Hresko Richard C, Mueckler Mike
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2005 Dec 9;280(49):40406-16. doi: 10.1074/jbc.M508361200. Epub 2005 Oct 11.
The insulin-signaling pathway leading to the activation of Akt/protein kinase B has been well characterized except for a single step, the phosphorylation of Akt at Ser-473. Double-stranded DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) gene product, integrin-linked kinase (ILK), protein kinase Calpha (PKCalpha), and mammalian target of rapamycin (mTOR), when complexed to rapamycin-insensitive companion of mTOR (RICTOR), have all been identified as playing a critical role in Akt Ser-473 phosphorylation. However, the apparently disparate results reported in these studies are difficult to evaluate, given that different stimuli and cell types were examined and that all of the candidate proteins have never been systematically studied in a single system. Additionally, none of these studies were performed in a classical insulin-responsive cell type or tissue such as muscle or fat. We therefore examined each of these candidates in 3T3-L1 adipocytes. In vitro kinase assays, using different subcellular fractions of 3T3-L1 adipocytes, revealed that phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 phosphorylation correlated well with the amount of DNA-PK, mTOR, and RICTOR but did not correlate with levels of ATM, ILK, and PKCalpha. PKCalpha was completely absent from compartments with Ser-473 phosphorylation activity. Although purified DNA-PK could phosphorylate a peptide derived from Akt that contains amino acid Ser-473, it could not phosphorylate full-length Akt2. Vesicles immunoprecipitated from low density microsomes using antibodies directed against mTOR or RICTOR had phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 activity that was sensitive to wortmannin but not staurosporine. In contrast, immunopurified low density microsome vesicles containing ILK could not phosphorylate Akt on Ser-473 in vitro. Small interference RNA knockdown of RICTOR, but not DNA-PK, ATM, or ILK, suppressed insulin-activated Ser-473 phosphorylation and, to a lesser extent, Thr-308 phosphorylation in 3T3-L1 adipocytes. Based on our cell-free kinase and small interference RNA results, we conclude that mTOR complexed to RICTOR is the Ser-473 kinase in 3T3-L1 adipocytes.
除了Akt在Ser-473位点的磷酸化这一步骤外，导致Akt/蛋白激酶B激活的胰岛素信号通路已得到充分表征。双链DNA依赖性蛋白激酶（DNA-PK）、共济失调毛细血管扩张症突变基因（ATM）产物、整合素连接激酶（ILK）、蛋白激酶Cα（PKCα）以及与雷帕霉素不敏感的mTOR伴侣（RICTOR）结合的哺乳动物雷帕霉素靶蛋白（mTOR），均已被确定在Akt Ser-473磷酸化过程中起关键作用。然而，鉴于这些研究中所检测的刺激因素和细胞类型各不相同，且所有候选蛋白从未在单一系统中进行过系统研究，因此这些研究报告中明显不同的结果难以评估。此外，这些研究均未在经典的胰岛素反应性细胞类型或组织（如肌肉或脂肪）中进行。因此，我们在3T3-L1脂肪细胞中对这些候选蛋白逐一进行了检测。利用3T3-L1脂肪细胞的不同亚细胞组分进行的体外激酶分析表明，磷脂酰肌醇3,4,5-三磷酸刺激的Ser-473磷酸化与DNA-PK、mTOR和RICTOR的量密切相关，但与ATM、ILK和PKCα的水平无关。具有Ser-473磷酸化活性的区室中完全没有PKCα。尽管纯化的DNA-PK能够磷酸化源自Akt且包含氨基酸Ser-473的肽段，但它无法磷酸化全长Akt2。使用针对mTOR或RICTOR的抗体从低密度微粒体中免疫沉淀的囊泡具有对wortmannin敏感但对星形孢菌素不敏感的磷脂酰肌醇3,4,5-三磷酸刺激的Ser-473活性。相比之下，免疫纯化的含有ILK的低密度微粒体囊泡在体外无法使Akt的Ser-473位点磷酸化。在3T3-L1脂肪细胞中，采用小干扰RNA敲低RICTOR而非DNA-PK、ATM或ILK，可抑制胰岛素激活的Ser-473磷酸化，并在较小程度上抑制Thr-308磷酸化。基于我们的无细胞激酶和小干扰RNA结果，我们得出结论，与RICTOR结合的mTOR是3T3-L1脂肪细胞中Ser-473位点的激酶。
