Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, National University Health Systems, Singapore, Singapore.
PLoS One. 2012;7(9):e45806. doi: 10.1371/journal.pone.0045806. Epub 2012 Sep 26.
Akt, a serine/threonine kinase has been shown to stimulate glycolysis in cancer cells but its role in mitochondrial respiration is unknown. Using PTEN-knockout mouse embryonic fibroblasts (MEF(PTEN-/-)) with hyper-activated Akt as a cell model, we observed a higher respiratory capacity in MEF(PTEN-/-) compared to the wildtype (MEF(WT)). The respiratory phenotype observed in MEF(PTEN-/-) was reproduced in MEF(WT) by gene silencing of PTEN which substantiated its role in regulating mitochondrial function. The increased activities of the respiratory complexes (RCs) I, III and IV were retained in the same relative proportions as those present in MEF(WT), alluding to a possible co-ordinated regulation by PTEN/Akt. Using LY294002 (a PI3K inhibitor) and Akt inhibitor IV, we showed that the regulation of enzyme activities and protein expressions of the RCs was dependent on PI3K/Akt. There was insignificant difference in the protein expressions of mitochondrial transcription factor: peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and its downstream targets, the nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (mtTFA) between MEF(PTEN-/-) and MEF(WT). Similarly, mRNA levels of the same subunits of the RCs detected in Western blots were not significantly different between MEF(PTEN-/-) and MEF(WT) suggesting that the regulation by Akt on mitochondrial function was probably not via gene transcription. On the other hand, a decrease of total 4E-BP1 with a higher expression of its phosphorylated form relative to total 4E-BP1 was found in MEF(PTEN-/-), which inferred that the regulation of mitochondrial respiratory activities by Akt was in part through this protein translation pathway. Notably, gene silencing of 4E-BP1 up-regulated the protein expressions of all RCs and the action of 4E-BP1 appeared to be specific to these mitochondrial proteins. In conclusion, PTEN inactivation bestowed a bioenergetic advantage to the cells by up-regulating mitochondrial respiratory capacity through the 4E-BP1-mediated protein translation pathway.
Akt 是一种丝氨酸/苏氨酸激酶，已被证明可刺激癌细胞中的糖酵解，但它在线粒体呼吸中的作用尚不清楚。我们使用 Akt 过度激活的 PTEN 敲除小鼠胚胎成纤维细胞 (MEF(PTEN-/-)) 作为细胞模型，观察到 MEF(PTEN-/-) 的呼吸能力高于野生型 (MEF(WT))。通过基因沉默 PTEN 在 MEF(WT) 中再现了 MEF(PTEN-/-) 的呼吸表型，证实了它在调节线粒体功能中的作用。呼吸复合物 (RC) I、III 和 IV 的活性增加保持与 MEF(WT) 中存在的相对比例相同，暗示了 PTEN/Akt 的可能协调调节。使用 LY294002（一种 PI3K 抑制剂）和 Akt 抑制剂 IV，我们表明 RC 的酶活性和蛋白表达的调节依赖于 PI3K/Akt。在 MEF(PTEN-/-) 和 MEF(WT) 之间，线粒体转录因子：过氧化物酶体增殖物激活受体 γ 共激活因子 1-α (PGC-1α) 及其下游靶标核呼吸因子 1 (NRF-1) 和线粒体转录因子 A (mtTFA) 的蛋白表达没有显著差异。同样，在 Western blot 中检测到的 RC 相同亚基的 mRNA 水平在 MEF(PTEN-/-) 和 MEF(WT) 之间也没有显著差异，这表明 Akt 对线粒体功能的调节可能不是通过基因转录。另一方面，在 MEF(PTEN-/-) 中发现总 4E-BP1 减少，而其磷酸化形式相对于总 4E-BP1 的表达增加，这表明 Akt 通过这条蛋白翻译途径调节线粒体呼吸活性。值得注意的是，基因沉默 4E-BP1 上调了所有 RC 的蛋白表达，并且 4E-BP1 的作用似乎是针对这些线粒体蛋白的。总之，PTEN 失活通过 4E-BP1 介导的蛋白翻译途径上调线粒体呼吸能力，为细胞赋予了生物能量优势。
