Control of cell cycle checkpoint mediated by mammalian target of rapamycin

雷帕霉素是一种亲脂大环内酯类化合物，在哺乳动物细胞中与FKBP12结合。mTOR（哺乳动物雷帕霉素靶蛋白）蛋白被鉴定为fkbp12 -雷帕霉素复合物的靶标。该蛋白计算分子量为289 kDa，在其羧基端含有激酶样结构域。在这项研究中，我们得到了以下发现。(1)mTOR在哺乳动物细胞中参与营养感知，特别是氨基酸感知，并调节翻译效应物，如p70S6激酶α (p70α)和eIF4E结合蛋白（4EBP）。(2)氨基酸- mtor信号通路的激活对于丝裂原诱导的蛋白质合成是必不可少的。换句话说，氨基酸- mtor信号是丝裂原诱导蛋白质合成的启动信号。(3)在氨基酸中，亮氨酸激活p70α磷酸化的能力最强。(4)从雷帕霉素处理过的细胞中以失活形式提取的p70α可以直接被mTOR激酶在雷帕霉素敏感位点Thr-412磷酸化。mtor催化的p70α体外磷酸化伴随着p70α激酶活性的大量恢复。(5) mTOR和3-磷酸肌醇依赖性蛋白激酶1在体外对p70α的顺序磷酸化可协同刺激p70α活性，达到与体内血清刺激相似的水平。(6)利用常规生化方法和酵母- 2杂交系统鉴定了mTOR的几个候选结合伙伴，并进行了详细的分析。(7)一些亮氨酸衍生物具有抑制p70α活化和T细胞增殖的能力。这种分析现在也在进行中。

Rapamycin is a lipophilic macrolide compound and binds to FKBP12 in mammalian cells. The mTOR (mammalian target of rapamycin) protein was identified as a target of FKBP12-rapamycin complex. This protein has a calculated molecular weight of 289 kDa and contains a kinase-like domain at its carboxyl-terminus. In this study, we obtained following findings.(1)mTOR participates in nutrient-sensing, especially amino acid-sensing, in mammalian cells and regulates translational effectors, such as p70S6 kinase α (p70α) and eIF4E binding protein (4EBP).(2)Activation of amino acid-mTOR signaling pathway is indispensable for mitogen-induced protein synthesis. In other words, amino acid-mTOR signaling is a priming signal for mitogen-induced protein synthesis.(3)Among amino acids, leucine has the most potent ability to activate phosphorylation of p70α.(4)p70α, extracted in inactive forms from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70α phosphorylation in vitro is accompanied by a substantial restoration in p70α kinase activity.(5)Sequential phosphorylation of p70α by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro results in a synergistic stimulation of p70α activity to levels similar to that attained by serum stimulation in vivo.(6)Several candidates of binding partners of mTOR were identified using conventional biochemical methods and yeast-two hybrid system and detailed analyses are now in progress.(7)Some of leucine derivatives were found to have the ability to inhibit p70α activation and T cell proliferation. This analysis is also now in progress.