Nakae J, Kitamura T, Ogawa W, Kasuga M, Accili D
Naomi Berrie Diabetes Center and Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
Biochemistry. 2001 Oct 2;40(39):11768-76. doi: 10.1021/bi015532m.
Insulin inhibits expression of certain liver genes through the phosphoinositol (PI) 3-kinase/Akt pathway. However, whether Akt activity is both necessary and sufficient to mediate these effects remains controversial. The forkhead proteins (Foxo1, Foxo3, and Foxo4, previously known as Fkhr or Afx) are transcriptional enhancers, the activity of which is inhibited by insulin through phosphorylation-dependent translocation and nuclear exclusion. Others and we have previously shown that the forkhead protein Foxo1 is phosphorylated at three different sites: S(253), T(24), and S(316). We have also shown that T(24) fails to be phosphorylated in hepatocytes lacking insulin receptors, and we have suggested that this residue is targeted by a kinase distinct from Akt. In this study, we have further analyzed the ability of Akt to phosphorylate different Foxo1 sites in control and insulin receptor-deficient hepatocytes. Expression of a dominant negative Akt (Akt-AA) in control hepatocytes led to complete inhibition of endogenous Akt, but failed to inhibit Foxo1 T(24) phosphorylation and, consequently, insulin suppression of IGFBP-1 promoter activity. Conversely, expression of a constitutively active Akt (Akt-Myr) in insulin receptor-deficient hepatocytes led to an overall increase in the level of Foxo1 phosphorylation, but failed to induce T(24) and S(316) phosphorylation. These data indicate that the Foxo1 T(24) and S(316) kinases are distinct from Akt, and suggest that the pathways required for insulin regulation of hepatic gene expression diverge downstream of PI 3-kinase.
胰岛素通过磷酸肌醇（PI）3激酶/Akt信号通路抑制某些肝脏基因的表达。然而，Akt活性对于介导这些效应是否既必要又充分仍存在争议。叉头蛋白（Foxo1、Foxo3和Foxo4，以前称为Fkhr或Afx）是转录增强子，其活性通过磷酸化依赖性易位和核排除而被胰岛素抑制。我们和其他人先前已表明，叉头蛋白Foxo1在三个不同位点被磷酸化：S(253)、T(24)和S(316)。我们还表明，在缺乏胰岛素受体的肝细胞中，T(24)未能被磷酸化，并且我们认为该残基是由一种不同于Akt的激酶靶向作用的。在本研究中，我们进一步分析了Akt在对照和胰岛素受体缺陷型肝细胞中磷酸化不同Foxo1位点的能力。在对照肝细胞中表达显性负性Akt（Akt-AA）导致内源性Akt完全抑制，但未能抑制Foxo1 T(24)磷酸化，因此也未能抑制胰岛素对IGFBP-1启动子活性的抑制作用。相反，在胰岛素受体缺陷型肝细胞中表达组成型活性Akt（Akt-Myr）导致Foxo1磷酸化水平总体增加，但未能诱导T(24)和S(316)磷酸化。这些数据表明，Foxo1 T(24)和S(316)激酶不同于Akt，并提示胰岛素调节肝脏基因表达所需的信号通路在PI 3激酶下游发生分歧。
