Molecular approach for the mechanism of insulin-stimulated glucose transport

为了探讨3-磷酸肌醇依赖的蛋白激酶1(PDK1)在Akt1磷酸化状态中的作用，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了野生型(Wt)PDK1及其突变体(Kd)。使用抗磷酸化Akt1抗体的免疫印迹显示，在胰岛素刺激下，Thr-308在1分钟时已达到最大程度的磷酸化，但在5分钟时完全去磷酸化，而胰岛素诱导的Akt1激活即使在Thr-308去磷酸化后仍保持不变。Wt-PDK1的过表达进一步增加了胰岛素刺激的Thr-308的磷酸化，随后也伴随着快速的去磷酸化。Wt-PDK1的表达也增强了胰岛素刺激的Akt1的活性，但即使在15min时仍保持不变。因此，Thr-308的磷酸化不是维持Akt1活性所必需的。有趣的是，在表达kd-PDK1的细胞中，胰岛素刺激的Thr-308的磷酸化状态即使在15min也保持不变，这表明kd-PDK1对Akt1的Thr-308的去磷酸化具有明显的负面影响。PP1和PP2A的抑制剂Calyculin A也延长了胰岛素刺激的Thr-308的磷酸化状态。此外，体外实验表明，PP2A，而不是PP1，可以完全使Akt1的Thr-308去磷酸化。这些发现表明，一种新的途径涉及Akt1在Thr-308处的去磷酸化，最初被鉴定为PP2A的磷酸酶是从Akt1激酶PDK1下游调控的。

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.