Cheng J Q, Altomare D A, Klein M A, Lee W C, Kruh G D, Lissy N A, Testa J R
Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Oncogene. 1997 Jun 12;14(23):2793-801. doi: 10.1038/sj.onc.1201121.
The AKT2 oncogene encodes a protein-serine/threonine kinase containing a pleckstrin homology domain characteristic of many signaling proteins. Recently, it was shown that AKT2 kinase activity can be induced by platelet-derived growth factor through phosphatidylinositol-3-OH kinase, suggesting that AKT2 may be an important signal mediator that contributes to the control of cell proliferation. We previously reported amplification and overexpression of AKT2 in human cancers. To investigate the transforming activity of AKT2, we used a retrovirus-based construct to express AKT2 in NIH3T3 cells. Overexpression of AKT2 was found to transform NIH3T3 cells, as determined by growth in soft agar and tumor formation in nude mice. The oncogenic activity of AKT2 was diminished by truncation of a 70-amino acid proline-rich region at the carboxyl-terminus. To facilitate the characterization of AKT2, we generated monoclonal and polyclonal antibodies against this protein. AKT2 was localized to the cytoplasm by cell fractionation experiments, immunocytochemistry, and immunofluorescence. Protein levels were more abundant in mitotic cells than in interphase cells. Western blot analysis of synchronized pancreatic cancer cells demonstrated that the expression level of AKT2 protein in mitotic cells is three to fivefold higher than in their interphase counterparts. A time-course study of phytohemagglutinin-stimulated lymphocytes revealed that AKT2 mRNA and AKT2 protein levels are highest 48-72 h after addition of mitogen, when cells are actively dividing. These data suggest that AKT2 could play a significant role in cell cycle progression and that the oncogenic activity of overexpressed AKT2 may be mediated by aberrant regulation of cellular proliferation.
AKT2癌基因编码一种蛋白质丝氨酸/苏氨酸激酶，其含有许多信号蛋白所特有的普列克底物蛋白同源结构域。最近研究表明，血小板衍生生长因子可通过磷脂酰肌醇-3-OH激酶诱导AKT2激酶活性，这表明AKT2可能是有助于控制细胞增殖的重要信号介质。我们之前报道过AKT2在人类癌症中存在扩增和过表达现象。为了研究AKT2的转化活性，我们使用基于逆转录病毒的构建体在NIH3T3细胞中表达AKT2。通过软琼脂生长实验和裸鼠肿瘤形成实验确定，AKT2的过表达可使NIH3T3细胞发生转化。通过截短羧基末端富含脯氨酸的70个氨基酸区域，AKT2的致癌活性减弱。为便于对AKT2进行特性分析，我们制备了针对该蛋白的单克隆抗体和多克隆抗体。通过细胞分级分离实验、免疫细胞化学和免疫荧光分析发现，AKT2定位于细胞质。有丝分裂细胞中的蛋白水平比间期细胞中的更丰富。对同步化的胰腺癌细胞进行蛋白质印迹分析表明，有丝分裂细胞中AKT2蛋白的表达水平比间期细胞中的高3至5倍。对植物血凝素刺激的淋巴细胞进行的时间进程研究显示，在添加促有丝分裂原后48 - 72小时，当细胞处于活跃分裂状态时，AKT2 mRNA和AKT2蛋白水平最高。这些数据表明，AKT2可能在细胞周期进程中发挥重要作用，而过表达的AKT2的致癌活性可能由细胞增殖的异常调节介导。
