Singh Paramjeet, Dar Mohd Saleem, Singh Gurjinder, Jamwal Gayatri, Sharma Parduman Raj, Ahmad Muzamil, Dar Mohd Jamal
Academy of Scientific and Innovative Research, New Delhi, India.
Division of Cancer Pharmacology, CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir, India.
J Cell Biochem. 2016 Dec;117(12):2864-2874. doi: 10.1002/jcb.25598. Epub 2016 Jun 3.
Cancer therapeutics is a hot subject and PI3K class 1A isoforms (p110α and p110β) are pursued as major targets. Genetic analysis, biochemical approaches, and structural studies have demonstrated crucial roles for these isoforms in several physiological processes. p110α is critical for insulin signaling, whereas p110β is essential for the growth and differs from p110α in many ways. Here, we have generated GFP-fusion clones of wildtype and mutant version of p110α and p110β and expressed them in HEK293 and cancer cells to examine their subcellular localization and their impact on downstream signaling. In HEK293 cells, p110β GFP-fusion protein is translocated into the nucleus, whereas p110α-GFP stays exclusively in the cytoplasm. This study demonstrates that p110α and p110β oncogenecity, kinase activity, and interaction with p85 regulatory subunit does not have any impact on their subcellular localization. PI3K pathway specific inhibitor, LY294002, abrogated PI3K signaling by reducing pAkt levels, however, the subcellular localization of p110α and p110β remained unchanged. Furthermore, we analyzed the expression of recombinant p110α and p110β in a panel of human cancer cells and observed remarkable differences in their expression levels. The differential expression of recombinant p110α and p110β was observed to be mainly regulated by the endogenous levels of pAkt. Unlike in HEK293, p110α showed nuclear localization in cancer cells in a similar fashion to p110β. Moreover, we observed the PI3K signaling activities in low pAkt expressing cells are mediated by PDK1 and S6K proteins. Finally, p110α and p110β were seen to play an essential role in promoting the cell cycle progression in MCF-7 and HCT-116 cells. J. Cell. Biochem. 117: 2864-2874, 2016. © 2016 Wiley Periodicals, Inc.
癌症治疗是一个热门话题，1A类PI3K亚型（p110α和p110β）被视为主要靶点。基因分析、生化方法和结构研究已证明这些亚型在多个生理过程中发挥关键作用。p110α对胰岛素信号传导至关重要，而p110β对生长至关重要，且在许多方面与p110α不同。在此，我们构建了p110α和p110β野生型及突变型的绿色荧光蛋白（GFP）融合克隆体，并在人胚肾293细胞（HEK293）和癌细胞中表达，以研究它们的亚细胞定位及其对下游信号传导的影响。在HEK293细胞中，p110β-GFP融合蛋白转位至细胞核，而p110α-GFP仅保留在细胞质中。本研究表明，p110α和p110β的致癌性、激酶活性以及与p85调节亚基的相互作用对其亚细胞定位均无影响。PI3K通路特异性抑制剂LY294002通过降低磷酸化Akt（pAkt）水平消除了PI3K信号传导，然而，p110α和p110β的亚细胞定位保持不变。此外，我们分析了重组p110α和p110β在一组人类癌细胞中的表达情况，观察到它们的表达水平存在显著差异。观察到重组p110α和p110β的差异表达主要受pAkt内源性水平的调节。与在HEK293细胞中不同，p110α在癌细胞中与p110β类似地呈现核定位。此外，我们观察到低pAkt表达细胞中的PI3K信号传导活性由磷酸肌醇依赖性蛋白激酶-1（PDK1）和核糖体蛋白S6激酶（S6K）蛋白介导。最后，发现p110α和p110β在促进MCF-7和HCT-116细胞的细胞周期进程中发挥重要作用。《细胞生物化学杂志》，第117卷，第2864 - 2874页，2016年。© 2016威利期刊公司
