Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Oncogene. 2014 Jun 26;33(26):3463-72. doi: 10.1038/onc.2013.301. Epub 2013 Aug 5.
Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated because of the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. Although basal AKT2 phosphorylation was sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K), basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus, NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation.
AKT 异常激活普遍存在于多种人类癌细胞系中，为治疗提供了一个重要的新靶点。在特定的 AKT 同工型上已经鉴定出 22 个独立的磷酸化位点，这些位点可能有助于不同同工型的调节。然而，由于缺乏能够评估单个 AKT 分子上多个位点磷酸化的稳健方法，因此尚未阐明调节单个 AKT 同工型分子磷酸化的机制。
我们使用纳米流体蛋白质组学免疫分析（NIA），包括等电聚焦和敏感的化学发光检测，证明在基础和配体诱导条件下，AKT1 和 AKT2 的磷酸化事件模式明显不同。事实上，至少有 12 个 AKT1 峰和至少 5 个 AKT2 峰，这与单个 AKT 分子上不同位点的磷酸化复杂组合一致。
胰岛素刺激后，AKT1 在 T 环中的 Thr308 和疏水结构域中的 Ser473 被磷酸化。相比之下，AKT2 仅在低水平下在等效位点（Thr309 和 Ser474）被磷酸化。此外，Thr308 和 Ser473 的磷酸化主要发生在相同的 AKT1 分子上，而 Thr309 和 Ser474 的磷酸化主要发生在不同的 AKT2 分子上。
虽然基础 AKT2 磷酸化对磷脂酰肌醇 3-激酶（PI3K）的抑制敏感，但基础 AKT1 磷酸化基本上是抵抗的。PI3K 抑制减少了 AKT2 上的 pThr451，但不减少 AKT1 上的 pThr450。因此，NIA 技术提供了一种能够对单个 AKT 分子的协调磷酸化进行特征描述的能力，为 AKT 同工型特异性磷酸化提供了重要信息，这对于靶向异常 AKT 激活的药物的最佳开发和实施是必需的。
