Diabetes and Obesity Research Program, The Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.
J Biol Chem. 2010 Jan 22;285(4):2245-57. doi: 10.1074/jbc.M109.060236. Epub 2009 Nov 6.
The phosphatidylinositol 3-kinase/Akt pathway regulates many biological processes, including insulin-regulated GLUT4 insertion into the plasma membrane. However, Akt operates well below its capacity to facilitate maximal GLUT4 translocation. Thus, reconciling modest changes in Akt expression or activity as a cause of metabolic dysfunction is complex. To resolve this, we examined insulin regulation of components within the signaling cascade in a quantitative kinetic and dose-response study combined with hierarchical cluster analysis. This revealed a strong relationship between phosphorylation of Akt substrates and GLUT4 translocation but not whole cell Akt phosphorylation. In contrast, Akt activity at the plasma membrane strongly correlated with GLUT4 translocation and Akt substrate phosphorylation. Additionally, two of the phosphorylated sites in the Akt substrate AS160 clustered separately, with Thr(P)-642 grouped with other Akt substrates. Further experiments suggested that atypical protein kinase Czeta phosphorylates AS160 at Ser-588 and that these two sites are mutually exclusive. These data indicate the utility of hierarchical cluster analysis for identifying functionally related biological nodes and highlight the importance of subcellular partitioning of key signaling components for biological specificity.
磷脂酰肌醇 3-激酶/蛋白激酶 B（Akt）途径调节许多生物过程，包括胰岛素调节的葡萄糖转运蛋白 4（GLUT4）向质膜的插入。然而，Akt 的作用远低于促进 GLUT4 转位的最大能力。因此，将 Akt 表达或活性的适度变化解释为代谢功能障碍的原因是复杂的。为了解决这个问题，我们在一项定量动力学和剂量反应研究中检查了信号转导级联中的成分，结合层次聚类分析。这表明 Akt 底物的磷酸化与 GLUT4 易位之间存在很强的关系，但与整个细胞 Akt 磷酸化无关。相比之下，质膜上 Akt 的活性与 GLUT4 易位和 Akt 底物磷酸化强烈相关。此外，Akt 底物 AS160 中的两个磷酸化位点分别聚类，Thr(P)-642 与其他 Akt 底物分组。进一步的实验表明，非典型蛋白激酶 Czeta 使 AS160 在 Ser-588 处磷酸化，并且这两个位点是相互排斥的。这些数据表明层次聚类分析可用于识别具有功能相关性的生物节点，并强调关键信号成分的亚细胞分区对于生物学特异性的重要性。
