Molecular approach for the mechanism of insulin-stimulated glucose transport

为了研究3-磷酸肌醇依赖性蛋白激酶1（PDK 1）在Akt 1磷酸化状态中的作用，利用腺病毒基因转导系统在稳定表达胰岛素受体的中国仓鼠卵巢细胞中表达野生型（wt）PDK 1及其激酶死亡（kd）突变体。使用抗磷酸化Akt 1抗体的免疫印迹显示，在胰岛素刺激下，Thr-308在1分钟时已被最大限度磷酸化，但在5分钟时完全去磷酸化，而即使在Thr-308去磷酸化后，胰岛素诱导的Akt 1活化仍得以维持。wt-PDK 1的过表达进一步增加胰岛素刺激的Thr-308磷酸化，随后也快速去磷酸化。胰岛素刺激的Akt 1活性也增强了wt-PDK 1的表达，但即使在15分钟仍维持不变。因此，一旦实现Akt 1活性，Thr-308的磷酸化对维持Akt 1活性并不重要。有趣的是，胰岛素刺激的Thr-308的磷酸化状态在表达kd-PDK 1的细胞中甚至在15分钟时也得以维持，这表明kd-PDK 1对Akt 1的Thr-308的去磷酸化具有显性负效应。Calyculin A是PP 1和PP 2A的抑制剂，也延长了胰岛素刺激的Thr-308磷酸化状态。此外，体外实验显示PP 2A，而不是PP 1，完全脱磷酸化Akt 1的Thr-308。这些研究结果表明，一种新的途径，涉及脱磷酸化的Akt 1在Thr-308的磷酸酶，可能PP 2A，最初确定为调节下游的PDK 1，Akt 1激酶。

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using anti-phosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulin-stimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK 1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulin-stimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.