【原】雷帕霉素如何发挥作用？

Dissociation of raptor from mTOR is a mechanism of rapamycin-induced inhibition of mTOR function.

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|核心内容：

哺乳动物的雷帕霉素靶标(MTOR)是一种丝氨酸/苏氨酸蛋白激酶，在调节细胞生长的营养敏感信号通路中起着至关重要的作用。

雷帕霉素通过将FK506结合蛋白12(FKBP12)/雷帕霉素复合体直接结合到TOR FRB结构域(一段氨基末端与激酶催化域)，有效地抑制了TOR信号转导。

在次研究之前，FKBP12/雷帕霉素抑制作用的分子基础尚不清楚。

Raptor(mTOR调节相关蛋白)是新近发现的一种mTOR结合伙伴，在体内mTOR信号转导中起重要作用，其与mTOR的结合在体外mTOR催化底物磷酸化过程中起关键作用。

在此，我们研究了内源性mTOR/Raptor复合体在体内对雷帕霉素的响应以及在体外直接加入FKBP12/Raptor复合体的稳定性。

雷帕霉素抑制内源性Raptor体内内源性或重组mTOR的恢复，这种抑制作用需要mTOR结合FKBP12/雷帕霉素复合体的能力，但这种抑制作用与mTOR激酶活性无关

在体外，在FKBP12存在下，雷帕霉素直接抑制Raptor与mTOR的结合，同时降低mTOR催化的Raptor依赖性底物的磷酸化，而不是Raptor非依赖性底物的磷酸化，mTOR自身磷酸化没有改变。

这些观察表明，雷帕霉素抑制mTOR功能，至少部分是通过抑制Raptor与mTOR的相互作用；

这一作用使mTOR与其底物解偶联，并在不改变mTOR固有催化活性的情况下抑制mTOR信号转导。

图1 fkbp12/雷帕霉素复合物对雷帕霉素与 mTOR 分离的体外作用 用含10% FBS 的 DMEM 培养的 HEK293细胞进行裂解，用抗 mtor Ab (第1ー4条)免疫沉淀等量的细胞裂解蛋白。将免疫沉淀 mTOR/raptor 复合物与 gst-fkbp125μm 培养基连同雷帕霉素(lanycin 1-4)指示浓度孵育90min，分离复合物和上清液。利用 SDS-PAGE 和免疫印迹技术对复合物(小球)进行了分析，并与抗 mtor 和抗 raptor 抗体进行了比较。同时用 SDSPAGE 和免疫印迹法检测上清液中raptor的游离量。这些结果是三个可重复的实验的代表。

原文摘要：

The mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that plays a crucial role in a nutrient-sensitive signalling pathway that regulates cell growth. TOR signalling is potently inhibited by rapamycin, through the direct binding of a FK506-binding protein 12 (FKBP12)/rapamycin complex to the TOR FRB domain, a segment amino terminal to the kinase catalytic domain. The molecular basis for the inhibitory action of FKBP12/rapamycin remains uncertain. Raptor (regulatory associated protein of mTOR) is a recently identified mTOR binding partner that is essential for mTOR signalling in vivo, and whose binding to mTOR is critical for mTOR-catalysed substrate phosphorylation in vitro. Here we investigated the stability of endogenous mTOR/raptor complex in response to rapamycin in vivo, and to the direct addition of a FKBP12/rapamycin complex in vitro. Rapamycin diminished the recovery of endogenous raptor with endogenous or recombinant mTOR in vivo; this inhibition required the ability of mTOR to bind the FKBP12/rapamycin complex, but was independent of mTOR kinase activity. Rapamycin, in the presence of FKBP12, inhibited the association of raptor with mTOR directly in vitro, and concomitantly reduced the mTOR-catalysed phosphorylation of raptor-dependent, but not raptor-independent substrates; mTOR autophosphorylation was unaltered. These observations indicate that rapamycin inhibits mTOR function, at least in part, by inhibiting the interaction of raptor with mTOR; this action uncouples mTOR from its substrates, and inhibits mTOR signalling without altering mTOR's intrinsic catalytic activity.

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参考文献：https://onlinelibrary./doi/pdf/10.1111/j.1356-9597.2004.00727.x

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