods Enzymol. 2006;407:46-54. doi: 10.1016/S0076-6879(05)07005-9.
Tuberous sclerosis complex (TSC) is a genetic disease caused by mutation in either the tsc1 or tsc2 tumor suppressor genes. TSC1 and TSC2 protein form a physical and functional complex in vivo. Recent studies have demonstrated that TSC2 displays GTPase activating protein (GAP) activity specifically toward the small G protein Rheb (Ras homolog enriched in brain) and inhibits its ability to stimulate the mammalian target of rapamycin (mTOR) signaling pathway. We have presented three methods to determine the activity of TSC2 as a GAP toward the Rheb GTPase. The first involves the isolation of TSC2 from cells and measurement of its activity toward Rheb substrate in vitro. The second involves the measurement of Rheb-associated guanine nucleotides as measure of TSC2 GAP activity on Rheb in vivo. The last method is to determine the phosphorylation of S6K1 (ribosomal S6 kinase), which is a downstream target of mTOR, as an indirect assay for TSC2 GAP activity in vivo.
结节性硬化症（TSC）是一种由tsc1或tsc2肿瘤抑制基因突变引起的遗传性疾病。TSC1和TSC2蛋白在体内形成一个物理和功能复合物。最近的研究表明，TSC2对小G蛋白Rheb（富含于脑中的Ras同源物）具有特异性的GTP酶激活蛋白（GAP）活性，并抑制其刺激哺乳动物雷帕霉素靶蛋白（mTOR）信号通路的能力。我们提出了三种方法来确定TSC2作为Rheb GTP酶的GAP活性。第一种方法是从细胞中分离TSC2，并在体外测量其对Rheb底物的活性。第二种方法是测量与Rheb相关的鸟嘌呤核苷酸，以此作为体内TSC2对Rheb的GAP活性的指标。最后一种方法是确定S6K1（核糖体S6激酶）的磷酸化，S6K1是mTOR的下游靶点，以此作为体内TSC2 GAP活性的间接检测方法。
