Attenuation of mammalian target of rapamycin activity by increased cAMP in 3T3-L1 adipocytes
Attenuation of mammalian target of rapamycin activity by increased cAMP in 3T3-L1 adipocytes
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DOI：
10.1074/jbc.273.51.34496
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发表时间：
1998-12-18
影响因子：
4.8
通讯作者：
Lawrence, JC
中科院分区：
文献类型：
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作者：
Scott, PH;Lawrence, JC
将3 T3-L1脂肪细胞与通过激活腺苷酸环化酶增加细胞内cAMP的毛喉素一起孵育，通过减弱胰岛素对刺激PHAS-I中四个（S/T）P位点磷酸化的作用来模拟雷帕霉素，PHAS-I是雷帕霉素靶蛋白（mTOR）信号通路的下游靶标。为了研究增加cAMP抑制mTOR的假设，在免疫复合物测定中用重组PHAS-I作为底物测量mTOR的蛋白激酶活性。毛喉素和8-（4-氯苯硫基）腺苷3 '-5'-单磷酸（CPT-cAMP）都能阻止脂肪细胞中胰岛素对mTOR的激活，但当直接加入免疫纯化蛋白时，两种药物都不影响mTOR活性。相比之下，cAMP磷酸二酯酶抑制剂茶碱不仅在添加到完整脂肪细胞中时，而且在添加到体外免疫纯化的mTOR中时抑制mTOR活性，这表明某些甲基黄嘌呤能够独立于增加cAMP而抑制mTOR。毛喉素和CPT-cAMP阻断了胰岛素对增加mTOR磷酸化的作用，这是使用mTAb 1评估的，mTAb 1是一种其结合被mTOR磷酸化抑制的抗体。尽管mTAb 1表位含有蛋白激酶B的共有位点，但两种药物都不能抑制胰岛素产生的蛋白激酶B的活化。这些发现支持这样的解释，即增加cAMP通过抑制mTOR的磷酸化和活化来减弱胰岛素对PHAS-I、p70（S6 K）和mTOR信号传导途径的其他下游靶点的作用。
Incubating 3T3-L1 adipocytes with forskolin, which increases intracellular cAMP by activating adenylate cyclase, mimicked rapamycin by attenuating the effect of insulin on stimulating the phosphorylation of four (S/T)P sites in PHAS-I, a downstream target of the mammalian target of rapamycin (mTOR) signaling pathway. To investigate the hypothesis that increasing cAMP inhibits mTOR, the protein kinase activity of mTOR was measured in an immune complex assay with recombinant PHAS-I as substrate. Both forskolin and 8-(4-chlorophenylthio) adenosine 3'-5'-monophosphate (CPT-cAMP) prevented the activation of mTOR by insulin in adipocytes, but neither agent affected mTOR activity when added directly to the immunopurified protein. In contrast, the cAMP phosphodiesterase inhibitor, theophylline, inhibited mTOR activity not only when added to intact adipocytes but also when added to immunopurified mTOR in vitro, demonstrating that certain methylxanthines are able to inhibit mTOR independently of increasing cAMP. Forskolin and CPT-cAMP blocked the effect of insulin on increasing mTOR phosphorylation, which was assessed using mTAb1, an antibody whose binding is inhibited by phosphorylation of mTOR. Although the mTAb1 epitope contains a consensus site for protein kinase B, neither agent inhibited the activation of protein kinase B produced by insulin, These findings support the interpretation that increasing cAMP attenuates the effects of insulin on PHAS-I, p70(S6K), and other downstream targets of the mTOR signaling pathway by inhibiting the phosphorylation and activation of mTOR.