A novel protein kinase B (PKB)/AKT-binding protein enhances PKB kinase activity and regulates DNA synthesis
A novel protein kinase B (PKB)/AKT-binding protein enhances PKB kinase activity and regulates DNA synthesis
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DOI：
10.1074/jbc.m500586200
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发表时间：
2005-05-06
影响因子：
4.8
通讯作者：
Asano, T
中科院分区：
文献类型：
--
作者：
Anai, M;Shojima, N;Asano, T
据报道，蛋白激酶B（PKB）/Akt在细胞的存活和/或增殖中起作用。我们利用酵母双杂交筛选系统鉴定出一种与PKB结合的新型蛋白质。这种结合不仅在体内通过过表达两种蛋白质或对内源性蛋白质进行免疫共沉淀得以证实，而且在体外利用谷胱甘肽S - 转移酶融合蛋白也得到了证实。重要的是，这种蛋白质特异性地与PKB的C末端结合，而不与其他AGC激酶结合，并且在没有生长因子刺激的情况下增强PKB的磷酸化和激酶活性。因此，我们将这种Akt特异性结合蛋白命名为APE（Akt - 磷酸化增强子）。由于在用渥曼青霉素或LY294002处理的细胞中未发生APE诱导的PKB磷酸化，所以APE本身不是一种激酶，但似乎增强或延长了磷脂酰肌醇3 - 激酶依赖的PKB磷酸化。在通过小干扰RNA抑制APE的细胞中，随着PKB磷酸化受到抑制，DNA合成显著减少，这表明APE在PKB诱导的增殖中具有协同作用。另一方面，在过表达PKB和APE的细胞中，尽管PKB的基础磷酸化显著增加，但出现了DNA重复复制以及随后的Chk2磷酸化和细胞凋亡，这表明APE参与细胞周期复制许可的调控。综合这些观察结果，APE似乎是PKB磷酸化的一种新型调节剂。此外，APE和PKB之间的相互作用（可能取决于两种蛋白质的表达水平）可能是一种导致增殖和/或细胞凋亡的新型分子机制。
Protein kinase B (PKB)/Akt reportedly plays a role in the survival and/or proliferation of cells. We identified a novel protein, which binds to PKB, using a yeast two-hybrid screening system. This association was demonstrated not only in vivo by overexpressing both proteins or by coimmunoprecipitation of the endogenous proteins, but also in vitro using glutathione S-transferase fusion proteins. Importantly, this protein specifically associates with the C terminus of PKB but not with other AGC kinases and enhances PKB phosphorylation and kinase activation without growth factor stimulation. Thus, we termed this Akt-specific binding protein APE (Akt-phosphorylation enhancer). Since APE-induced phosphorylation of PKB did not occur in cells treated with wortmannin or LY294002, APE itself is not a kinase but seems to enhance or prolong the phosphoinositide 3-kinase-dependent phosphorylation of PKB. In cells in which APE was suppressed by small interfering RNA, DNA synthesis was significantly reduced with suppression of PKB phosphorylation, suggesting a synergistic role of APE in PKB-induced proliferation. On the other hand, in cells overexpressing both PKB and APE, despite markedly increased basal phosphorylation of PKB, both DNA rereplication and subsequent Chk2 phosphorylation and apoptosis were seen, suggesting the involvement of APE in the regulation of cell cycling replication licensing. Taking these observations together, APE appears to be a novel regulator of PKB phosphorylation. Furthermore, the interaction between APE and PKB, possibly dependent on the expression levels of both proteins, may be a novel molecular mechanism leading to proliferation and/or apoptosis.