Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway
Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway
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DOI：
10.1073/pnas.95.13.7772
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发表时间：
1998-06-23
影响因子：
11.1
通讯作者：
Lawrence, JC
中科院分区：
文献类型：
--
作者：
Scott, PH;Brunn, GJ;Lawrence, JC
在3 T3-L1脂肪细胞中研究了胰岛素对雷帕霉素的哺乳动物靶标mTOR的影响，在免疫复合物测定中以重组PHAS-I作为底物测量mTOR蛋白激酶活性，当用mTOR抗体mTAb 2进行免疫沉淀时，清楚地观察到胰岛素刺激的激酶活性。胰岛素还使mTOR中P-32含量增加数倍，这是在用雷帕霉素. FKBP 12琼脂糖珠从P-32标记的脂肪细胞中纯化蛋白质后测定的。胰岛素既不影响mTOR免疫沉淀的量，也不影响mTAb 2免疫印迹检测到的mTOR的量。然而，该激素显著降低mTOR与mTAb 1（一种激活mTOR蛋白激酶的抗体）的反应性。通过将mTOR与蛋白磷酸酶I孵育，胰岛素对增加mTOR蛋白激酶活性和降低mTAb 1反应性的作用被消除。有趣的是，mTAb 1的表位位于mTOR的COOH末端附近的20个氨基酸区域中，该区域包括蛋白激酶B（PK B）磷酸化的共有位点。在MER-Akt细胞中进行实验以研究PK B在控制mTOR中的作用。这些细胞表达当细胞暴露于4-羟基他莫昔芬时被激活的PKB突变型雌激素受体融合蛋白。激活Pk; B与4-羟基他莫昔芬通过降低mTOR与mTAb 1的反应性和通过增加mTOR的PHAS-I激酶活性来模拟胰岛素我们的发现支持胰岛素通过经由包含PK B的信号通路促进蛋白质的磷酸化来激活mTOR的结论。
The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes, mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate, Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the P-32 content of mTOR that was determined after purifying the protein from P-32-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase, The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase I. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB), Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating Pk;B with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.